Science of the Total Environment 946 (2024) 174020

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Isolation of biocrust cyanobacteria and evaluation of Cu, Pb, and Zn

immobilisation potential for soil restoration and sustainable agriculture
Carlotta Pagli a, b, c, * , Sonia Chamizo d, b , Giada Migliore e , Lorenza Rugnini a ,
Giovanni De Giudici f , Roberto Braglia a , Antonella Canini a , Yolanda Cantón b, g
a
Department of Biology, University of Rome Tor Vergata, Italy
b
Department of Agronomy, University of Almería, Spain
c
PhD Program in Evolutionary Biology and Ecology, University of Rome Tor Vergata, Italy
d
Department of Desertification and Geo-Ecology, Experimental Station of Arid Zones (EEZA-CSIC), Almería, Spain
e
ENEA, Territorial and Production Systems Sustainability Department, Italy
f
Department of Chemical and Geological Sciences, University of Cagliari, Italy
g
Center for Research on Scientific Collections of the University of Almeria (CECOUAL), Spain

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Cyanobacteria comprised 26 % of the

bacterial community in metal-
contaminated soils.
• Nostoc commune strains from ex-mining
sites varied in growth rates and EPS
production.
• N. commune strains were able to effec­
tively immobilise copper, lead, and zinc.
• Cu immobilisation was strongly associ­
ated to cyanobacterial EPS production. Cyanobacteria Biomass production
a) Nostoc commune RI23
• Results suggest potential for soil biore­
Extracellular polymeric substances (EPS) production
b) Nostoc commune AB55 Heavy metals uptake

mediation with indigenous

c) Nostoc commune NR64

cyanobacteria.

Copper (Cu) Lead (Pb) Z inc (Z n)

A R T I C L E I N F O A B S T R A C T

Editor: Christopher Rensing Soil contamination by heavy metals represents an important environmental and public health problem of global
concern. Biocrust-forming cyanobacteria offer promise for heavy metal immobilisation in contaminated soils due
Keywords: to their unique characteristics, including their ability to grow in contaminated soils and produce exopoly­
Bioremediation saccharides (EPS). However, limited research has analysed the representativeness of cyanobacteria in metal-
Nostoc commune
contaminated soils. Additionally, there is a lack of studies examining how cyanobacteria adaptation to spe­
Soil contamination
cific environments can impact their metal-binding capacity. To address this research gap, we conducted a study
Exopolysaccharides
Microbial community analysing the bacterial communities of cyanobacteria-dominated biocrusts in a contaminated area from South
Metal-binding capacity Sardinia (Italy). Additionally, by using two distinct approaches, we isolated three Nostoc commune strains from
cyanobacteria-dominated biocrust and we also evaluated their potential to immobilise heavy metals. The first
isolation method involved acclimatizing biocrust samples in liquid medium while, in the second method, biocrust
samples were directly seeded onto agar plates. The microbial community analysis revealed Cyanobacteria,

* Corresponding author at: Department of Biology, University of Rome Tor Vergata, Italy.
E-mail addresses: [email protected], [email protected] (C. Pagli).

https://doi.org/10.1016/j.scitotenv.2024.174020
Received 23 March 2024; Received in revised form 30 April 2024; Accepted 13 June 2024
Available online 17 June 2024
0048-9697/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
C. Pagli et al. Science of the Total Environment 946 (2024) 174020

Bacteroidota, Proteobacteria, and Actinobacteria as the predominant groups, with cyanobacteria representing
between 13.3 % and 26.0 % of the total community. Despite belonging to the same species, these strains
exhibited different growth rates (1.1–2.2 g L− 1 of biomass) and capacities for EPS production (400–1786 mg
L− 1). The three strains demonstrated a notable ability for metal immobilisation, removing up to 88.9 % of Cu,
86.2 % of Pb, and 45.3 % of Zn from liquid medium. Cyanobacteria EPS production showed a strong correlation
with the removal of Cu, indicating its role in facilitating metal immobilisation. Furthermore, differences in Pb
immobilisation (40–86.2 %) suggest possible environmental adaptation mechanisms of the strains. This study
highlights the promising application of N. commune strains for metal immobilisation in soils, offering a potential
bioremediation tool to combat the adverse effects of soil contamination and promote environmental
sustainability.

1. Introduction solutions to address heavy metal contamination in agricultural soil is

emerging as an increasingly pressing concern.
Over the last decades soil contamination by heavy metals, primarily The remediation of metal-contaminated sites has traditionally relied
caused by industrial activities, agricultural practices, and natural sour­ on physical remediation methods such as soil thermal desorption and
ces, has become a prominent and concerning environmental and public soil replacement, as well as chemical remediation approaches including
health issue on a global scale (Ilyin et al., 2004; Lu et al., 2012; Zhang chemical leaching, chemical stabilization, electrokinetic remediation-
and Wang, 2020; Alengebawy et al., 2021; European Environment permeable reactive barrier, and chemical oxidation/reduction (Bolan
Agency, 2023). Different levels of soil pollution due to heavy metals et al., 2014; Dhaliwal et al., 2020; Song et al., 2022). However, these
have been detected all around the globe (Su, 2014; Hou et al., 2020; Liu techniques sometimes lack selectivity and often render metal ions more
et al., 2022). For instance, in China, about 10 % of agricultural soils have soluble and mobile, leading to groundwater pollution and negatively
been found to be affected by heavy metal pollution (Kou et al., 2018). In affecting soil quality. Chemical/physical methods are also laborious and
other countries, including France, Sweden, Slovakia, Hungary, and expensive, and many times provide limited results for heavy metal
Austria, over 200,000 sites have been categorized as heavy metal contamination removal (Wu et al., 2010). Therefore, considering re­
contaminated. Meanwhile, Greece and Poland have reported about agent toxicity, unselectivity, inefficacy and high cost of traditional
contaminated 10,000 sites (Su, 2014). In Italy, despite the absence of remediation techniques, bioremediation techniques have emerged as
comprehensive studies on heavy metal contamination of soils, this issue more environmentally friendly and economically viable alternatives.
affects many localities in various regions of the country (Buccheri et al., These include phytoremediation and microbial remediation, which are
2018; Agrelli et al., 2020; Cicchella et al., 2020). In contrast to organic respectful of both the environment and human health (Mohapatra et al.,
pollutants, metal(loids) are not easily degraded by microbes or chem­ 2019; Priya et al., 2022; Rizvi et al., 2020; Shen et al., 2022). Among
icals (Raffa et al., 2021). Consequently, after their introduction into the various microorganisms, the employment of cyanobacteria, a widely
soil, the total concentration of these metal(loids) can remain for an distributed phylum of photoautotrophic bacteria, has attracted scientific
extended period depending on the type of soil and nature of the metal attention. Cyanobacteria offer potential for bioremediation of heavy
(Bolan et al., 2014). For example, in the Montevecchio-Ingurtosu mining metals both by biosorption and bioaccumulation processes (Al-Amin
district in Southern Sardinia (Italy), >30 years after mining activity et al., 2021). The effectiveness of cyanobacteria in heavy metal immo­
ceased, we observe that the landfill materials still exhibit substantial, bilisation has primarily focused on water cyanobacteria strains for
and at times remarkably high, concentrations of Pb (up to 77,681 ppm), application in wastewater treatment (Ahmad, 2022). However, inves­
Zn (up to 48,194 ppm), and to a lesser extent, Cu (up to 3154 ppm), far tigation of soil cyanobacteria for application in heavy metal immobili­
exceeding the permissible limit values set by European Directive 2008/ sation has been much more limited, despite their potential for treating
50/CE (Fanfani et al., 2001). contaminated soils in both natural and agricultural environments (Ciani
It is widely recognized that the overuse of fertilizers and pesticides and Adessi, 2023; Cui et al., 2020; Dutta et al., 2022; Potnis et al., 2021).
contributes to heavy metal contamination in the soil. Indeed, fertilizers In soils, cyanobacteria usually form assemblages with other photo­
may contain traces of heavy metals such as Cd, Cu, Zn, Pb, or As (Hou autotrophic organisms (algae, lichens, mosses) and heterotrophic or­
et al., 2020; Lu et al., 2012; Nziguheba and Smolders, 2008). These el­ ganisms (fungi, bacteria) residing on top of the uppermost millimetres of
ements are frequently incorporated into micronutrient fertilizers or as soil, forming the community known as biological soils crust or biocrust
components of balanced fertilizer formulations. For instance, Cu has (Weber et al., 2022). Biocrusts cover approximately 12 % of the Earth's
several applications in agriculture due to its use as an essential micro­ terrestrial surface (Rodriguez-Caballero et al., 2018), including
nutrient for plants and as an effective fungicide (Lamichhane et al., contaminated areas and regions with extreme temperatures and salt
2018; Moreira et al., 2022). Cu-based compounds are extensively concentrations (Xiao et al., 2022). These communities exhibit remark­
applied as treatments to protect seeds and plants from fungal and bac­ able resistance to harsh conditions and may survive in situations with
terial pathogens, particularly for olive groves and fruit trees (Rehman high levels of heavy metal contamination (Fan et al., 2023). Addition­
et al., 2019). In these specific crops, it is common for Cu concentration ally, biocrusts are important for soil health and ecosystem resilience,
levels to exceed established limits (Ballabio et al., 2018; Provenzano due to their recognized effects improving soil water availability,
et al., 2010; Steinmetz et al., 2017; European Commission, 2018). Also, fertility, biodiversity and on erosion control (Chamizo et al., 2016;
phosphate fertilizers can be associated with the presence of metals, Antoninka et al., 2020; Román et al., 2021). Biocrust-forming cyano­
including Ni, Cd, Zn, Pb, As, and Cr. This correlation stems from the bacteria could be a promising solution for soil heavy metal immobili­
natural presence of these metals in phosphate rock, a common phos­ sation due to their unique characteristics, such as the ability to grow in
phorus source for fertilizer production (Gupta et al., 2014; Nziguheba contaminated soils and the production of extracellular polymeric sub­
and Smolders, 2008). The introduction of Pb from fertilizers can result in stances (EPS). Cyanobacteria possess a cell wall characterized by a
soil contamination, hazarding crop health and food safety (Nacke et al., thicker peptidoglycan layer and with a higher degree of cross-linking
2013; Khan et al., 2018). Once Pb is present in the soil, it tends to remain among the polysaccharidic chains in comparison with other Gram-
there for an extended period, potentially persisting for decades or even negative bacteria (Hoiczyk and Hansel, 2000). Due to the presence of
centuries, and may eventually continue to pollute the soil, putting at risk these negatively charged EPS cell layers, cyanobacteria are capable of
the ecosystem due to its high persistence (Weissengruber et al., 2018; passively absorbing high amounts of solubilized metals (De Philippis
Frank et al., 2019). Considering these data, the search for effective et al., 2011; Ciani and Adessi, 2023). Indeed, the synthesis of EPS by

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C. Pagli et al. Science of the Total Environment 946 (2024) 174020

cyanobacteria plays a crucial role in interfering with the mobility and intense mining activity (Fig. 1). The localities and GPS coordinates are
bioavailability of heavy metal ions in the soil. Cyanobacteria EPS chelate listed in Table 1.
and immobilise heavy metals, mitigating their harmful effects and Mean annual precipitation ranges approximately from 600 to 800
facilitating their removal from the environment (De Philippis et al., mm, and average annual temperatures fluctuate between 15 and 17 ◦ C.
2011). To further improve the efficacy of cyanobacteria in heavy metal Climate is characterized by dry spring and summer and wet autumn and
remediation, it is necessary to identify suitable species with key func­ winter (Dore et al., 2020).
tional characteristics, such as high metal immobilisation capacity, easy Soils show sandy-loam texture, with pH ranging from 5.4 to 6.7, soil
and rapid ex-situ culture, and soil survival and colonisation. Native organic carbon content is low and varies from 1 to 16 g kg− 1, and
species collected and isolated from contaminated environments, already average total nitrogen content is ~2 g kg− 1 (Vacca et al., 2012; Nicolas
adapted to the site-specific conditions to be restored, potentially can et al., 2022). These mining sites are significantly affected by heavy metal
show these traits. However, there is limited research focusing on how contamination, especially from Cd, Cr, Pb, and Zn (Satta et al., 2012; Boi
the adaptation of cyanobacteria to their specific environment can in­ et al., 2021). Additionally, mine wastes in the area exhibit significant
fluence their metal-binding capacity. heterogeneity in terms of metal concentrations. This heterogeneity is
To fill in this gap, in this study, the bacterial communities of primarily attributed to the diverse extraction methods employed
cyanobacteria-dominated biocrusts inhabiting a contaminated area in throughout the history of mining operations in the region (Bacchetta
South Sardinia (Italy) were analysed at the phylum level and different et al., 2017; Boi et al., 2021). Vegetation cover is low and the most
cyanobacteria strains were isolated and tested for their ability to frequent plant species in the area are Rumex bucephalophorus, Pistacia
immobilise heavy metals. Therefore, the objectives of this work were: i) lentiscus, Helichrysum microphyllum subsp. Tyrrhenicum, Euphorbia cupani
to examine the bacterial community composition and diversity of bio­ pithyusa, Juncus acutus, and Carlina corymbose (Angius et al., 2011;
crust communities in four heavy metal contaminated sites; ii) to isolate Sprocati et al., 2014; De Giudici et al., 2015; Medas et al., 2019; Fois
and identify cyanobacteria strains from contaminated soils, and thus et al., 2023). Biocrusts are present forming small patches in the inter­
potentially applicable for soil bioremediation; and iii) to evaluate the shrub spaces and mainly consist of well-developed cyanobacteria
growth in liquid medium, the EPS production and the heavy metal biocrusts.
removal of the isolated strains. Therefore, this research directly ad­ The soil samples were collected in April 2022. At each site, three soil
dresses the global issue of soil contamination by heavy metals, with a samples at 1 cm of depth were randomly taken from cyanobacteria-
specific focus on exploring the use of native cyanobacteria in soil dominated biocrust communities with a sterile trowel and directly
restoration and sustainable agriculture. stored in 50 mL sterile Falcon® tubes and kept dry in the dark until their
use. Part of the samples (~1 g) was weighed and stored at − 20 ◦ C for
2. Material and methods further metagenomic analysis. The resting soil was air-dried, sieved to 2
mm, and ground for the determination of Cu, Pb, and Zn concentrations,
2.1. Study sites and biocrust sampling using ICP-MS (iCAP TQ, Thermo Scientific). These metals have been
selected due to their significant impact on the phenomenon of agricul­
The study sites consist of ex-mining areas, located in the Medio tural soil contamination. Before the analysis, the samples underwent
Campidano and Carbonia-Iglesias provinces in Southwest Sardinia, Italy acid digestion using a microwave oven, specifically employing a single
(Fig. 1).
South Sardinia region is rich in mineral resources, and mining ac­
tivities have significantly contributed to the local economy during the Table 1
20th century (Manca et al., 2019). The minerals extracted in this area Sites and coordinates of biocrust samples.
include Pb, Zn, Fe, Mn, Cu, coal, salt, and other minerals (Fanfani et al., Site Coordinates Biocrust sample
2001; Bacchetta et al., 2015; Manca et al., 2019). Lead and zinc were
Mine waste Sanna 39◦ 32′03.7″N 8◦ 31′41.8″E CS
particularly important due to their historical significance and economic Rio Irvi banks 39◦ 32′47.9″N 8◦ 28′40.6″E RI
value, with abundant ore deposits supporting a thriving mining industry Naracauli 39◦ 31′09.2″N 8◦ 29′23.5″E NR
(Vacca et al., 2012). Ex-mine S. Acqua Bona 39◦ 28′36.3″N 8◦ 30′07.1″E AB
For this study, we selected four localities that have experienced

Fig. 1. General view of the study area (a). Location of sampling sites: Mine waste Sanna, CS (b), Rio Irvi banks, RI (c), Naracauli, NR (d), and Ex-mine S. Acqua Bona,
AB (e).

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reaction chamber digestion system (UltraWAVE “ERC”, MILESTONE). nlm.nih.gov) of cyanobacteria genera (Hauer and Komárek, 2022).
The results of the morphological identification were further confirmed
2.2. Analyses of microbial community composition by the genetic identification. Genomic DNA was extracted from pure
cultures using the GeneMATRIX Plant & Fungi DNA Purification Kit. The
Biocrust samples were first pre-treated with three freeze–thaw cycles 16S rRNA gene was amplified using the forward primer 359F (5′- GGG
and ground in a mortar using a pestle (Loza et al., 2013). DNA extraction GAA TYT TCC GCA ATG GG -3′) and reverse primer 781Ra/781Rb (5′-
from biocrust was performed using the QIAGEN DNeasy PowerSoil kit GAC TAC TGG GGT ATC TAA TCC CAT T -3′/5′- GAC TAC AGG GGT ATC
(Qiagen, Germantown, MD, USA) following the manufacturer's protocol. TAA TCC CTT T-3′) (Nübel et al., 1997). The products were purified
The microbial community composition was analysed using Next Gen­ using the Wizard® SV Gel and PCR Clean-Up System Purification Kit and
eration Sequencing (NGS). For library preparation, a “two-step PCR sequenced in both directions using the Sanger method by AB 3500 DNA
protocol” was employed, which involved DNA amplification, using analyser (Life Technologies, Thermo Fisher Scientific Inc., US). For the
primers modified with universal tags, followed by enzymatic purifica­ 16S rRNA gene sequences, the closest matching taxa were searched
tion using Thermostable Exonuclease I. Subsequently, a second PCR step using the National Center for Biotechnology Information (NCBI)
was carried out where Illumina Nextera XT indexes were attached to the (https://www.ncbi.nlm.nih.gov), and nucleotide sequences were
universal tags. The samples were then normalized, multiplexed, and deposited under the accession numbers P256154, PP256155, and
sequenced using the MiSeq platform (Illumina, San Diego, CA, USA). PP256156.
The sequence data were processed with Qiime2 tools version 4.0.
Initially, adapter trimming was performed utilizing Cutadapt. To ach­ 2.4. Growth curves and EPS production
ieve this, raw sequences from the Illumina MiSeq platform were pre-
filtered, which entailed removing primer sequences from both forward Once isolated, cyanobacteria strains were first inoculated in multi­
and reverse reads. This operation was executed with the default pa­ well plates and then relocated in flasks with BG11 liquid medium and
rameters of Cutadapt. After adapter sequence removal, read denoising incubated for 14–19 days at room temperature (T = 25 ± 1 ◦ C), irra­
was carried out utilizing the DADA2 toolset. This encompassed a series diance of 200 μmol photons m− 2 s− 1 and aerated with air sterilized by
of steps including read quality filtering and trimming, estimation of filtering (0.22 μm, Millex EMD Millipore™). Cyanobacteria strains were
error rates, dereplication, read merging, and the detection of chimeric grown in 100 mL Erlenmeyer flasks, in triplicate, starting from an initial
sequences. The resulting outcome of these preprocessing steps yielded a concentration of ~0.5 g L− 1. During incubation, cyanobacteria growth
feature table that served as the foundational dataset for subsequent was monitored through measurements of dry weight and chlorophyll a.
analyses. In the following steps, each sample was analysed individually. In addition, the production of total and released exopolysaccharides
To refine the dataset, a feature filtering approach was applied, leading to (EPS) at the beginning and at the end of the experiment (after 14 days)
the exclusion of features representing <0.01 % of the total data. Addi­ were analysed.
tionally, singleton features were removed. Taxonomy was assigned The dry weight of culture was determined by filtering 5 mL of the
using trained sequences (OTUs at 99 %) from Silva database version culture onto pre-weighed filter papers with 0.45 μm pore size. The filter
138. The alpha biodiversity within the biocrust samples was quantified was then dried at a temperature of 80 ◦ C for 24 h (Roncero-Ramos et al.,
by the Shannon and the Simpson diversity index (1-D), and the Inverse 2022). The concentration of chlorophyll a was determined according to
Simpson diversity index (1/D) by using R Statistical Software (v4.2.1; R Singh et al. (2016). 5 mL of culture was centrifuged at 2500 ×g at 9 ◦ C of
Core Team, 2021). These indices are commonly used to assess and temperature for 7 min. The supernatant was discarded, then 5 mL of
compare the richness and evenness of species in ecological communities, methanol was added to the pellet, vortexed and heated at 70 ◦ C of
providing valuable insights into the overall diversity of the biocrust temperature for 5 min. The sample was then centrifuged again at 2500g,
samples. and the absorbance of the supernatant was measured using a spectro­
photometer at 665 nm and 750 nm wavelengths. The chlorophyll a
2.3. Cyanobacteria strains isolation and identification content was determined using the equation of Ritchie (2006):
Vm
Two different isolation methods were employed to obtain cyano­ Chl a = 12.9447 × (Abs665 − Abs750) × (1)
Vs
bacteria isolates from the biocrust samples. According to Rippka et al.
(1979), small biocrust fragments (~ 0.5 g) were placed in Eppendorf Chl a is Chlorophyll a (μg mL− 1), Abs665 and Abs750 are the values
tubes® with BG110 medium, shaken, and kept at 19 ± 1 ◦ C and at 40 of absorbance at 665 nm and 750 nm wavelengths, respectively, Vm is
μmol photon m-2 s-1 for 4 h to allow cyanobacteria acclimatization. the volume of methanol and Vs is the sample volume (mL).
Then, 400 μL of medium was seeded onto agar-solidified Petri dishes The content of both total and released EPS was determined using the
with BG11 and BG110 media at 1.5 % (w/v), added with cycloheximide phenol‑sulfuric acid method (Dubois et al., 1956). For the released EPS,
(0.1 mg mL− 1) to avoid fungal contamination. BG11 medium contains 5 mL of culture was previously centrifuged at 4000g for 30 min and 1 mL
nitrogen, while BG110 medium, without nitrogen source, is specifically of the supernatant was taken to quantify carbohydrate content (Chamizo
used to select nitrogen-fixing cyanobacteria (Stanier et al., 1971). et al., 2020). When EPS amount exceeded the detection limit, 1:5 or 1:10
Furthermore, biocrust samples of ~1 cm2 were positioned directly in dilutions were applied to the samples, after which the phenol‑sulfuric
both BG11 and BG110 agar medium in the centre of the dish. The Petri assay was applied on 1 mL aliquots of the samples. This method is based
dishes were kept at the temperature of 19 ± 1 ◦ C, with a 12:12 photo­ on the principle that carbohydrates, when reacted with concentrated
period, and at 40 μmol photon m− 2 s− 1 of irradiance for 6 weeks. After sulfuric acid, undergo dehydration and form furfural derivatives. The
this time, single colonies were re-streaked on fresh agar plates and this absorbance was measured at 488 nm, with glucose used as standard for
process was repeated until a single morphotype was obtained (Roncero- comparison.
Ramos et al., 2019).
Identification of cyanobacteria strains was carried out using a 2.5. Heavy metal removal experiments
multiphase approach that combined morphological characterization by
microscope observation and molecular analyses (Roncero-Ramos et al., The isolated cyanobacteria strains were tested for their ability to
2019). The isolated strains were observed under a light microscope at a immobilise Zn, Pb, and Cu, which were selected due to their presence at
magnification of 40 X and the morphological identification was based on high concentrations at the selected study sites as well as their note­
literature (Komárek and Anagnostidis, 2005; Komárek, 2018; Singh worthy relevance in the context of agricultural soil pollution resulting
et al., 2020) combined with the use of GenBank (https://submit.ncbi. from fertilizer inputs. A preliminary treatment of the cultures was car

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ried out using 0.1 M HCl to remove the metal ions bound to the nega­
tively charged groups (Micheletti et al., 2008). After 30 min, the cultures
were centrifuged at 2500 ×g for 10 min and resuspended 3 times in
deionized water to remove residual HCl. The metal solutions of Cu, Pb,
and Zn were respectively prepared using copper nitrate (VWR Chem­
icals), lead (II) nitrate (VWR Chemicals), and zinc sulfure (Scharlau).
Each cyanobacterial strain was incubated at the concentration of 2 g L− 1.
Individual metals were added to the culture with a concentration of 10
mg L− 1. For each treatment, 3 replicates were set up, each with a volume
of 100 mL. The negative control was a solution of the selected metal
without cyanobacteria. The cultures were incubated for 90 min at 100
rpm shaking at the temperature of 25 ± 1 ◦ C, providing an optimal
timeframe for the accumulation of heavy metals by cyanobacteria
(Chojnacka et al., 2005; Micheletti et al., 2008). During the experiment,
the cultures were maintained at pH 4. After 90 min, the cultures were Fig. 2. Relative abundance of dominant bacteria phyla (>1.0 % of total
filtered through a 0.45 μm membrane. The final metal concentration was reading) for the four study sites: Ex-mine S. Acqua Bona (AB), Mine waste
detected with ICP-MS (iCAP TQ model, Thermo Scientific), and the Sanna (CS), Naracauli (NR), and Rio Irvi banks (RI).
metal removal (mg metal removed g− 1 dry weight) was calculated with
the following formula: in CS, NR, and RI. Crenarchaeota, RCP2-54, WPS-2, Methylomirabilota,
and Sumerlaeota were exclusively detected in RI, while Dependentiae
Ci − Ct
q=V (2) was identified in AB, CS, and NR. Nitrospirota, SAR324_clade, and
m
Fibrobacterota were uniquely found in AB, NR, and CS, respectively.
Where V is the sample volume (mL), Ci and Ct are the initial and final According to Shannon, Simpson and Inverse Simpson index, the
heavy metal concentrations, respectively, and m is the dry weight value biocrust bacterial biodiversity was highest for AB and CS sites, followed
for sample (g) (Volesky and May-Phillips, 1995). by RI and finally by NR (Fig. 3). The Shannon index ranged between 4.4
and 5, the Simpson index between 0.95 and 0.99, and the Inverse
Simpson between 20 and 70.
2.6. Statistical analysis
3.2. Identification of cyanobacterial strains
Repeated measures ANOVA was used to examine variations among
cyanobacteria strains in dry weight and chlorophyll a values over time. We isolated 56 cyanobacterial morphotypes from the study sites: 10
One-way ANOVA was employed to analyse differences in released EPS from CS, 14 from RI, 13 from NR, and 19 from AB. Of all the isolated
production, total EPS production, and metal removal among cyano­ strains, we focused on the Nostocaceae family, which proved to have
bacteria strains, and when statistically significant, the Tukey post-hoc strong bioremediation properties (Morales et al., 2017; Williams et al.,
test was applied for multiple comparisons. All variables were previ­ 2019). Notably, members of the Nostocaceae family were found to be
ously tested for normality and homogeneity of variance using the Sha­ present in all sampled sites, emphasizing their widespread distribution.
piro–Wilk and Levene's test. Non-metric multidimensional scaling After this screening, three Nostoc-like cyanobacteria strains (RI23, AB55,
(NMDS) was used to visually detect relations between cyanobacteria's and NR64), filamentous with interconnected cells, heterocysts, and
capacity to immobilise heavy metals and the presence of heavy metal in gelatinous mucilage surrounding the filaments, were selected (Fig. 4).
the place of isolation. All statistical analyses were performed using R 16S rRNA molecular analysis confirmed the three isolated cyanobacte­
Statistical Software (v4.2.1; R Core Team, 2021) and IBM SPSS Statistics rial strains as Nostoc commune with similarity values ranging between
(v29.0; IBM Corp). Results were considered significant when p value < 99.2 and 99.75 % according to NCBI database.
0.05.
3.3. Growth curves and EPS production
3. Results
Growth curves of the three isolated cyanobacteria strains were
3.1. Biocrust bacterial community composition and diversity assessed by measurement of dry weight and chlorophyll a overtime.
Both time and strain had a significant effect (p < 0.001) on dry weight
By NGS sequencing we obtained 133,890 high-quality reads, classi­ and chlorophyll a content (Fig. 5). Nostoc commune AB55 strain showed
fied into 1202 operational taxonomic units (OTUs). We found nine main the highest biomass and chlorophyll a content, reaching its maximum
phyla across all biocrust samples. Cyanobacteria, Bacteroidetes, Pro­ growth between 7 and 9 days and resulting in a biomass of 2.2 g L− 1 and
teobacteria and Actinobacteria were the most abundant phyla, with chlorophyll a content of 12.4 g L− 1, whereas strains RI23 and NR64
different proportions depending on the biocrust sample (Fig. 2). Cya­ showed significantly lower biomass and chlorophyll a content (Fig. 5).
nobacteria were prevalent in CS (26.0 %), RI (19.9 %), and NR (22.5 %), The maximum dry weight recorded for these strains was between 1.1
while they exhibited a lower relative abundance (13.3 %) in AB. Bac­ and 1.3 g L− 1, while the maximum chlorophyll a content was approxi­
teroidota and Proteobacteria respectively accounted for 14.4 % and mately 2.7 g L− 1. The growth peak was reached after 9 days for strain
21.8 % of the relative abundance in CS, 15.5 % and 14.3 % in RI, 22.7 % RI23, after 12 days for strain AB55, and after 14 days for strain NR64.
and 14.1 % in NR, and 23.8 % and 19.3 % in AB. Actinobacteria There were significant differences in released EPS (p = 0.000329 at
exhibited a similar abundance in CS, RI, and AB (around 17 %), but day 0 and p = 0.00675 at day 14) and total EPS (p = 0.000171 at day
showed a lower abundance in NR (11.2 %). Other phyla characterizing 0 and p = 0.000942 at day 14) between strains. The strains AB55 and
biocrust samples included Acidobacteriota, Chloroflexi, Planctomyce­ NR64 showed the highest released EPS concentrations, both at the
tota, and Verrucomicrobiota. beginning (day 0) of the experiment and after 14 days. The strain RI23
Among the taxa with lower representation (<2 %), the phyla Gem­ showed a significantly lower released EPS content (Fig. 6). There were
matimonadota, Armatimonadota, Myxococcota, Bdellovibrionota, no significant differences in the production of released EPS between
Patescibacteria, Firmicutes, and Deinococcota were present in all bio­ AB55 and NR64. The strain AB55 also exhibited the highest total EPS
crust samples, while Chlorophyta and Abditibacteriota were found only content, with an average value of 1786 mg L− 1 after 14 days. The strains

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Fig. 3. α-diversity indices (Shannon, Simpson, Inverse Simpson) calculated for the four study sites: Ex-mine S. Acqua Bona (AB), Mine waste Sanna (CS), Naracauli
(NR), and Rio Irvi banks (RI).

Fig. 4. Microphotographs of cyanobacterial strains isolated from biocrust: a) Nostoc commune RI23, b) Nostoc commune AB55, c) Nostoc commune NR64. Scale Bar
= 10 μm.

Fig. 5. Growth curves based on dry weight (a) and chlorophyll a (b) measurements. Means and standard deviations are presented along with p-values from repeated
measures ANOVA for both “Between” and “Within” subject comparison. Different letters are included only in case of significant differences between strains at
each day.

NR64 and RI23 showed a significantly lower total EPS content, with cyanobacteria strain (Fig. 7). The strains showed significant differences
values consistently lower, both at the beginning and after 14 days of in Cu (p = 0.000367), Pb (p = 0.000298), and Zn (p = 0.0355) removal
incubation. from the liquid medium. The AB55 and NR64 strains demonstrated the
ability to immobilise all tested metals (Cu, Pb, Zn), whereas strain RI23
did not exhibit any Cu removal capacity. For Cu immobilisation, strain
3.4. Heavy metal immobilisation AB55 showed the highest removal capability, removing up to 88.9 % of
the metal from the liquid medium, and strain NR64 immobilised up to
The metal removal capacity differed according to the metal and the

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Fig. 6. Released (a and b) and total EPS (c and d) content in cyanobacterial cultures on day 0 and day 14. Letters mark significant differences (p < 0.05) in EPS
production among different strains. All strains showed a significant increase in EPS production after 14 days.

Fig. 7. Heavy metal immobilisation (q), expressed as mg of metal removed per g of biomass dry weight, by the three isolated cyanobacterial strains: Nostoc commune
RI23, Nostoc commune AB55, and Nostoc commune NR64. Letters mark significant differences (p < 0.05) in q among different strains.

25.5 % of Cu. In the case of Pb, the strain RI23 showed the highest Pb 4. Discussion
removal, up to 86.2 % of Pb in solution. Strains AB55 and NR64 had a
lower capacity for Pb immobilisation, at 40 % and 50 % respectively. All 4.1. Biocrust bacterial community composition
strains were less effective in Zn uptake, with percentages below 50 %.
NR64 was the most efficient, removing 45.3 % of the Zn in solution, The microbial communities of biocrust samples from four heavy
while AB55 was the least efficient, with only 33 % removed. metal-contaminated sites in Sardinia were characterized by the presence
Fig. 8 illustrates the NMDS showing the different cyanobacteria of four predominant bacterial phyla: Cyanobacteria, Bacteroidota, Pro­
strains, the content of both released and total EPS by the cyanobacterial teobacteria and Actinobacteria. These phyla were found in varying
strains, cyanobacteria capability to immobilise the selected heavy abundances across the samples, in agreement with previous studies on
metals, and the content of these metals in the isolation environment. biocrust from arid soils (Moreira-Grez et al., 2019; Gabay et al., 2022;
Average heavy metal concentrations on each study site are reported in Vega-Cofre et al., 2023). Furthermore, while some less prevalent phyla
Table 2. The NMDS ordination indicates an association between Pb were consistently present in all samples, others were exclusive to spe­
content in the soil from which the cyanobacteria was isolated and the cific samples, such as Crenarchaeota, RCP2–54, WPS-2, Methylomir­
ability of cyanobacteria to immobilise this metal. abilota, and Sumerlaeota, which were found solely in the RI sample. All
Such association between metal content in the cyanobacteria isola­ these phyla collaborate to support the structure and functionality of
tion sites and cyanobacteria removal capability was not observed for Cu biocrusts, contributing to nutrient cycling, soil conservation, and the
and Zn. Meanwhile, Cu removal was associated with high contents of maintenance of the ecosystem (Wang et al., 2023). Among these mi­
total and released EPS. croorganisms, Cyanobacteria play a pivotal role as primary producers
and pioneers in the establishment of biocrust communities (Román
et al., 2018; Deng et al., 2020; Rubio and Lázaro, 2023). The relative
abundances of cyanobacteria in the analysed samples, ranging from
13.3 % to 26 %, are relatively high compared to other phyla. This
pattern is characteristic of biocrusts in their early stages of development,

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Fig. 8. Non-metric multidimensional scaling (NMDS) ordination of Biocrust cyanobacteria strains. Vectors indicate the magnitude and direction of correlations
between the strains, EPS production, metal removal, and heavy metal content in the soil from which the strains were isolated.

Table 2
Heavy metal values for the four sampling sites.
1 1 1 1 1 1 1 1 1 1
Study sites Al g kg− Cr mg kg− Mn g kg− Fe g kg− Ni mg kg− Cu mg kg− Zn g kg− As mg kg− Cd mg kg− Pb g kg−

CS 15.02 34.97 2.33 42.51 23.92 89.89 8.54 44.07 33.06 1.78
RI 7.05 36.47 3.43 62.7 30.88 303.25 12.27 115.68 40.42 16.04
NR 4.39 27.21 3.99 69.25 31.97 178.59 24.77 129.41 82.85 2.32
AB 4.42 18.72 32.56 42.55 395 234.57 229.57 119.88 233.47 2.51

where cyanobacteria are the primary colonizers and dominant (Miralles Limited research has been conducted on the analysis of biocrust
et al., 2020; Lan et al., 2022). The decrease in the proportion of cya­ composition in contaminated sites. Previous studies examining the effect
nobacterial abundances in AB and RI was accompanied by an increased of various disturbance factors on biocrusts also demonstrate that
proportion of other soil bacterial phyla, such as Bacteroidota, Actino­ disruptive agents such as heavy metal contamination, grazing distur­
bacteriota, Proteobacteria, and Acidobacteriota. All these phyla play a bance, and other human activities, lead to a decrease in the number of
key role in nutrient cycling and are involved in the decomposition of soil species and functional traits within both plant and bacteria forming
organic matter. Actinobacteria break down complex organic matter, biocrust communities (Bao et al., 2019; Gabay et al., 2022; Sánchez
such as cellulose and lignin (Bao et al., 2021), Acidobacteria are adapted et al., 2022). On the whole, analysis of microbial community composi­
to acidic pH conditions and play a role in soil stabilization (Kim et al., tion and diversity in biocrusts from metal-contaminated sites contrib­
2016; Kalam et al., 2020). Proteobacteria have been recognized for their utes to a better understanding of how the presence and concentration of
significant contribution to combating wind erosion and facilitating ni­ different metals may affect biocrust structure and composition as well as
trogen fixation (Gundlapally and Garcia-Pichel, 2006). The presence of identify potential microorganisms able to grow under these conditions
these phyla in contaminated sites suggests their potential tolerance to and potentially applicable as inoculum for soil bioremediation.
heavy metals. It is also hypothesized that some members of these phyla
may be actively involved in either the bioaccumulation or the reduction 4.2. Growth and EPS production capabilities of isolated cyanobacterial
of heavy metals (Jroundi et al., 2020; Fathollahi et al., 2021). strains
Regarding the biological diversity of the studied biocrusts, the higher
Shannon index for CS and AB in comparison to RI and NR indicates a The isolation of the cyanobacterium N. commune from the three
greater diversity within the community of microorganisms. This sug­ different study sites confirms its ability to adapt to terrestrial environ­
gests that there are likely many different species present, and these ments and its cosmopolitan distribution. Indeed, its presence extends
species are distributed relatively evenly within the biocrust samples. across diverse climates, ranging from tropical zones to polar regions, as
Meanwhile, the lower Simpson values also found in NR and RI, indicate reported in other studies (Tamaru et al., 2005; Sand-Jensen and Jes­
a reduced dominance of specific species within the biocrust samples. persen, 2012; Roncero-Ramos et al., 2019).
Compared to other studies assessing microbial community diversity in Our results reveal distinct growth capabilities of isolated N. commune
cyanobacteria-dominant biocrusts (Miralles et al., 2020; Wei et al., strains (AB55, RI23, and NR64) as shown by the substantial variability
2022; Barrón-Sandoval et al., 2023), biocrusts from our study sites in biomass production and chlorophyll levels across the three strains.
exhibit lower α-diversity values. This lower α-diversity can be explained Indeed, strain AB55 stands out for its productivity, while strains RI23
by the heavy metal contamination in the study sites. Metal toxicity harm and NR64 exhibit lower performance (Fig. 5). The results obtained
sensitive species, and competitive interactions between species could match with recent studies on N. commune growth in the same medium
benefit resistant ones leading to alterations in soil community re­ and similar growth conditions (Diao and Yang, 2014), where different
lationships (Li et al., 2020; Shuaib et al., 2021). strains have demonstrated varying in growth ranges. Cyanobacterial

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biomass increases in parallel with an increase in EPS synthesis (Chamizo production, such as active bioaccumulation, intracellular complexation
et al., 2020). The AB55 strain showed the highest growth and the highest with ligands, and uptake through active processes (Al-Amin et al.,
total and released EPS amount. Nonetheless, strain NR64, which showed 2021).
a limited growth in the liquid medium, synthesized a high amount of According to the NMDS analysis, which demonstrates an association
released EPS, comparable to that of strain AB55. This variability in EPS between the presence of Pb in the site of cyanobacterial isolation and the
synthesis among strains is consistent with values previously reported for removal capacity of this metal by the strain isolated from the site in
cyanobacteria where strains belonging to the same species exhibited question (RI23), the observed differences in Pb immobilisation could
differences in terms of polysaccharide production (Cruz et al., 2020; arise from environmental adaptation. Cyanobacterial adaptation to
Alvarez et al., 2021). Variability in both biomass and EPS production specific Pb-contaminated environments may enhance their abilities to
among different strains of N. commune may be attributed to intraspecific immobilise Pb through a range of physiological and biochemical adap­
genetic variations that affect their ability to adapt to specific culturing tations that enable them to survive and thrive under stressful conditions
conditions. Genetic mutations or differences in genetic composition (Chakdar et al., 2022). These changes may include the expression of
could lead to variations in biomass, chlorophyll, and EPS production specific genes involved in Pb resistance and detoxification, as well as the
(Wright et al., 2001; Jungblut et al., 2021). Another explanation could activation of metabolic pathways that promote lead absorption and
be that some strains might be adapted to grow and produce EPS in reduction within bacterial cells (Tripathi and Poluri, 2021; Priya et al.,
specific environmental conditions, showing increased growth and EPS 2022).
production only in particular contexts. Indeed, N. commune strains have Our findings are consistent with earlier studies that have identified
exhibited variations in growth and EPS production in response to the ability of N. commune strains to retain heavy metals. Specifically, the
changes in temperature and light conditions, as well as differences in infrared analysis of cyanobacterial biomass revealed the presence of
composition and pH of the growth medium (Pereyra and Ferrari, 2016; essential functional groups such as amino, carboxyl, hydroxyl, and
Tripathi et al., 2019). In the examined sites, the soil's physico-chemical carbonyl groups, demonstrating their pivotal role in the biosorption of
alterations resulting from contamination may stimulate EPS production, Cd and Zn (Morsy et al., 2011). Additionally, in another research, due to
thereby potentially augmenting their capacity for heavy metal its proficiency in accumulating heavy metals, N. commune was suggested
immobilisation. as a model species for monitoring heavy metal pollution (Patova et al.,
This information can be valuable for selecting suitable strains for 2000). All these studies have been conducted in aquatic environments
large-scale cyanobacterial biomass production for biotechnological ap­ and focused on their application for wastewater decontamination.
plications, particularly applicable in agriculture and bioremediation. However, they also open the possibility for their use in the immobili­
sation of metals from the soil, an aspect that has been barely explored to
4.3. Cyanobacteria potential for heavy metal immobilisation date.
The demonstrated capabilities of the analysed cyanobacteria strains
Cyanobacteria have been regarded as a potential tool for heavy metal for heavy metals removal suggest them as promising candidates for
removal due to their efficient metal-binding capabilities and imple­ application in the bioremediation of both agricultural soils and water.
mentation of mechanisms such as biosorption, bioaccumulation, and
biotransformation (Chakdar et al., 2022). Our experimental findings 5. Conclusions
indicate that all three isolated strains exhibit the capability to immo­
bilise the selected heavy metals, except for strain RI23, which appears The present study offers insightful information about the possible
ineffective in removing Cu. Notably, there are substantial variations in application of native cyanobacteria for metal immobilisation as a
immobilisation capacity, particularly for Cu. Among the tested metals, bioremediation technique for sustainable agriculture and soil restora­
Cu stands out with the highest absorption capacity, and strain AB55 tion. By comprehending the intricate relationship among microbial
demonstrates remarkable efficiency by removing up to 88.9 % of this communities, strain diversity, and metal immobilisation processes, we
metal from the liquid solution. These results are consistent with previous facilitate the creation of novel bioremediation approaches customized to
studies conducted on cyanobacteria from different environments, not environmental settings. From the findings of this study, it is evident that
associated with biocrusts, which described variations in heavy metal the diversity of cyanobacteria in biocrusts from contaminated soils is
absorption across different cyanobacterial species and strains within the lower than in uncontaminated soils, suggesting that only some adapted
same species (Micheletti et al., 2008; Roy et al., 2015; Atoku et al., species can survive in these environments. Consequently, three strains of
2021). These significant differences in Cu immobilisation could be Nostoc commune have been isolated, showcasing remarkable metal-
attributed to the differential production of EPS by N.commune strains. binding capabilities. These strains can absorb up to 88.9 % of copper
Indeed, the three analysed strains exhibit a correlation between both (Cu), 86.2 % of lead (Pb), and 45.3 % of zinc (Zn) in liquid medium. The
total and released EPS production and Cu removal, a relationship that is high metal removal capability of the isolated native cyanobacteria
absent for the other analysed heavy metals (Fig. 8). supports their potential use as soil inoculants to immobilise a wide range
The presence of EPS produced by cyanobacteria enhances the bio­ of heavy metals, thus representing an environmental-friendly tool for
sorption process, which is considered the major mechanism for the bioremediation of metal-contaminated sites to combat their harmful
removal of heavy metals, involving several mechanisms, such as ion effects on agricultural productivity and ecosystem health. Future
exchange, adsorption, surface complexation, precipitation, and chela­ research on the bioremediation capability of native cyanobacteria, in
tion (Bhatt et al., 2022; Ciani and Adessi, 2023). The EPS matrix con­ conjunction with developments in biotechnological applications, holds
tains abundant ionizable functional groups, such as carboxyl and significant promise for reducing the negative consequences of soil
hydroxyl groups. These groups can interact with cationic species in a contamination and advancing environmental sustainability.
solution, indicating the potential for serving as metal-binding sites (De
Philippis and Micheletti, 2017; Priya et al., 2022). A recent investigation CRediT authorship contribution statement
showed that EPS extracted from sewage sludge had higher efficacy in Cu
removal compared to other heavy metals examined in the study (Pb, Ni, Carlotta Pagli: Writing – original draft, Visualization, Investigation,
and Zn) (Pagliaccia et al., 2022). This finding aligns with the outcomes Formal analysis, Data curation, Conceptualization. Sonia Chamizo:
obtained in our research. For the other two tested metals (Pb and Zn), no Writing – review & editing, Supervision, Investigation, Formal analysis,
clear correlation was found between EPS production and metal immo­ Conceptualization. Giada Migliore: Writing – review & editing, Re­
bilisation (Fig. 8). Pb and Zn removal by N.commune strains may be sources, Investigation. Lorenza Rugnini: Writing – review & editing,
attributed to different mechanisms or factors not directly related to EPS Supervision. Giovanni De Giudici: Writing – review & editing,

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C. Pagli et al. Science of the Total Environment 946 (2024) 174020

Resources. Roberto Braglia: Writing – review & editing, Supervision. Bao, Y., Dolfing, J., Guo, Z., Chen, R., Wu, M., Li, Z., Feng, Y., 2021. Important
ecophysiological roles of non-dominant Actinobacteria in plant residue
Antonella Canini: Writing – review & editing, Supervision. Yolanda
decomposition, especially in less fertile soils. Microbiome 9, 1–17. https://doi.org/
Cantón: Writing – review & editing, Visualization, Supervision, Re­ 10.1186/s40168-021-01032-x.
sources, Funding acquisition, Conceptualization. Barrón-Sandoval, A., Martiny, J.B., Pérez-Carbajal, T., Bullock, S.H., Leija, A.,
Hernández, G., Escalante, A.E., 2023. Functional significance of microbial diversity
in arid soils: biological soil crusts and nitrogen fixation as a model system. FEMS
Declaration of competing interest Microbiol. Ecol. 99 (2), fiad009 https://doi.org/10.1093/femsec/fiad009.
Bhatt, P., Bhandari, G., Bhatt, K., Simsek, H., 2022. Microalgae-based removal of
pollutants from wastewaters: occurrence, toxicity and circular economy.
The authors declare that they have no known competing financial Chemosphere 306, 135576. https://doi.org/10.1016/j.chemosphere.2022.135576.
interests or personal relationships that could have appeared to influence Boi, M.E., Cappai, G., De Giudici, G., Medas, D., Piredda, M., Porceddu, M., Bacchetta, G.,
2021. Ex situ phytoremediation trial of Sardinian mine waste using a pioneer plant
the work reported in this paper.
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s11356-021-14710-y.
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Kirkham, M.B., Scheckel, K., 2014. Remediation of heavy metal(loid)s contaminated
soils - to mobilize or to immobilize? J. Hazard. Mater. 266, 141–166. https://doi.
Data will be made available on request. org/10.1016/j.jhazmat.2013.12.018.
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Acknowledgements
Montan. Slovaca 23 (3).
Chakdar, H., Thapa, S., Srivastava, A., Shukla, P., 2022. Genomic and proteomic insights
This study is part of a Doctoral Thesis funded thanks to the “National into the heavy metal bioremediation by cyanobacteria. J. Hazard. Mater. 424,
Operational Program for Research and Innovation 2014-2020” (PON 127609 https://doi.org/10.1016/j.jhazmat.2021.127609.
Chamizo, S., Cantón, Y., Rodríguez-Caballero, E., Domingo, F., 2016. Biocrusts positively
R&I 2014-2020) of the Italian Ministry of Education, University and affect the soil water balance in semiarid ecosystems. Ecohydrology 9 (7),
Research. This research is part of the projects TED2021-132332B-C21 1208–1221. https://doi.org/10.1002/eco.1719.
and BIOPLANTFLUX (CNS2023-144749), funded by MCIN/AEI/ Chamizo, S., Adessi, A., Torzillo, G., De Philippis, R., 2020. Exopolysaccharide features
influence growth success in biocrust-forming cyanobacteria, moving from liquid
10.13039/501100011033 and by the European Union “NextGener­ culture to sand microcosms. Front. Microbiol. 11, 568224 https://doi.org/10.3389/
ationEU”/“PRTR”. Moreover, the EMERGIA program (EMC21_00424), fmicb.2020.568224.
funded by the Council of Economic Transformation, Industry, Knowl­ Chojnacka, K., Chojnacki, A., Gorecka, H., 2005. Biosorption of Cr3+, Cd2+ and Cu2+
ions by blue–green algae Spirulina sp.: kinetics, equilibrium and the mechanism of
edge and Universities of the Andalusian regional government contrib­ the process. Chemosphere 59 (1), 75–84. https://doi.org/10.1016/j.
uted to fund this work. We extend our gratitude to Lisa Maggioli for her chemosphere.2004.10.005.
invaluable assistance in laboratory analyses, and to Beatriz Roncero and Ciani, M., Adessi, A., 2023. Cyanoremediation and phyconanotechnology: cyanobacteria
for metal biosorption toward a circular economy. Front. Microbiol. 14, 1166612
Emilio Rodríguez for their contributions to data analyses.
https://doi.org/10.3389/fmicb.2023.1166612.
Cicchella, D., Zuzolo, D., Albanese, S., Fedele, L., Di Tota, I., Guagliardi, I., Lima, A.,
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