Science of the Total Environment 802 (2022) 149755

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Science of the Total Environment

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

Isolation and identification of microalgal strains with potential as

carotenoids producers from a municipal solid waste landfill
David Suarez-Montes a,d,⁎, Yaisel Juan Borrell b, Jose Manuel Gonzalez c, Jose Manuel Rico d
a
Neoalgae Micro Seaweed Products, C/ Carmen Leal Mata 191, 33211 Gijón, Spain
b
Department of Functional Biology, University of Oviedo, C/ Catedrático Valentín Andrés Álvarez s/n, 33006 Oviedo, Spain
c
R&D, COGERSA SAU, Carretera de Cogersa 1125, 33697 Gijón, Spain
d
Department of Organisms and Systems Biology, University of Oviedo, C/ Catedrático Valentín Andrés Álvarez s/n, 33006 Oviedo, 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

• The microalgal diversity within the

water bodies of a municipal solid waste
(MSW) landfill was investigated.
• Fourteen (14) mono-cultures strains
were identified and isolated through
the use of morphological and genetic
tools.
• Coelastrella cogersae was proposed as a
new species based on both morphologi-
cal and molecular results.
• Three strains exhibited caretogenic ac-
tivity, observed by the colour change
under nutrient deprivation.

a r t i c l e i n f o a b s t r a c t

Article history: Derived from their great capacity of adaptation, microalgae have several industrial applications, including pig-
Received 3 June 2021 ment production for nutraceutical sector. However, the scarcity of studies on the diversity and life histories
Received in revised form 12 August 2021 from several environments, highlight the need for more research on new species and habitats. Based on this,
Accepted 15 August 2021
the present study assessed the microalgal diversity in water bodies of a municipal solid waste (MSW) landfill
Available online 24 August 2021
in Asturias (Spain). A total of 14 strains were successfully isolated and scaled up in liquid monocultures. They
Editor: Huu Hao Ngo were identified through a combination of morphologic features with molecular assignation by DNA barcoding
via the 18S and ITS1-5.8S-ITS2 genes. The results of the genetic procedures (BLAST assignments and the 18S
and ITS1-5.8S-ITS2 genealogies) showed that 10 of the 14 assayed isolates were identified at the species level.
Keywords: The available genetic data were not sufficient for species classifications of the remaining isolates. It is possible
Diversity that some might be new species not previously studied or described. Indeed, a new species, Coelastrella cogersae,
18S rRNA gen marker was proposed in this study. Moreover, 3 of the 14 isolates (including the newly proposed species) exhibited
ITS1-5.8S-ITS2 gen marker caretogenic activity under specific conditions during the culture. These results are a great step forward in both
Scanning electron microscopy (SEM)
the screening of lesser-known environments and the discovery of new sources of bioactive compounds. The
DNA barcoding
study could be of great value to the nutraceutical industries and markets.
Carotenoids
© 2021 Elsevier B.V. All rights reserved.

1. Introduction

⁎ Corresponding author at: Calle Carmen Leal Mata, 191, 33211 Gijón, Asturias, Spain. Microalgae are a widely distributed group of microorganisms (Qiao
E-mail addresses: dsuarezmon@gmail.com, dsuarez@neoalgae.es (D. Suarez-Montes). et al., 2015) that take efficiently sunlight energy and CO2 generating

https://doi.org/10.1016/j.scitotenv.2021.149755
0048-9697/© 2021 Elsevier B.V. All rights reserved.
D. Suarez-Montes, Y.J. Borrell, J.M. Gonzalez et al. Science of the Total Environment 802 (2022) 149755

carbon compounds in primary metabolism (Lynch et al., 2015). Asturian Consortium for the Management of Urban Residues), at
Approximately, they have between 1 and 50 time more carbon fixation which the main activity is the sustainable management of solid urban
yield in comparison to higher plants (Ruiz et al., 2016). Despite the wastes. A preliminary aerial survey was performed by a drone to iden-
complexities of determining the number of taxonomic groups in algae, tify the landfill ground. Specifically, there were two kind of water bod-
it has been suggested that there are between 70,000 and 1 million ies: spontaneous little ponds (natural) produced by settlement of the
groups (De Clerck et al., 2013). Apart from those features, several landfill which were spread across the surface (the leachate is highly di-
compounds derived from the microalgae metabolisms could be luted because of the rainfall); and man-made ponds (artificial) where
exploited, being lipids the most versatile group of biomolecules. They most of the landfill leachate was collected prior depuration process
are present in high amounts constitutively or accumulated under (the ratio leachate/rainfall was lower). The sampling was designed to
specific stress conditions (Aratboni et al., 2019). Carotenoids, such as represent all of the types of water bodies present in the landfill to assess
astaxanthin from Haematococcus pluvialis (Machado et al., 2014) and β- the quality of the microalgae and cyanobacteria (Table 1). It took place
carotene from Dunaliella salina (Wolf et al., 2020), are well established in autumn 2017. Samples of 100 mL were collected in triplicate and sub-
in the nutraceutical market. Furthermore, their varied metabolic features sequently stored at 4°C in sterile test tubes (Fig. 1). In addition, photo-
make microalgae a ubiquitous group that can colonise a variety of envi- graphs were taken to document the features of each sample site. The
ronments. For instance, these organisms have been found in environ- stored samples were prepared for serial dilutions from 10−1 to 10−5
ments with extreme pH, temperatures, alkalinity and salinity (Varshey and subsequently streaked in Petri plates of 100 × 15 mm with an
et al., 2014). agar solid medium. The plates were inoculated with approximately
The precise identification of microalgae found in any environment is 100 μL of each dilution (Andersen, 2005).
key to uncovering the possibilities for their uses. Traditionally, the char- An agar solid medium was made following the protocol proposed by
acterisation of microalgae has been based on morphological features Lee et al. (2015) with few modifications. The nutrient media BG-11
and life cycles via light or scanning/transmission electron microscopy (Allen, 1968) and f/2 (Guillard and Ryther, 1962) were used as general
(Carmelo and Grethe, 1997; Fon-Sing et al., 2013). Nevertheless, envi- media for the microalgae and the cyanobacteria, respectively. The plates
ronmental parameters and their changes may alter the morphology were placed in a chamber with standard conditions: 25–30 μE m−2 s−1
and physiology of microalgae, thus making it difficult to discern the dis- of photon flux density, 25 ± 2 °C and a 16:8 photoperiod. Approxi-
tinctions among groups that have similar characteristics (Darienko mately 9 days after inoculation, small colonies were grown in Petri
et al., 2015). Molecular techniques, such as DNA barcoding, have facili- plates. They were re-streaked every 15 days until the cultures were
tated the construction of phylogenies and the identification of problem- unialgal.
atic groups, thus eliminating the possibility of morphological errors
(Radha et al., 2013; Gong et al., 2018). Among the gene markers that 2.2. Morphological identification by microscopy
have been used for species identification (COI-5P, rbcL, tufA and many
others), sequences belonging to ribosomal subunits are the most com- Preliminary observations of cultures were made by using inverted
mon in microalgal characterisation. Specifically, some studies have ob- phase-contrast optical microscopy (BioBlue, S/N-EU-1612189,
tained extraordinary results in the identification of microalgal groups Euromex, Holland). A small sample was taken and characterised based
based on the 18S marker (Durvasula et al., 2015) and the ITS1-5.8S- on the previously described morphological features (Qiao et al., 2015).
ITS2 fragment (Liu et al., 2014a; Certnerová and Skaloud, 2020). More- All of the images were made at 1000× magnification, and the descrip-
over, species identification was accomplished by combining morpho- tions and identifications were based on several morphological charac-
logical and molecular analyses (Qiao et al., 2015). Therefore, increased teristics: shape, size (width and length), pyrenoid position, flagella
exploration and screening of specific and unexplored environments (presence or absence of motility) and cell arrangement. The images
could lead to the discovery of new species and strains with improved (with or without phase contrast) were modified with Adobe Photoshop,
or even new biotechnological potentials (Malavasi et al., 2020). with tools such as cloning buffer, plaster and selective focus. Adobe
By following the same line, a prime example of unexplored and specific Lightroom was used to adjust the clarity, exposure and light as well as
environment is a municipal solid waste (MSW) landfill. There are different to change the contrast.
works focused on landfill leachate mitigation by microalgae, such as nitro- Scanning electron microscopy photomicrographs were taken to
gen and phosphorous removal by Chlorella vulgaris (Pereira et al., 2016) check the details on some of the characters that had been examined.
and Chlorella zofingiensis (Zhou et al., 2018). However, strains use for Specifically, the external ultrastructure of the cells was determined in
this biotechnological purpose came from certified algae collections. In con- order to facilitate the shape and texture analysis. The pre-treatment of
trast, microalgae which have grown inside natural and anthropogenic the samples was done in accordance with the protocol of Collins et al.
water ponds surrounding MSW landfill located in northwest of Spain (1993) with few modifications. Culture volumes of 300–500 μL
were screened. Thus, the hypothesis expected is that, in spite of the an- (depending on the apparent cellular density) were filtered in Whatman
thropogenic environment which will be investigated, a collection of native GF/F filters of 25 mm diameter and 0.22 μm pore size. The filters were
microalgae could be isolated. This group of microalgae would be adapted continually subjected to a serial acetone: water gradient for 10 min
to the local conditions (climate, sunlight, high levels of CO2 and even for dehydration. The dilution percentages were 25, 50, 75, 95 and
nutrients of landfill leachate), being suitable for cultivation nearby the 100% v/v. The test tubes were stored at 4 °C. After dehydration, the fil-
landfill and the waste incinerator. Moreover, biotechnological uses ters were dried by the critical point dry (CPD) methodology to eliminate
outside focusing on carotenoid production, could be investigated. the acetone and to avoid cell damage. Furthermore, the total extension
In the present study, the main objective was the characterisation and
isolation of the diverse microalgae living in a singular environment (land- Table 1
fill) and the assessment of their biotechnological potential. Additionally, Type, location and specific codes for sampling sites in the study.
the potential carotenogenic activity of the isolates, was explored.
Name Origin Latitude (N) Longitude (O) Code

Seasonal pond 1 Natural 43°29′32.6028″ 5°49′6.8353″ CE1

2. Materials and methods
Seasonal pond 2 Natural 43°29′31.1883″ 5°49′12.4417″ CE2
Permanent pond 1 Artificial 43°29′50.2496″ 5°48′57.5999″ CP1
2.1. Sampling site and the microalgae isolation process Permanent pond 2 Artificial 43°29′16.0390″ 5°49′4.6490″ CP2
Artificial container 1 Artificial 43°29′57.2808″ 5°49′3.3986″ CA1
Samples were taken from various locations inside the Asturian cen- Artificial container 2 Artificial 43°29′57.9675″ 5°49′3.9152″ CA2
Pond G Artificial BG
tral landfill (referred to as COGERSA, the Spanish acronym for the

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D. Suarez-Montes, Y.J. Borrell, J.M. Gonzalez et al. Science of the Total Environment 802 (2022) 149755

Fig. 1. Bird's eye view of the Central Landfill of COGERSA. The sampling locations are indicated by coloured triangles and diamonds (http://sigpac.mapa.es/fega/visor/).

of the filters was achieved without any wrinkles. The filters were then taken and put in an electrophoresis gel (1% w/v) to verify the presence
sputtered by a gold cover to start the morphological analysis. of DNA. In the molecular identification of the isolates, five different gene
To make a tentative identification of the isolates prior to the genetic markers were assayed: COI-5P, rbcL, 18S rRNA, ITS (ITS1-5.8S-ITS2) and
analyses, this morphological study was used to find matches in the tuf A (Table S1).
AlgaeBase database (Guiry and Guiry, 2012) and the taxonomic key by PCRs were carried out in a final volume of 20 μL composed (final
Bellinger and Sigee (2010). concentrations) by a specific 1× reaction buffer, 2.5 mM of MgCl2,
0.5 mM of dNTPs, 0.2 μM of each primer and 0.5 U of Taq
2.3. Scale-up and maintenance of specific cultures (mono-cultures) polymerase. The remaining volume of mixture was filled with
distilled water, and an adequate quantity of extracted DNA (approx.
Single microalgae colonies were grown after 8 medium renewals. A 20 pg) was added to each PCR tube. The conditions for the PCR were
few (2–4 colonies) were picked up with a modified Pasteur pipette and initial denaturation of the double strand at 95 °C for 5 min and 35 cy-
put in 30-mL glass vessels (3 replicates/culture) with 10 mL liquid cul- cles divided in 30″ at 95 °C, an annealing temperature of 55 °C for 30″
ture. Both f/2 and BG-11 media were used. The cultures reached the sta- and 60″ at 72 °C. A final extension of 5′ at 72 °C was carried out as the
tionary phase in approximately 18 days. last step. All of the reactions were verified through electrophoresis
Likewise, 10 mL were scaled up in 250-mL Erlenmeyer flasks (3 rep- gels (2% of agarose w/v), along with SimplySafe to observe the
licates/culture) with 50 mL and 200 mL of liquid culture. The average band pattern under ultraviolet light (5 μL dye/100 g agarose).
inoculum/medium was 1:5 (1 liquid culture:5 medium) or 1:10 de-
pending on culture growth. The flasks were placed in a chamber with 2.5. Sequence analysis and phylogenetic trees
standard conditions: 70–100 μE m−2 s−1 of irradiance, 25 ± 1 °C of tem-
perature and a 16:8 photoperiod (16 h light:8 h dark) (Andersen, 2005). PCR bands were purified from the electrophoresis gel by the
To determine the stationary phase in the growth curves, the NO− 3 GeneMATRIX Agarose-Out DNA purification kit (Roboklon GmbH,
dissolved in the cultures was measured by a reagent test (NO− 3 Test Berlin, Germany). Fourteen samples of each marker gene were sent to
SERA, GmbH, Berlin, Germany). Previously, 5–10 mL of liquid cultures Macrogen Inc. Spain for sequencing using the standard Sanger sequenc-
had been taken and centrifuged during 7 min at 4320g to submit the su- ing method (Sanger et al., 1977). The sequences were manually revised
pernatant to the kit reagents. using the BioEdit (Hall, 1999) and aligned using ClustalW multiple se-
quence alignment (Thompson et al., 1994). Subsequently, species iden-
2.4. DNA extraction, primers and PCR conditions tification was performed, making BLAST attempts against the GenBank
sequence database to identify all of the isolates. The genetic identifica-
After the centrifugation of 30 mL of liquid culture (4320g, 7 min), the tions were performed using comparisons based on the BLAST proce-
supernatant was discarded, and the pellets were stored at −20 °C. Usu- dures (option highly similar sequences, MegaBLAST) to find the best
ally, the quantity of raw material stored as pellets was approximately matched sequences (highest alignment scores after evaluating the E
20–40 mg in every isolate. DNA was extracted with a GeneMATRIX value, query cover and percentages of identity) in the GenBank database
Plant and Fungi DNA purification kit (Roboklon GmbH, Berlin, sequences. Valid genetic assignments were considered only when the
Germany) kept at −20 °C until analysis. Small volumes (5 μL) were identity percentages were above 97% for the best hits found. Another

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D. Suarez-Montes, Y.J. Borrell, J.M. Gonzalez et al. Science of the Total Environment 802 (2022) 149755

consideration for the assignment validations was that the matched da- coccoid microalgae. As was explained in the Materials and Methods
tabase sequences (best hit) found had to have already been published, section, the plate streak or inoculation was made with a serial dilution
thus having undergone a rigorous peer-review scientific process. The protocol with successful results regarding the green coccoid algae. Never-
analysis and phylogenetic tree were done using the MEGA v6 software theless, the large sizes of the cyanobacteria and filaments, in general,
(Tamura et al., 2013). The neighbour-joining (NJ) method (Saitou and could interfere with the development of the protocol, thus breaking the
Nei, 1987) with a bootstrap of 10,000 (Felsenstein, 1985) was used to filaments or even not to be taken in pipetting. Another reason whereby
infer the evolutionary history. The models were done using the cyanobacteria were not isolated could be related to the agar solid me-
ModelTest application in MEGA 6v. The phylogenetic trees were modi- dium. It has been asserted that there are various growth inhibitors inside
fied with CorelDRAW X8. gelled agar (Castenholz, 1988), and this makes the development of con-
tinuous cyanobacteria solid cultures more difficult. It is possible that the
3. Results and discussion potential isolates in the raw samples would not grow because of the in-
ability to adapt to the new environment, which is sometimes very dry de-
3.1. Isolation, scale-up and maintenance of cultures pending on the amount of agar per litre of water (Lee et al., 2015).
Because the BG-11 and f/2 culture media were satisfactory choices, the
In the present study, samples from solid waste (MSW) landfill of scaled-up liquid cultures were completed successfully in each volume.
COGERSA in Asturias were collected and plated in solid agar with a spe- This result was in accordance with the results of previous studies in
cific nutritional medium. However, a total of 9 re-inoculations in a fresh which microalgae had been isolated from various environments
medium were necessary because of contamination, mainly by bacteria (Skaloud et al., 2012; Lynch et al., 2015).
and yeast.
After the previous steps had been completed, 14 mono-culture 3.2. Morphological and genetic identification
plates were obtained. They were maintained in the specific conditions
explained above. The scale-up of the cultures was successful, resulting The morphological characterisation of the 14 isolates was performed
in 14 liquid cultures in different volumes: 10 mL, 50 mL and 200 mL mainly by light microscopy to achieve a group of diagnostic features
(Fig. 2). that allowed for the tentative or preliminary identification of the iso-
The use of a traditional serial dilution protocol for the raw samples lates (Table 2). As it can be seen in Fig. 3, the diversity at the sampling
followed by agar Petri dish plating (Andersen, 2005), was the most sites was moderate despite the presence of various structures and cellu-
cost-effective decision despite the time that elapsed until the final isola- lar arrangements, such as solitary cells and coenobic forms. Four groups
tion. Alternative methods are being used to overcome the length of the or genera were identified. However, isolating the microalga from the
isolation period. For instance, enrichment cultures can be performed be- CP1, CP2 and CA2 locations or the cyanobacteria was not possible at
fore plating the sample in solid agar in order to increase the dominance any of the sampling sites. The scanning electron microscopy (SEM)
of microalgae over the remaining organisms (Pryvil et al., 2015; technique yielded important data about the surface characteristics
Thompson et al., 2018). More sophisticated techniques, such as auto- (Fig. 4). As was indicated in Table 2, longitudinal grooves (one or
mated single cell isolation through flow cytometry (Neofotis et al., more), rifts and star-shaped structures on the surface facilitated the in-
2016) and fluorescence-activated cell sorting (Terashima et al., 2018), trinsic identification of Scenedesmus-related isolates although it was not
can drastically reduce the time needed for isolation. However, they are determinant.
expensive and technically demanding. Moreover, environmental sam- Inside molecular results, a total of 9 PCR amplifications from 5 genes
ples are normally in varying life cycle stages. This implies that they are were attempted in a preliminary assessment in this study. Two gene
of different sizes and shapes, and this makes the technique less reliable. fragments, the 18S rRNA gene and the ITS1-5.8S-ITS2 gene cluster,
The isolation of the cyanobacteria and filamentous microalgae was not were subsequently used for the molecular identifications of the 14 iso-
achieved despite their presence in the raw samples. The cyanobacteria, lates because they shed consistent and reliable PCR results (GenBank ac-
being macroscopically distinguished, were several times larger than the cession numbers for 18S gene marker from MH307942 to MH307955

Fig. 2. Sampling, isolation, scale-up and maintenance of 14 isolates.

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D. Suarez-Montes, Y.J. Borrell, J.M. Gonzalez et al. Science of the Total Environment 802 (2022) 149755

Table 2
Preliminary identification of 14 microalgae isolates at the Asturian landfill.

Code Shape Width Length Motility Pyrenoid Cell arrangement Special feature (SEM) Tentative identification
(μm) (μm)

BG.600 Spherical 13–15 13–15 No No No Chlorococcum sp.

BG.601 Ovoid 10.89 13.18 No No No “Chlorella” sp.
BG.602 Spherical 13–15 13–15 No No No Chlorococcum sp.
BG.603 Ovoid 9.5–10.8 9.5–9.8 Yes (but lost) No No Chlamydomonas sp.
CE1.500 Ovoid 4.2–4.3 5.7–6 No Yes Yes (sometimes, 2–3–4-cell coenobia) Longitudinal grooves Scenedesmus sp.
CE1.501 Ovoid 2.4–2.7 3.1–3.3 No Yes Yes (2–4-cell coenobia or free cells) Desmodesmus sp./Scenedesmus sp.
CE2.319 Spindel-acute 2.4–2.8 3.8–3.9 No Yes Yes (4-cell coenobia or free cells) One longitudinal wrinkle Scenedesmus sp.
CE2.320 Ovoid 6.7–8.2 8.6–9.1 No Yes (sometimes, 2-cell coenobia) Longitudinal soft grooves Scenedesmus sp.
CE2.401 Ovoid 3.22 5.2–5.4 No Yes Yes (sometimes, 2–3–4-cell coenobia) Longitudinal grooves Scenedesmus sp.
CE2.402 Spindel-acute 2.3–2.8 3.5–3.6 No Yes Yes (4-cell coenobia or free cells) One longitudinal wrinkle Scenedesmus sp.
CA1.122 Ovoid 2.3–2.8 3.4–3.6 No Yes Yes (2–4-cell coenobia or free cells) Desmodesmus sp./Scenedesmus sp.
CA1.123 Ovoid 2.1–2.3 3.2–3.3 No Yes Yes (2–4-cell coenobia or free cells) Desmodesmus sp./Scenedesmus sp.
CA1.321 Spherical 2.5–3.5 2.5–3.5 No Yes No Chlorella sp.
CA1.322 Spherical 2.5–3.5 2.5–3.5 No Yes No Chlorella sp.

and from MH311536-MH311547 for the ITS1-5.8S-ITS2 gene cluster). percentages (95%). Surprisingly, the BG.601 isolate was related to a new
The results of the matched database sequences, the accession numbers reference sequence, Chlorella vulgaris (99%). There was confusion in the
and the identity percentages for each of the isolates are shown in assignments of the CE2.320 isolate because it was near to both the
Table 3. In sum, the genetic assignments using BLAST against the Chlorella sp. and the Scenedesmus sp. database sequences (Table 3). On
GenBank microalgae sequences revealed the identification, at the spe- the other hand, phylogenetic trees were constructed using all the
cies level, of 9 of the 14 isolates under study (64%) using the 18S gene. isolates' sequences and published reference sequences from each of
Lower percentages (58%) were found using the ITS cluster instead. The the main microalgae taxonomic groups. Most of those reference se-
global picture showed 8 cases where one of the markers failed to quences belonged to prestigious culture collections such as The Culture
allow species classification, 2 contradictory results (same genus but dif- Collection of Algae and Protozoa (CCAP), University of Texas Culture
ferent species) and 4 matching results isolates CE2.319 and CE2.402 Collection of Algae (UTEX) or The Culture Collection of Algae at
(Acutodesmus obliquus), and isolates CA1.321 and CA1.322 (Chlorella Göttingen University (SAG). The 18S NJ tree of microalgae clearly distin-
sorokiniana) (Table 3). The sequences for the ITS1-5.8S-ITS2 gene clus- guished by two big clades that were well supported by the bootstrap
ter obtained here for 12 of the isolates showed a slight drop in efficacy values (Fig. 5). The clade at the top of the tree encompassed 2 subclades
regarding the identity percentages, leading to less reliable genetic as- and 3 orders where 12 of the 14 isolates were nested: Chlorellales (Class
signments. Nevertheless, the correspondence between the best hits Trebouxiophyceae) Sphaeropleales and Chlamydomonadales (Class
using the ITS gene and the previous 18S results was high (Table 3). It Chlorophyceae). The published reference ITS1-5.8S-ITS2 sequences
was even new isolates assignments to a species level since the from each of the main taxonomic microalgae groups were downloaded
CE1.501 isolate was matched now to a Desmodesmus multivariablisis and aligned with the 12 sequences obtained in this study. Unfortu-
sub. turkiensis reference sequence with a high identity percentage nately, the BG.600 and BG.602 sequences were not obtained. It lacked
(99%). The CA1.321 and CA1.322 isolates were also found to be close the Chlorococcales clade, but it exhibited the previously seen two
to the Chlorella sorokiniana reference sequences but with lower identity subclades and 3 microalgae groups. In spite of the ITS1-5.8S-ITS2 NJ

Fig. 3. Images of strains under both optical microscopy and scanning electron microscopy and their green/red-to-orange phases in glass recipients. A: Acutodesmus obliquus CE2.402 strain;
B: Coelastrella sp. CE1.320 strain; C: Coelastrella sp. CE2.401 strain, with a strong colour change under stress conditions. Photographs were taken after 8 days of cultivation.

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D. Suarez-Montes, Y.J. Borrell, J.M. Gonzalez et al. Science of the Total Environment 802 (2022) 149755

Fig. 4. Light microscopy photographs (with phase contrast) of the 14 microalgae isolated in the landfill; the codes are indicated. A: Chlorococcum sp. (BG.600); B: Chlorella sp. (BG.601); C:
Chlorococcum sp.; D: Chlamydomonas sp. (BG.603); E: Desmodesmus sp./Scenedesmus sp. (CA.122); F: Scenedesmus sp. (CE2.401); G: Desmodesmus sp./Scenedesmus sp. (CA1.122); H:
Desmodesmus sp./Scenedesmus sp. (CA1.123); I: Scenedesmus sp. (CE2.319); J: Scenedesmus sp. (CE2.320); K: Scenedesmus sp. (CE2.402); L: Desmodesmus sp./Scenedesmus sp.
(CE1.501); M: Chlorella sp. (CA1.321); N: Chlorella sp. (CA1.322). Scale bars can be seen in the lower section of each photograph.

tree was supported by low bootstrap values, its topology matched the (Coestastrella sp. and Desmodesmus sp.). More work is needed to assess
18S gene tree (Fig. 6). the taxonomic and genetic characteristics of the genus. Specifically,
The genetic procedures (BLAST assignments and the 18S and ITS1- CE2.320, BG.600 and BG.602 could possibly be new species not yet stud-
5.8S-ITS2 genealogies) showed that 10 of the 14 isolates assayed were ied or described. A major problem with microalgae taxonomy has been
indeed identified (species level). The available genetic data were not the difficulty in fully understanding the life cycles of the microalgae and
sufficient for species classifications for the remaining isolates, but for the various cell shapes and sizes they can assume in response to envi-
the CE1.500, CE2.401 and CA1.122 isolates, the genus was clear ronmental changes (Radha et al., 2013; Qiao et al., 2015). Therefore, a

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D. Suarez-Montes, Y.J. Borrell, J.M. Gonzalez et al. Science of the Total Environment 802 (2022) 149755

Table 3
Molecular identification of strains isolated in the Asturian landfill including both gene markers.

Strain 18S (GenBank) ITS1-5.8S-ITS2 (GenBank)

(ID)
Closest match % Accession New accession Closest match species % Accession number New accession
species similarity number number similarity (reference) number
(this work) (This work)

BG.600 Tetracystis tetraspora 97 JN968582.1 MH307952 No sequence

BG.601 Chlorella sorokiniana 99 KP726221.1 MH307953 Chlorella vulgaris 99 KP645229.1 MH311546
BG.602 Tetracystis tetraspora 97 JN968582.1 MH307954 No sequence
BG.603 Chlamydomonas 99 AB511839.1 MH307955 Chlamydomonas zebra 91 AF033294.1 MH311547
orbicularis
CE1.500 Coelastrella sp. 99 KM020087.1 MH307950 Coelastrella sp. 99 KM061472.1 MH311544
CE1.501 Uncultured chlorophyte 99 EU910612.1 MH307951 Desmodesmus multivariabilis var. 99 DQ417525.1 MH311545
turkiensis
CE2.319 Acutodesmus obliquus 100 KP726267.1 MH307944 Acutodesmus obliquus 100 KP645234.1 MH311538
CE2.320 Chlorella sp. (not 99 KM985412.1 MH307945 Scenedesmus sp./Chlorella sp. 97 KJ676125.1/KJ696124 MH311539
published)
CE2.401 Coelastrella sp. 99 KM020087.1 MH307948 Coelastrella sp. 99 KM061472.1 MH311542
CE2.402 Acutodesmus obliquus 99 KP726267.1 MH307949 Acutodesmus obliquus 99 KP645234.1 MH311543
CA1.122 Desmodesmus sp. 100 AB917128.1 MH307942 Desmodesmus sp. 98 AB917128.1 MH311536
CA1.123 Desmodesmus abundans 100 KF673371.1 MH307943 Desmodesmus sp. 96 AB917128.1 MH311537
CA1.321 Chlorella sorokiniana 99 KP726221.1 MH307946 Chlorella sorokiniana 95 KP645222.1 MH311540
CA1.322 Chlorella sorokiniana 99 KP726221.1 MH307947 Chlorella sorokiniana 95 KP645222.1 MH311541

great deal of effort has been focused on the development of DNA the same species. Errors in species classifications or low representation
barcoding to support the morphological data needed for microalgae of some species or groups within the data deposited in databases such
identification (Kwong et al., 2012; Darienko et al., 2015). While the as GenBank are common (Krienitz and Bock, 2012), not only in
BLAST assignment is based on the mere matching of scores (identity) microalgae, but in other groups such as fungi (Chowdhary et al.,
to sequences previously deposited in databases, genealogies can pro- 2019). It is crucial that combined studies be conducted to assess the spe-
vide a more complete picture about the origin and evolution of these cies taxonomy within this genus.
complex organisms. There are major gaps in our knowledge about
microalgae taxonomy given the genetic divergences. The molecular 3.2.2. Order Sphaeropleales (Class Chlorophyceae)
phylogeny of this group is incomplete, and many of the records go Several isolates were properly encompassed within the Scenedesmus/
only as far as the genus level. Errors in species classification in the Desmodesmus genus based on the cell arrangement in different coenobia
GenBank database are somewhat usual (Ardura et al., 2013). The lack forms, their oval or spindle shapes and their size. Consistent results were
of reference sequences or the ambiguity of these sequences in the data- also obtained by BLAST. The CA1.122 and CA1.123 isolates showed a 100%
bases can hinder species identifications (Ahmad et al., 2013; Ardura identity with the referenced sequences for Desmodesmus sp. and
et al., 2013). In this study, the morphological and genetic results were Desmodesmus abundans, respectively. The CE1.500 and CE2.401 isolates
combined to enable the identification and classification of the new were found to be close to a Coelastrella sp. database sequence (99%). A
microalga isolates. high correlation was also found between the morphological and molecu-
lar assignments of the CE2.319 and CE2.402 isolates, which were related
3.2.1. Order Chlamydomonadales (Class Chlorophyceae) to 2 different Acutodesmus obliquus reference sequences (100 and 99%
The BG.603 isolate was especially difficult to include in a group be- similarity, respectively). In phylogenies based on 18S gene, the
cause of the number of morphological changes that it had undergone Sphaeropleales order was divided into two main groups. The first group
during the acclimation from the landfill environment to the laboratory; covered the Desmodesmus genus, with 3 isolates (CE1.501, CA1.122 and
nevertheless, it was assigned to the Chlamydomonas genus (Table 2). As- CA1.123) allocated near to one another. The second included predomi-
signation results revealed that the isolated BG.603 had a high percent- nantly the Coelastrella and the Scenedesmus (Acutodesmus) genus. In
age of identity (99%) with the Chlamydomonas orbicularis sequence ITS1-5.8S-ITS2 phylogenetic tree, the 8 isolates belonged to the last
database. Supporting database results, phylogenies based on the 18S order were allocated on the top covering in a big subclade (3 different or-
gene appeared the last order, comprising the Chlamydomonas genus ders). They were also divided into 3 clear genera: Scenedesmus (with
into which the BG.603 fell. Also, in phylogenies based on ITS1-5.8S- CE2.319, 402 isolates and questionably CE2.320 isolate), Coelastrella
ITS2 gene cluster, sequences subclade at the top clustering the (with CE1.500 and CE2.401 isolates) and Desmodesmus (with CA1.122,
Chlamydomonadales order (Chlorophyceae class). The BG.603 isolate 123 and CE1.501).
fell into this group with high bootstrap values. The BG.603 was the Based on the final outcomes, Sphaeroplelales were the most abun-
only isolate related to the Chlamydomonas genera that was found after dant group in the sampling locations. They appeared in many types of
the genetic analyses using two different gene markers (18S and ITS1- sites except Pond G (BG). First, morphological studies have explained
5.8S-ITS2). In the first observation of the BG location sample, the most the dominance of Scenedesmus-type morphotypes with a lower pres-
abundant morphotype was an active flagellated cell closely related mor- ence of Desmodesmus types in the case of the CE1.501, CA1.122 and
phologically to Chlamydomonas. However, the changes in the environ- CA1.123 isolates. Few diagnostic characteristics (Uzunov et al., 2008;
mental conditions between the pond and the laboratory during the Qiao et al., 2015), such as specific coenobia structures for both
isolation and scaled-up processes produced alterations in the morphol- Scenedesmus (spindle-shaped without spines) and Desmodesmus
ogy and physiology (e.g., loss of flagella and increase in size). These (ovoid- or cylindric-shaped with spine ornamentations) forms, were
kinds of conflicts were discussed in previous studies (Collins et al., found in the raw samples or after the isolation process. Nevertheless,
2012). In this work, DNA barcoding was necessary for identifying the the cells lost their coenobia forms (free cells) during the re-
BG.603 isolate, which seemed to belong clearly to Chlamydomonas. inoculations in the liquid cultures. Specifically, the morphology of the
The genetic evidence suggests that it is probably Chlamydomonas Desmodesmus types was altered, acquiring ovoid shapes and increased
orbicularis (99% 18S identity). In sum, the phylogenetic trees indicate in size. The SEM images later showed major differences in the surfaces
that Chlamydomonas zebra and Chlamydomonas orbicularis are probably of the CE1.500, CE2.401 and CE2.320 isolates. A series of longitudinally

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D. Suarez-Montes, Y.J. Borrell, J.M. Gonzalez et al. Science of the Total Environment 802 (2022) 149755

Fig. 5. Scanning electron microscopy photographs of 13 of the 14 microalgae isolated in the landfill; the codes are indicated. A: Chlorococcum sp. (BG.600); B: Chlorococcum sp. (BG.602); C:
Chlamydomonas sp. (BG.603); D: Desmodesmus sp./Scenedesmus sp. (CA1.122); E: Desmodesmus sp./Scenedesmus sp. (CA1.123); F: Coelastrella sp. (CE2.401); G: Coelastrella sp. (CE2.320);
H: Scenedesmus sp. (CE2.402); I: Coelastrella sp. (CE1.500); J: Scenedesmus sp. (CE2.319); K: Desmodesmus sp./Scenedesmus sp. (CE1.501); L: Chlorella sp. (CA1.321); M: Chlorella sp.
(CA1.322). Scale bars can be seen in the lower part of each photograph.

disposed grooves or ribs were observed. This special feature was noticed the BLAST application, 3 groups or genera were differentiated when
by Uzunov et al. (2008) in the first European description of Coelastrella using 2 different gene markers showing high identity percentages
sp. strains, following by current studies of new Coelastrella species and (96–100%; see Table 3): Desmodesmus sp., Coelastrella sp. and, specifi-
varieties (Wang et al., 2019); thus, it is possible that the results of the cally, Acutodesmus (Scenedesmus) obliquus.
analyses performed under light microscopy might have contained er- The CA1.122 and CA1.123 isolates were identified as Desmodesmus
rors. A more precise assignation of the isolates was achieved during sp., based on the BLAST and the phylogenies results. The same results
the DNA barcoding procedure. Based on the preliminary results using were found for the CE1.501 isolate. This indicates that they are two

8
D. Suarez-Montes, Y.J. Borrell, J.M. Gonzalez et al. Science of the Total Environment 802 (2022) 149755

Fig. 6. NJ consensus tree based on 18S rRNA gene. The new sequences derived from this study appear as coloured dots based on the taxonomic order.

9
D. Suarez-Montes, Y.J. Borrell, J.M. Gonzalez et al. Science of the Total Environment 802 (2022) 149755

different species. It is likely that CA1.122 and CA1.123 are Desmodesmus Wolf, 2015). Fortunately, it can be concluded that CA1.321 and CA1.322
abundans (100% identity using 18S gene). Again, few reference se- correspond to the Chlorella sorokiniana species, but a deeper analysis
quences were found, and the taxonomical status of the genus was not will be required to determine whether BG.601 should be assigned to
clear. The Acutodesmus (Scenedesmus) group had a high percentage of Chlorella vulgaris or Chlorella sorokiniana.
homology (99–100%) as a result of BLAST analysis in both CE2.319 and
CE2.402. Moreover, they fell into the same cluster inside the two phylo- 3.3. A special case: CE2.320 strain (new species description)
genetic trees even though the lower support of their branches; conse-
quently, they belong to Acutodesmus obliquus. Finally, the third group The CE2.320 seemed to be a particular case. The genetic analyses lo-
was Coelastrella sp. with 3 representatives: CE1.500, CE2.401 and cated it in the Coelastrella subclade in both phylogenetic trees but
CA1.320. Based on the genetic results, CE2.401 and CE1.500 seem to completely isolated in an independent group. Fortunately, the SEM
be Coelastrella saipanensis. A special case (CA1.320) will be explained morphological observations on its surface revealed longitudinal ribs,
in following sections. which was a specific feature. Thus, the CE2.320 isolate is proposed as a
new species inside the Coelastrella genus: Coelastrella cogersae. The
3.2.3. Order Chlorococcales (Class Chlorophyceae) proposition is explained by specific characteristics (Darienko et al.,
The BG.600 and BG.602 isolates were initially assigned inside the 2015):
Chlorococcum genus because of their spherical shape, solitary forms, Coelastrella cogersae Suarez, Borrell et Rico sp. nov.
large number of cytoplasm starch granules and size. In terms of genetic Diagnosis: Solitary or few-celled aggregation, oval-shaped and sym-
assignation, isolates were related to the same sequence of Tetracystis metric mature cells. The width was 6.7–8.2 μm, and the length was
tetraspora (99%) using 18S sequences. No sequence was obtained 8.6–9.1. The chloroplast appeared single and parietal with polygonal-
using ITS1-5.8S-ITS2 gene cluster. In phylogenies based on 18S gene, a shaped plates. There was 1 pyrenoid per cell. The cell wall was normal
big clade covered the Chlorococcales order resulted, with a relatively to gross. It contained 10–12 longitudinal rifts, and the polar thickenings
high robustness among their subclades and branches. Isolates were lo- were moderately developed. Asexual reproduction occurred through
cated inside this group although they were far from the main the division of the parental cells and a final release resulting from the
Chlorococcum and Tetracystis genus (Fig. 5). Morphologically, BG.600 rupture of the cell wall (liberation of approximately 2–8 spindle-
and BG.602 exhibited various forms in solid and liquid media (including shaped cells that were smaller than mature cells). The exact identifica-
4 or 8 aplanospores formations). The most abundant was a big spherical tion was possible only by using DNA barcoding techniques and molecu-
cell with many cytoplasm granules of starch, very close to lar markers.
Haematococcus. However, flagellate morphotypes, which are the active Habitat: Water pond inside a landfill.
form in this group, were not found. AlgaeBase contains a great deal of Type locality: 43°29′31.1883″ N, 5°49′12.4417″ O, COGERSA facilities
combination research (Bellinger and Sigee, 2010; Guiry and Guiry, (landfill), Asturias, Spain.
2012) on microalgae freshwater key related to Chlorococcum sp. Holotype: Coelastrella cogersae.
forms. Genetic studies using the 18S sequences indicated a strong iden- Iconotype: Figs. 3J, 4G and 7B
tity (97%) of these isolates with the Tetracystis tetraspora species. The Etymology: The name assigned to the species was based on the acro-
18S phylogenetic tree was slightly uncertain because both isolates nym by which the landfill site is known.
seem to be isolated units close to both the Chlorococcum and Tetracystis
genera. A further analysis of the life cycle is needed because, based on 3.4. Carotenoid potential
the AlgaeBase data, the main difference is the ability of Tetracystis to
produce tetrads. From this study, it can be concluded that more infor- A preliminary experiment with an added volume of the BG-11 me-
mation is needed within the group (the isolates identified in this dium was performed for all of the cultures growing in the Erlenmeyer
study might be new and different from those previously described in flasks. The cultures in the stationary phase were refreshed with a me-
this group). dium containing 20-fold less than the standard nutrient levels.
CE2.401, CE2.402 and CE2.320 isolates showed a change in colour
3.2.4. Order Chlorellales (Class Trebouxiophyceae) from green to yellow-orange in a short period (3–6 days) (Fig. 7). Cyto-
The CA1.321 and CA.322 isolates were consistently related to the plasm inclusions could be observed in CE2.401 yellow-orange cells,
Chlorella genus based on their small size, special green colour and spher- making difficult the distinction of different organelles such as pyrenoid
ical shape. However, the most specific features were the girdle-, cup- or and chloroplast in contrast to green cells without nutrient medium
saucer-shaped chloroplast and the parietal pyrenoid. The BG.601 could modification.
be included in the Chlorella group, but its large size made assignment Carotenoid production has been detected in some microalgae
difficult. Moreover, the isolates BG.601, CA1.321 and CA1.322 were groups, being dependent on the environmental conditions (Ambati
matched to the same reference sequence of Chlorella sorokiniana with et al., 2018). Nutrient limitation appeared as a key factor to induce met-
99% identity using the 18S gene marker. The CA1.321 and CA1.322 iso- abolic pathways to generate few high-value compounds, such as β-
lates were also found to be close to the Chlorella sorokiniana reference carotene from Asterarcys quadricellulare, usually through N and P deple-
sequences but with lower identity percentages (95%) in the case of tion and/or starvation (Singh et al., 2019). For that reason, carotenoid
ITS1-5.8S-ITS2 gene cluster. Surprisingly, the BG.601 isolate was related potential production from all strains isolated in this study was
to a new reference sequence, Chlorella vulgaris (99%). The Chlorellales discussed. The Sphaeroplelales strains isolated in this study have
order clustered the CA1.321, CA1.322 (both in the same branch) and yielded promising results related to carotenoid production in the
BG.601 isolates in 18S phylogenetic tree (Fig. 5). Similarly, the Coelastrella and Scenedesmus genera. Aburai et al. (2015) demonstrated
CA1.321 and 322 isolates were joined in this cluster with the BG.601 iso- that high irradiance and salinity had a dramatic effect on carotenoid ac-
late in ITS1-5.8S-ITS2 phylogenetic tree (Fig. 6), Hence, and despite the cumulation in Scenedesmus sp. KGU-Y002. Moreover, various classes of
doubts about the morphological assignation of BG.601 sequence, the carotenoids were identified (e.g., astaxanthin, lutein and zeaxanthin).
BLAST results confirmed the morphological identification with a high Similarly, another strain of Scenedesmus sp. yielded a high accumulation
percentage of homology (99%) in both gene markers. Also, the distribu- of total carotenoids without any treatment (Neofotis et al., 2016), or
tion inside the phylogenetic trees was precise, given that it was clus- even by the addition of low concentrations of brassinosteroids, which
tered close to the Chlorella sorokiniana reference sequences (CA1.321 could lead to an increase of carotenoids profile (Talarek-Karwell et al.,
and CA1.322). However, further studies based on evolutionary diversity 2018). During this study, the Acutodesmus obliquus strain (CE2.402) ex-
of evolutionary diversity Chlorella-related species have arisen (Heeg and hibited a change of phase from green to orange. This was likely caused

10
D. Suarez-Montes, Y.J. Borrell, J.M. Gonzalez et al. Science of the Total Environment 802 (2022) 149755

Fig. 7. NJ consensus tree based on the ITS1-5.8S-ITS2 gene cluster. The new sequences derived from this study appear as coloured dots based on the taxonomic order.

by the dramatic decrease in the main nutrients when they were (Saeki et al., 2017) could be stressed by different salts and phytohor-
renewed. Coelastrella genus was the least studied of the isolates in this mones to increase the number of carotenoids.
work. Surprisingly, 75% of the studies reviewed in the literature have There are few references to biotechnological applications of the
explained its high capacity for accumulating carotenoid compounds Chlorococcum genera. Promising results have been achieved with regard
(Hu et al., 2013; Neofotis et al., 2016). In fact, the CE2.401 and CE2.320 to carotenoid production from Chlorococcum humicola species, showing
strains isolated in this study could change its colour from green to an interesting carotenoid profile where lutein, astaxanthin and β-
yellow-orange. Not only is the Coelastrella genus capable of producing carotene appeared as main high-value compounds (Sivanathu and
and accumulating carotenoid, but it could also be a relevant diagnostic Paliniswamy, 2012). Moreover, up-scaling cultures in both different
feature of the group. Certain Coelastrella sp. strains, such as KGU-Y002 PBR systems and cultivation modes were analysed for chlorophylls

11
D. Suarez-Montes, Y.J. Borrell, J.M. Gonzalez et al. Science of the Total Environment 802 (2022) 149755

and carotenoids production (Wannachod et al., 2018). More recent Acknowledgments

studies have been performed in the same species, not only focusing
on cultivation or carotenoid production, but in extraction by innova- The authors acknowledge ReCO2very project support by INGEMAS,
tive organic solvents such as liquefied dimethyl ether (Babadi et al., NEOALGAE and COGERSA SAU, as well as Ministry of Economy and
2020). Competitiveness funding (MINECO RETOS-COLABORACION RTC-2014-
Carotenoid production in the Chlorella genera is possible. For exam- 2109-5), and participation of the University of Oviedo, ITMA Materials
ple, for sources of astaxanthin, Chlorella zofingiensis has been proposed Technology and Instituto Nacional del Carbón (INCAR-CSIC). To
as an alternative to Haematococcus pluvialis, the main producer (Liu Dr. Mauro Sanna for the inestimable help in revising the manuscript.
et al., 2014a, 2014b). Also, the species was cultured in both heterotro-
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