Environmental Advances 4 (2021) 100062

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Environmental Advances
journal homepage: www.elsevier.com/locate/envadv

Biostimulant and biopesticide potential of microalgae growing in piggery

wastewater
Alice Ferreira a, Lusine Melkonyan b,c, Sofia Carapinha a, Belina Ribeiro a, Daniel Figueiredo d,
Gayane Avetisova b,c, Luisa Gouveia a,d,∗
a
LNEG, National Laboratory of Energy and Geology I.P., Bioenergy and Biorefineries Unit, Estrada do Paço do Lumiar 22, 1649-038 Lisbon, Portugal
b
SPC “Armbiotechnology” NAS RA, 14 Gyurjyan Str., 0056 Yerevan, Armenia
c
Yerevan State University, 1 Alex Manoogian, 0025 Yerevan, Armenia
d
Green CoLab - Green Ocean Technologies and Products Collaborative Laboratory, Centro de Ciências do Mar do Algarve, Universidade do Algarve, Campus Gambelas,
Edifício 7, 8005-139, Faro, Portugal

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

Keywords: Pig farming generates highly polluting wastewaters which entail serious environmental issues when not ade-
Tetradesmus obliquus quately managed. Microalgae systems can be promising for cost, energy and environment-efficient treatment of
Cyanobacteria piggery wastewater (PWW). Aside from clean water, the produced biomass can be used as biostimulants and
Swine wastewater
biopesticides contributing to a more sustainable agriculture.
Germination index
Plant protection Three microalgae (Tetradesmus obliquus, Chlorella protothecoides, Chlorella vulgaris) and one cyanobacterium (Syne-
chocystis sp.) were selected after a preliminary screening in diluted wastewater (1:20) to treat PWW. The nutrient
removals were 62-79% for COD (chemical oxygen demand), 84-92% for TKN (total Kjeldahl nitrogen), 79-92%
for NH4 + and over 96% for PO4 3− . T. obliquus and C. protothecoides were the most efficient ones.
After treating PWW, the produced biomass, at 0.5 g L−1 , was assessed as a biostimulant for seed germination,
root/shoot growth, and pigment content for tomato, watercress, cucumber, soybean, wheat, and barley seeds. We
observed an overall increase on germination index (GI) of microalgae-treated seeds, owing to the development
of longer roots, especially in T. obliquus and C. vulgaris treatments. The microalgae treatments were especially
effective in cucumber seeds (75-138% GI increase).
The biopesticide activity against Fusarium oxysporum was also evaluated at 1, 2.5 and 5 g L−1 of microalgae
culture. Except for Synechocystis sp., all the microalgae tested inhibited the fungus growth, with T. obliquus and
C. vulgaris achieving inhibitions above 40% for all concentrations.

1. Introduction generated from pig excreta and water used clean the hog housing sheds,
containing a high organic load, ammoniacal nitrogen, and phosphorus.
The ever-growing population has put an extreme pressure on agricul- While these pollutants are a problem for pig farms to handle, they
ture to produce more food (Searchinger et al., 2019). Livestock farming can be valuable as low-cost and readily available nutrient and water
practices have largely shifted to intensive animal farming to assure high sources for microalgae growth. The use of wastewater allows the re-
yields of animal-derived products, but have led to negative impacts on duction of microalgae biomass production below 5 €/kg at large scale
the environment and public health (Anomaly, 2015). (Acién et al. 2016). Microalgal-bacterial systems have already been used
In the European Union (EU), the majority of the protein consumed to treat PWW (e.g. Ferreira et al. 2018; García et al. 2017). They are com-
comes from animal sources (European Environment Agency, 2017). EU monly described as cost-efficient for nutrient recovery, providing a free
is currently one of the largest pig producers, with an average of 148 mil- process oxygenation, with reduced energy requirements and environ-
lion pig heads over the last 10 years, according to Eurostat (2020). Con- mental impacts (Cuellar-Bermudez et al., 2017; Ferreira et al., 2018). In
sequently, this industry is estimated to generate 215 – 430 m3 /year (4-8 a perspective of circular bioeconomy, microalgae can recover the nutri-
L/day/pig) of piggery wastewater (PWW) (García et al., 2017). PWW is ents from piggery wastewaters, which can generate further income for
the pig production facilities, as a source of biofuels (Batista et al., 2015;
Ferreira et al., 2018, 2017), animal feed, fertilizers, stimulants and/or

∗
Corresponding author.
E-mail address: luisa.gouveia@lneg.pt (L. Gouveia).

https://doi.org/10.1016/j.envadv.2021.100062
Received 23 March 2021; Received in revised form 15 April 2021; Accepted 23 April 2021
2666-7657/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
A. Ferreira, L. Melkonyan, S. Carapinha et al. Environmental Advances 4 (2021) 100062

pesticides (Ferreira et al., 2019; García et al., 2017, 2018; Navarro- The microalgae tested were Synechocystis sp. PCC 6803 (Amster-
López et al., 2020; Posadas et al., 2017). dam University, Netherlands), Tetradesmus obliquus (formerly known
There is a growing trend on sustainable agriculture to promote low as Scenedesmus obliquus) (ACOI 204/07, ACOI Culture Collection,
pesticide-input and the application of natural products, in detriment of Coimbra University, Portugal), Chlorella protothecoides (also known
minerals and chemicals, which are not only limited but can bring se- as Auxenochlorella protothecoides) (strain 25,UTEX Culture Collection,
vere environmental problems (e.g., eutrophication, soil infertility, and Austin University, USA), Chlorella vulgaris (INETI 58, 90 LNEG_UB, Por-
biodiversity loss) (Bulgari et al., 2015; Calvo et al., 2014; Sharma et al., tugal), Neochloris oleoabundans (UTEX #1185, UTEX Culture Collection,
2014). Catching this new wave, the use of biofertilizers, biostimulants, Austin University, USA), and Nostoc sp. PCC 9202 (Instituto de Bio-
and biopesticides derived from microorganisms can promote seed ger- química Vegetal y Fotosíntesis, Seville, Spain).
mination, plant growth, flower set and fruit production (Bulgari et al.,
2015; Colla and Rouphael, 2020; du Jardin, 2015; Singh et al., 2016), 2.2. Microalgae/Cyanobacteria screening
and expand the tolerance to abiotic (e.g. high salinity, drought, and
frost) and biotic stresses (e.g. pathogens, pests, and insects) (Carvajal- A screening was carried out to select the microalgae or cyanobacteria
Muñoz and Carmona-Garcia, 2012; Costa et al., 2019). All these aspects which were able to grow in PWW. The different species tested were
could be fulfilled by microalgae. They contain valuable compounds, inoculated in small flasks using different dilutions (1:20, 1:10, 1:5, 1:2,
such as amino acids, carbohydrates, minerals, trace elements, and phy- 1:1) of PWW with tap water as the cultivation medium and were kept at
tohormones, among others (Colla and Rouphael, 2020; Górka et al., room temperature (23-25°C), under continuous artificial light conditions
2015; Khan et al., 2009). They can enhance plant growth by acting as (3 fluorescent lamps of 18W, Philips TL-D) at light intensity of 41 μE
an organic slow-release fertilizer to supply nutrients assimilated from m−2 . s−1 , and orbital agitation at 150 rpm (G-25 incubator shaker (New
wastewater and avoid the contamination of soils and water bodies with Brunswick Scientific Co, USA).
extreme nutrient loads (Coppens et al., 2016). Microalgae-based bios-
timulants can also improve nutrient uptake by plants and the soil struc-
2.3. Wastewater treatment experiments
ture and aeration, which may stimulate root growth (Bumandalai and
Tserennadmid, 2019). Microalgae and cyanobacteria have also been
The microalgae and cyanobacteria capable of growing in 1:20 PWW
shown to have antibacterial and antifungal activity (Costa et al., 2019;
- Synechocystis sp., T. obliquus, C. protothecoides, and C. vulgaris – were
Renuka et al., 2018; Singh et al., 2016). However, deeper investigation is
used for further treatment experiments to evaluate their performance
required on this agricultural biotechnological field (Costa et al., 2019).
on nutrient removal. Because most species are microalgae, from this
Europe is currently the biggest market for biostimulants, with around
point forward, Synechocystis sp. (cyanobacterium) will be referred to
8.5 million hectares of area treated in 2016 (Liebig et al., 2020). This
as a microalga as well when mentioning all the species tested, just to
has amplified the need for a harmonized European Regulation for plac-
simplify the writing.
ing biostimulants on the market. Thus, on 2019, a new Fertilizing Prod-
The microalgae cultures were cultivated in 5 L bubble columns pho-
ucts Regulation (FPR) (EU) 2019/1009 was published including bios-
tobioreactors (PBRs) using the same 1:20 PWW as medium, at a working
timulants for the first time as CE-marked fertilizing products on 2022
volume of 4 L. The cultures were maintained at room temperature (23-
(Regulation (EU) 2019/1009, 2019). The Global Biostimulant Market
25°C) under continuous illumination (3 fluorescent lamps of 36 W and
was estimated to be valued at USD 2.6 billion in 2019 and is expected
6 of 18 W, Philips TL-D) at an average light intensity of 53 μE m−2 s−1 .
to grow 11.24% through 2025 (MarketsandMarkets, 2020).
The aeration was supplied at 0.15 vvm (air volume (L) per volume of
Considering all the aspects presented, the research on microalgae
culture medium (L) per minute (m)) through aquarium pumps. After 19
for agriculture is a very relevant and promising topic. Our work aimed
days of cultivation, the microalgae cultures were left to settle for 24 h
to combine microalgae cultivation with piggery wastewater treatment
at room temperature to concentrate the biomass. The supernatant was
to generate clean water and bioproducts (bio-fertilizers, stimulants,
collected for further analysis. The microalgal biomass was further con-
and pesticides) to respond to an eco-friendlier approach for sustain-
centrated by settling for more 24 h at 4°C for germination, plant growth
able agriculture. For this, we did a screening of several microalgae
and pesticide trials.
(Tetradesmus obliquus, Chlorella protothecoides, Chlorella vulgaris, and
Neochloris oleoabundans) and cyanobacteria (Synechocystis sp. and Nos-
toc sp.) to treat piggery wastewater to select the most successful one(s) 2.3.1. Microalgae growth
in nutrient removal efficiency, and with the best biomass quality for The assessment of microalgae growth was monitored by measuring
agricultural products. The obtained microalgal biomass was evaluated the optical density of the culture samples, at 540 nm (Rocha et al.,
for germination, root and shoot growth, and pigment content in dif- 2003), against distilled water, using a Hitachi U-2000 spectrophotome-
ferent seeds, such as watercress, tomato, cucumber, barley, wheat, and ter. In addition, the biomass dry weight and the ash free dry weight
soybean. Their biopesticide effect was also investigated against the fun- (AFDW) were determined through gravimetry by drying the samples at
gus Fusarium oxysporum. The production of biostimulants and biopesti- 105°C overnight and incinerating at 550°C for 1 h, respectively. The
cides from microalgae cultivated in wastewaters is yet an unexplored biomass productivity was calculated from the final biomass concentra-
approach, and to the best of our knowledge, few studies address this. tion, given by the AFDW at the end of the cultivation period of 19 days.
Thus, we believe our work can offer an important contribution to better
understand the potential of microalgae for agricultural purposes. 2.3.2. Nutrient removal
The initial raw and diluted (1:20) PWW were characterized in terms
of ammonia and total Kjeldahl nitrogen (TKN), chemical oxygen demand
2. Materials and methods (COD), and phosphorus, according to the standard methods by APHA
(2005), as previously described by Ferreira et al. (2017). Ammonium
2.1. Effluent and microalgae nitrogen was quantified using an ion selective electrode Crison code:
96 63 (Crison-HACH). TKN was determined by the standard method
The piggery wastewater was collected from a stabilization pond in a 4500-Norg B with adaptation. The COD determination was carried out
local pig farm from Valorgado in Herdade do Pessegueiro (39°00′09.0"N by the Open Reflux method – Method 5220-B (APHA, 2005). A commer-
8°38′45.5"W) (Glória do Ribatejo, Portugal) during the month of May. cial kit was used for the measurement of phosphorus (Phosver 3-Powder
This PWW corresponds to the liquid fraction of pig slurry after separa- Pillows, Cat. 2125-99, Hach) at 890 nm, using a HACH DR/2010 spec-
tion from solid manure. trophotometer.

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A. Ferreira, L. Melkonyan, S. Carapinha et al. Environmental Advances 4 (2021) 100062

To evaluate the efficiency of microalgal-based treatment, the same

analyses were performed for the final effluent at the end of the cultiva- 𝐶𝑏 (𝜇𝑔∕𝑚𝐿) = 21.5 × 𝐴646 − 5.1 × 𝐴663 (3)
tion runs, after settling and filtration.
1000 × 𝐴470 − 1.82 × 𝐶𝑎 − 85.02 × 𝐶𝑏
2.3.3. Microalgae biomass characterization 𝐶𝑎𝑟𝑜𝑡 (𝜇𝑔∕𝑚𝐿) = (4)
198
The biochemical composition of the microalgal biomass was deter-
mined in terms of proteins, sugars, and fatty acids. Total sugars (carbo- 2.5. Biopesticide trials
hydrates) content was determined through the phenol-sulfuric method
(DuBois et al., 1956), following quantitative acid hydrolysis extraction The biopesticide bioassays were done against the fungus Fusarium
(Hoebler et al., 1989). Protein content was estimated through the Kjel- oxysporum in sterile Petri dishes. Potato Dextrose Agar (PDA) was used
dahl method and calculations were conducted applying the conversion as culture medium (4 mg L−1 potato starch, 20 mg L−1 dextrose, 15 mg
factor 5.95 (López et al., 2010; Waghmare et al., 2016). A detailed de- L−1 agar). Due to the vast bacterial load coming from the effluent, tar-
scription of the methods was already made by Ferreira et al. (2017). taric acid (10% w/v) was used to decrease the medium’s pH to 3.5 and,
The elemental composition of the microalgae/cyanobacteria biomass thus, inhibit the bacterial growth, according to manufacturer instruc-
was analyzed by x-ray fluorescence (XRF) spectroscopy, using a Ni- tions. First, agar was poured into the Petri dishes until half, and 4 holes
tonTM XL3t analyzer (Thermo Fischer Scientific, USA). The analysis was were done using Oxford towers. After the agar solidification, the PDA
conducted exposing samples of freeze-dried biomass in proper cuvettes was added. The microalgae suspensions were poured into the holes and
to XRF, under helium-rich atmosphere. the fungus was placed in the middle. Sterile distilled water was used as
control. The Petri dishes were then incubated in the dark at 25°C for 10
2.4. Seed germination study days.
The inhibition percentage is calculated using the Eq. 5, where PD and
The seed germination/plant growth experiments were carried out in CD correspond, respectively, to the diameter of the fungi growth with
Petri dishes with Whatmann filter paper, with 8 seeds of each plant, microalgae suspension and in the control (distilled water), respectively.
in duplicate. The plants tested were cucumber (Cucumis sativus), bar- ( )
ley (Hordeum vulgare), wheat (Triticum aestivum), soybean (Glycine max), 𝑃𝐷
𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 (%) = 100 − × 100 (5)
watercress (Nasturium officinale), and tomato (Licopersicon esculentum). 𝐶𝐷
For each plant, five treatments were done: control (distilled water) and
3. Results and discussion
four microalgae cultures of 0.5 g L−1 each (Synechocystis sp., T. obliquus,
C. protothecoides, and C. vulgaris). The microalgae cultures were adjusted
3.1. Microalgae screening
to the desired concentration (0.5 g L−1 for germination and growth ex-
periments, and 1, 2.5 and 5 g L−1 for biopesticide trials) by adding dis-
To choose the best microalga(e) for the PWW treatment, a prelim-
tilled water. All samples were incubated at room temperature (25 ºC) in
inary screening was done with six microalgae - Synechocystis sp., T.
the dark for 5 days followed by sunlight in the remaining 5 days. During
obliquus, C. protothecoides, C. vulgaris, N. oleoabundans, and Nostoc sp.
the experiment, the samples were watered daily with the same amount
cultivated in diluted PWW (1:20). Several dilution factors were tested
of distilled water to keep the filter paper humid.
(1:20, 1:10, 1:5, 1:2, 1:1). However, 1:20 was the only one that pro-
vided the adequate nutrient content (especially for ammonium) for the
2.4.1. Root and shoot growth
microalgae growth as well as a suitable light penetration. Their growth
At the end of 10 days, the seedlings were carefully separated and
was monitored through optical density for 23 days (Fig. 1). Only N.
measured with a ruler and the results registered for comparison between
oleoabundans and Nostoc sp. were not able to grow in the diluted PWW,
the microalgae treatments and the control with the distilled water.
and consequently they were excluded for the following experiments.
Except for Synechocystis sp., which started to grow right after being in-
2.4.2. Germination index oculated, the other microalgae took around 10 days to acclimatize to
The germination index (GI) of each sample was determined accord- the effluent conditions (lag phase).
ing to Zucconi et al. (1981) by the following equation:
𝐺×𝐿 3.2. Treatment performance
𝐺𝐼 (%) = × 100 (1)
𝐺𝑊 × 𝐿𝑊
Where G and L are the number of germinated seeds and the root Table 1 presents the initial composition of raw and diluted (1:20)
length in the case of the microalgae extracts and Gw and Lw are the PWW. This wastewater has very high levels of COD (7232 mg O2 L−1 )
same parameters for the control (distilled water). and ammonia (3150 mg NH4 + L−1 ), which are inhibitory for microalgae
growth (Collos and Harrison, 2014). The values are much higher than
ones usually reported in studies using PWW for microalgae cultivation,
2.4.3. Chlorophyll and carotenoid contents
mainly because PWW underwent anaerobic digestion before algae cul-
The chlorophyll a, b and carotenoid contents were evaluated spec-
tivation (Ayre et al., 2017; Uggetti et al., 2014; Wang et al., 2013). Fur-
trophotometrically according to Sumanta et al. (2014) using 80% ace-
thermore, the PWW has a very dark brown color which can hinder the
tone as the solvent. The grown sprout leaves, from each plant were col-
light penetration and thus the photosynthetic growth. A dilution of 1:20
lected and grinded in 5 mL of acetone; the samples were homogenized
was then required to significantly decrease ammonium and color until
for 2 min in vortex followed by 20 min centrifugation 13000 × g in a
levels that are adequate for microalgae growth.
2-6E centrifuge (Sigma, Switzerland); the resulting supernatant was sep-
Synechocystis sp., T. obliquus, C. protothecoides, and C. vulgaris al-
arated and a volume of 0.5 mL of supernatant was mixed with 4.5 mL of
lowed nutrient removal efficiencies that are depicted in Table 2.
80% acetone; this solution was then measured in a U-2000 spectropho-
All microalgae were able to efficiently treat the wastewater since
tometer (Hitachi, Japan).
they all achieved high removal efficiencies, with ammonium removals
The chlorophyll a (Ca ), chlorophyll b (Cb ) and total carotenoids
above 79% and near complete removal of phosphate. Regarding COD
(Carot ) were calculated through the following equations (Sumanta et al.,
removal, C. vulgaris and T. obliquus gave the best results (79 and 73%,
2014):
respectively). Furthermore, T. obliquus and C. protothecoides achieved
𝐶𝑎 (𝜇𝑔∕𝑚𝐿) = 12.25 × 𝐴663 − 279 × 𝐴646 (2) productivities higher than 30 mg L−1 d−1 , while Synechocystis sp. and

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A. Ferreira, L. Melkonyan, S. Carapinha et al. Environmental Advances 4 (2021) 100062

Fig. 1. Growth curves of Synechocystis sp. ( ),

Tetradesmus obliquus ( ), Chlorella protothe-
coides ( ), Chlorella vulgaris ( ), Neochloris
oleoabundans ( ), and Nostoc sp. ( ) cultivated
in diluted piggery wastewater (1:20) for 23
days (mean ± standard deviation, n=2).

Table 1
Piggery wastewater (PWW) composition in terms of pH, chemical oxygen demand (COD), total
Kjeldahl nitrogen (TKN), ammonium nitrogen (NH4 + ) and phosphate (PO4 3− ) (mean ± stan-
dard deviation, n=2). Legislation values are depicted in Portuguese law (Decree-Law No 236/98,
1998).

PWW pH COD (mg O2 L−1 ) TKN (mg L−1 ) NH4 + (mg L−1 ) PO4 3− (mg L−1 )

Raw 7.70 7232±89 3500±420 3150±56 117.2±2.3

Diluted (1:20) 7.90 335±0 175±21 158±3 5.86±0.11
Legislation 6-10 150 15 10 10

Table 2
Productivity (in ash free dry weight) and nutrient removal efficiency (mean ± standard deviation, n=2) after 19 days of wastewater treatment with the microalgae
Synechocystis sp., Tetradesmus obliquus, Chlorella protothecoides and Chlorella vulgaris in 5L PBRs.

Productivity Nutrient Removal efficiency (%)

Microalgae (mg L−1 d−1 ) COD TKN NH4 + PO4 3−

Synechocystis sp. 23.7±2.6 61.6±5.5 88.0±5.7 92.4±0.1 90.1±0.0

T. obliquus 31.6±0.0 73.1±3.3 89.6±4.7 87.5±0.4 98.1±0.0
C. protothecoides 36.8±7.9 68.4±2.2 92.0±1.6 92.0±0.0 98.5±0.0
C. vulgaris 22.4±3.9 79.2±3.5 84.0±2.3 79.4±0.1 98.6±0.3

C. vulgaris were around 22-23 mg L−1 d−1 . The final pollutants compo- 3.3. Microalgae biomass composition
sition of the treated water after microalgae cultivation was still slightly
above the permitted discharged limits (Table 1) for nitrogen pollutants The biochemical and mineral composition of the microalgae biomass
TKN (14-28 mg L−1 ) and NH4 + (12-32 mg L−1 ), while COD levels are is available in Table 3. All microalgae grown in PWW are rich in proteins
under the limits (70-128 mg O2 L−1 ). Thus, it would be necessary a owing to the higher nitrogen content of the wastewater, which is used
longer treatment period to fully treat the wastewater to comply with by microalgae for protein synthesis. Moreover, it indicates that they are
the Portuguese legislation (Decree-Law No 236/98, 1998). growing in adequate conditions. Synechocystis sp. presents the highest
Nonetheless, it is important not to forget that the effluent was pre- protein content as expected (47.3%), but also the other strains presented
viously diluted with a significant amount of water to adjust its compo- significant contents (above 35%). These protein rich wastewater-grown
sition to microalgae growth. However, this is not a viable strategy for microalgae could then be a key source of amino acids, such as trypto-
large scale application, from the economic and environmental point of phan and arginine which are metabolic precursors of phytohormones
view. However, this strategy is adequate for the purpose of the present (Chiaiese et al., 2018). Hence, the microalgae are expected to have a
work, which was to select the microalgae that could simultaneously stimulating effect on the growth and yield of plants. On the other hand,
grow by treating PWW, and have effect on plant germination, growth, C. protothecoides has the highest carbohydrate content (32.7%), while
and protection. the others had very similar contents (25-27%). Some studies have al-
To upgrade the present work, we are looking for alternative strate- ready evidenced that microalgae polysaccharides promote plant growth,
gies to avoid the use of fresh water, which is a scarce resource. A nutrient uptake, and extend plant tolerance to stress (El-Naggar et al.,
stronger inoculum to start the microalga culture as well as the injec- 2020; EL Arroussi et al., 2018, 2016; Farid et al., 2019).
tion of CO2 could be used to control the pH range (6-7) and shift the All the listed macro- and microelements are essential minerals for
chemical equilibrium from NH3 to NH4 + , which is less toxic for mi- plant physiology and development, being part of several cellular mech-
croalgae (Ayre et al., 2017). Moreover, pre-treatment processes could anisms, such as ion fluxes, osmosis, salt tolerance and even as co-factors
be applied aiming to reduce the ammonia toxicity and decolorize the for enzymes. Macronutrients are normally found in plants within a range
effluent, to avoid the need of using water for dilution (Depraetere et al., of 1000 to 15000 ppm (dry weight) and micronutrients concentrations
2013; Kim et al., 2014). 100 to 10000 times lower (Delhaize et al., 2015). Considering these val-

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A. Ferreira, L. Melkonyan, S. Carapinha et al. Environmental Advances 4 (2021) 100062

Table 3
Biochemical (protein and carbohydrates) and mineral composition (mean ± standard deviation,
n=2) of Synechocystis sp., Tetradesmus obliquus, Chlorella protothecoides, and Chlorella vulgaris
grown in diluted (1:20) piggery wastewater.

Composition Synechocystis sp. T. obliquus C. protothecoides C. vulgaris

Protein (%) 47.3±2.5 34.5±2.1 34.4±0.8 38.3±0.9

Carbohydrates (%) 25.1±0.2 25.5±0.1 32.7±0.6 26.8±2.7
Mineral content (ppm)
N 77000±1400 57960±280 57750±910 64330±1050
P 3193±89 3634±91 4104±90 3571±100
K 26249±252 17957±199 16394±185 16007±285
Ca 9331±227 42303±405 30901±338 24268±461
S 12415±144 13392±148 13991±146 11451±193
Mg <LOD <LOD <LOD <LOD
Fe 222±27 74.8±25 273±28 350±28
Cl 1211±32 5173±57 2391±50 781±27
Mn <LOD <LOD <LOD <LOD
Zn 434±12 188±8 447±14 561±13
Cu 124±13 78.5±13 120±13 135±13
Mo 6.4±1.0 7.3±1.0 6.6±1.0 7.7±1.0
Ni <LOD <LOD <LOD <LOD

LOD: limit of detection

Fig. 2. Germination index (%), considering

distilled water as the control (100%) for the 6
different seeds treated with different microal-
gae suspensions (Synechocystis sp., Tetradesmus
obliquus, Chlorella protothecoides, and Chlorella
vulgaris) grown in diluted piggery wastewater
at a concentration of 0.5 g L−1 . Error bars indi-
cate standard deviation (n=2).

ues, the mineral contents of the cultivated microalgae are within the out from all the other microalgae. Because soybean seeds have a high
specified range and can, therefore, meet the plant necessities, which is protein contents, the demand for N is extremely high during seed for-
an indication of the potential of these microalgae to act as biofertilizers. mation. To fulfill this, they can fixate N2 in a symbiotic process with soil
borne rhizobia bacteria (Mcgrath et al., 2013). Synechocystis sp. can also
3.4. Effect of microalgae as biostimulants for seed germination, plant fixate N2 , and thus, can explain why it had a major effect on soybean
growth, and pigment content seeds.
Microalgae and cyanobacteria can synthesize a remarkable diver-
3.4.1. Germination index sity of biologically active molecules, such as fatty acids, phytohormones
It is considered that a germination index (GI) of 100% corresponds to (e.g. auxins, cytokinins, gibberellins, etc.), polysaccharides and pheno-
control samples, where seeds were treated with distilled water. There- lics (Cuellar-Bermudez et al., 2017; Renuka et al., 2018; Ronga et al.,
fore, only microalgae suspensions leading to GI higher than 100%, 2019). However, the action mode of these compounds in plant devel-
are considered to have biostimulant activity. The results are shown in opment is still not well explored. Thus, it is not completely clear why
Fig. 2a. T. obliquus and C. vulgaris promoted best results than Synechocystis sp.
In general, it is possible to observe that all microalgae studied have in the present work, when the latter has a higher N content. Never-
shown a positive effect on the germination index for the tested seeds. theless, we can hypothesize that the first two may have optimal con-
This effect seems to be especially clear in cucumber seeds, where GI tents in some components that might provide a higher effect of seed
values were at least 99% higher compared to control (distilled water). germination and plant growth. For example, Plaza et al. (2018) saw
On the other hand, the positive effect was lower in the case of tomato that Scenedesmus spp. showed a higher concentration of phytohormones
seeds. It is also important to notice that T. obliquus generated GI values compared to Arthrospira spp., promoting a higher increase in root dry
higher than 100% in all the tested seeds, and the highest GI average val- weight of Petunia x hybrid plants.
ues. C. vulgaris gave the best results for cucumber, wheat, and tomato
seeds, while T. obliquus had the greatest impact on barley and watercress
seeds. This is in accordance with results obtained previously by Navarro- 3.4.2. Shoot and root growths
López et al. (2020), where T. obliquus grown in brewery wastewater was The results for shoot and root development of each type of seed tested
also shown to have biostimulant potential in watercress seeds. Likewise, are displayed in Fig. 3. The microalgae studied had an overall positive
the same was shown in barley seeds (Ferreira et al., 2019). For soybean, effect on plant roots, with lengths higher than the control. However, the
Synechocystis sp. increased 65% the GI compared to the control, standing effect of microalgae on plant shoots was not significant.

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A. Ferreira, L. Melkonyan, S. Carapinha et al. Environmental Advances 4 (2021) 100062

Fig. 3. Average shoot and root length for (a)

cucumber, (b) barley, (c) wheat, (d) soybean,
(e) watercress and (f) tomato seeds with dis-
tilled water (control) and treated with microal-
gae biomass suspensions of Synechocystis sp.,
Tetradesmus obliquus, Chlorella protothecoides,
and Chlorella vulgaris, after a 10-day cultivation
period. Error bars indicate standard deviation
(n=2).

The positive effect on roots were especially evident in cucum- Regarding shoot lengths, only in soybean plants the increase in
ber and wheat (100% and 33.5% average length increase, respec- treated seeds was more perceptible, especially in the case of Synechocys-
tively). Furthermore, most seeds treated with C. vulgaris and T. obliquus tis sp. and C. protothecoides (above 90% increase). In the case of bar-
originated plants with longer roots. Similar trends were obtained by ley, seeds treated with T. obliquus stood out from other microalgae (al-
Bumandalai and Tserennadmid (2019) using C. vulgaris suspensions, at most 9% increase in shoot length). This is accordance with the previ-
different concentrations, to treat cucumber seeds. They highlighted that ous study done by Ferreira et al. (2019), where T. obliquus grown in
0.25 g L−1 of algal suspension is the best treatment for root and shoot brewery wastewater also showed a promising effect on barley seeds. In
lengths, being more effective in the root, like in the present study. Never- wheat shoots, the microalgae had a negative effect which could also be
theless, the same authors show up an increased germination for tomato explained by the reasons presented before regarding the high concentra-
seeds, being the best results obtained at 0.17 g L−1 with C. vulgaris tion of microalgae in the present study, just like showed by Kumar and
biomass. For higher concentration, they observed an inhibitory effect Sahoo (2011) for Triticum aestivum var. Pusa Gold (wheat) seeds treated
on the plant growth. This last result might suggest that the microalga with seaweed extract at concentrations above the optimum. Moreover,
concentration used (0.5 g L−1 ) might be excessive for tomato plants, Rachidi et al. (2020) obtained significant differences in shoot length of
negatively affecting their growth. This is especially clear in the case of tomato seeds treated with microalgae (Arthrospira platensis, Dunaliella
Synechocystis sp. treatment, which has a higher protein concentration salina, and Phorphorydium sp.), unlike the present results, but not for
(47.3%) and, consequently, of amino acids and/or polyamines, which the root lengths, similar to the present study.
could inhibit seed growth at concentrations exceeding the optimum These results could be expected due to the application method,
(Navarro-López et al., 2020; Tarakhovskaya et al., 2007). Nonetheless, where the seed is soaked in the microalgae suspensions, reach-
in the case of cucumber seeds, it can be said that the concentration ap- ing the roots first and slowing spreading to the other parts of
plied (0.5 g L−1 ) was beneficial for the plant roots as shown by Navarro- the plant. In addition, the cultivation period of 10 days could be
López et al. (2020) with T. obliquus treating cucumber seeds. short for some of the plants tested. Moreover, foliar application

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A. Ferreira, L. Melkonyan, S. Carapinha et al. Environmental Advances 4 (2021) 100062

Fig. 4. Biopesticide activity of the microal-

gae suspensions (Synechocystis sp., Tetradesmus
obliquus, Chlorella protothecoides, and Chlorella
vulgaris), at different concentrations (1, 2.5 and
5 g L−1 ), against pathogen Fusarium oxysporum.

of microalgae could promote a better development in plant shoots Table 4

(Plaza et al., 2018). Inhibition percentage of Fusarium oxysporum by Syne-
chocystis sp., Tetradesmus obliquus, Chlorella protothecoides
and Chlorella vulgaris at different concentrations (1, 2.5,
3.4.3. Chlorophyll and carotenoid contents and 5 g L−1 ).
The application of microalgae to enhance pigment content was eval-
uated in all plants tested and the results are shown in Fig. S1 (see Sup- Concentration (g L−1 )
Microalgae 1 2.5 5
plementary Material).
Considering the overall results, the chlorophyll b and total Synechocystis sp. 10.5±6.5 3.2±0.4 0.0±0.0
carotenoid concentrations in the six plants studied were not affected T. obliquus 43.5±0.0 43.5±1.6 46.8±1.6
C. protothecoides 35.1±10.9 36.7±0.4 53.2±4.0
by the microalgae suspensions in comparison with distilled water sam-
C. vulgaris 45.6±3.6 48.8±0.4 49.6±0.4
ples, unlike the results shown by Mutale-joan et al.(2020) (for both)
and Rachidi et al. (2020) (for carotenoids) in tomato plants. However,
for chlorophyll a there was a slight increment in plants treated with
microalgae, similar to the results obtained by Jimenez et al. (2020) and lated to the microalgal concentration. Even at the lowest concentration
Rachidi et al. (2020) using microalga Monoraphidium sp. and Arthrospira (1 g L−1 ), C. vulgaris and T. obliquus allow an inhibition percentage
platensis, respectively, to treat tomato seeds. This was more noticeable higher than 40%, which were maintained with the increased biomass
for cucumber, where there was an enhancement of chlorophyll a in the concentration (except for C. protothecoides which increased from 35 to
plants treated with microalgae. For soybean and wheat, the seeds treated 53% when the concentration increased from 1 to 5 g L−1 ). The inhibi-
with Synechocystis sp. revealed higher chlorophyll a content than the tion effect of C. vulgaris, grown in different media, against F. oxyspo-
control, and in tomato, T. obliquus stood out from the others. Thus, the rum was already reported by Vehapi et al. (2018). In another study by
results suggest that the microalgae tested will probably have a more Vehapi et al. (2020), C. vulgaris followed by C. protothecoides showed
pronounced effect on chlorophyll rather than in carotenoids. However, the strongest antifungal effect against various apple-infecting fungi,
more detailed analysis is needed to further conclude the potential effect such as Aspergillus niger, Alternaria alternata, and Penicillium expansum.
of microalgae biomass on the photosynthetic pigments of the plants. Moreover, the authors attributed the major antifungal activity to com-
pounds like terpenes, alkaloids and polypeptides found in C. vulgaris
(Vehapi et al., 2020).
3.5. Effect of microalgae as biopesticide
Given these results, the assessment of their biopesticide potential
should be extended to other pathogenic microorganisms (fungi and bac-
Having been found several cyanobacteria strains, macroalgae
teria) that could have a greater impact on crop productivity. Moreover,
and some microalgae with pesticide activity (Costa et al., 2019;
lower concentrations should be studied to determine if microalgae sus-
Renuka et al., 2018), trials were conducted to understand if any of the
pensions can be used at the same concentrations for both biostimulant
four microalgae used in this work – Synechocystis sp., T. obliquus, C.
and biopesticide activities.
protothecoides and C. vulgaris – could have an impact on the growth of
the fungus Fusarium oxysporum. Different concentrations of microalgae
suspension were studied (1, 2.5 and 5 g L−1 ). The results are shown in 4. Conclusions (233)
Fig. 4.
In Fig. 4, it is possible to observe the inhibition of fungi growth Microalgae can recover nutrients and water from livestock wastewa-
in most samples containing microalgae suspensions, when compared to ter to promote a more sustainable use of these resources on agriculture,
control. The only microalga that seemed to have an insignificant effect with full respect for public health and the environment. The introduc-
on the fungi growth is Synechocystis sp. tion of microalgae as biofertilizers, biostimulants and biopesticides is a
These observations can be confirmed by calculating the inhibition promising approach to reduce or even replace the use of non-renewable
halo, which is presented in Table 4. As expected, the inhibition is re- chemicals, without compromising plant productivity.

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A. Ferreira, L. Melkonyan, S. Carapinha et al. Environmental Advances 4 (2021) 100062

On this perspective, our study offers some insights on how microal- Batista, A.P., Ambrosano, L., Graça, S., Sousa, C., Marques, P.A.S.S., Ribeiro, B.,
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Declaration of Competing Interest croalgae as biopesticides to contribute to sustainable agriculture and environmen-
tal development. J. Environ. Sci. Heal. - Part B Pestic. Food Contam. Agric. Wastes.
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The authors declare that they have no known competing financial Cuellar-Bermudez, S.P., Aleman-Nava, G.S., Chandra, R., Garcia-Perez, J.S., Contreras-
interests or personal relationships that could have appeared to influence Angulo, J.R., Markou, G., Muylaert, K., Rittmann, B.E., Parra-Saldivar, R.,
2017. Nutrients utilization and contaminants removal. A review of two ap-
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indicators/13.2-development-in-consumption-of-2/assessment-1 (accessed 1.31.20).
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Farid, R., Mutale-joan, C., Redouane, B., Mernissi Najib, E., Abderahime, A., Laila, S., Ar-
access to their facilities to collect the effluent. The authors would also
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like to thank Graça Gomes and Natércia Sousa (LNEG) for laboratorial metabolomics pathways related to plant defense in Solanum lycopersicum. Appl.
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