ORIGINAL RESEARCH
published: 25 October 2016
doi: 10.3389/fpls.2016.01591
Edited by:
Amanullah Khan,
University of Agriculture, Peshawar,
Pakistan
Reviewed by:
Xiaojin Song,
Qingdao Institute of Bioenergy and
Bioprocess Technology (CAS), China
Luis Guillermo Ramírez Mérida,
University of Carabobo, Venezuela
*Correspondence:
Izabela Michalak
izabela.michalak@pwr.edu.pl
Specialty section:
This article was submitted to
Plant Nutrition,
a section of the journal
Frontiers in Plant Science
Received: 05 October 2016
Accepted: 07 October 2016
Published: 25 October 2016
Citation:
Michalak I, Chojnacka K, Dmytryk A,
Wilk R, Gramza M and Rój E (2016)
Evaluation of Supercritical Extracts
of Algae as Biostimulants of Plant
Growth in Field Trials.
Front. Plant Sci. 7:1591.
doi: 10.3389/fpls.2016.01591
Evaluation of Supercritical Extracts
of Algae as Biostimulants of Plant
Growth in Field Trials
Izabela Michalak
1
*
, Katarzyna Chojnacka
1
, Agnieszka Dmytryk
1
, Radosław Wilk
1
,
Mateusz Gramza
2
and Edward Rój
3
1
Department of Advanced Material Technologies, Faculty of Chemistry, Wrocław University of Science and Technology,
Wrocław, Poland,
2
AGRECO Ltd, Wrocław, Poland,
3
Supercritical Extraction Department, New Chemical Syntheses
Institute, Puławy, Poland
The aim of the field trials was to determine the influence of supercritical algal extracts
on the growth and development of winter wheat (variety Akteur). As a raw material for
the supercritical fluid extraction, the biomass of microalga Spirulina plantensis, brown
seaweed – Ascophyllum nodosum and Baltic green macroalgae was used. Forthial and
Asahi SL constituted the reference products. It was found that the tested biostimulants
did not influence statistically significantly the plant height, length of ear, and shank length.
The ear number per m
2
was the highest in the group where the Baltic macroalgae
extract was applied in the dose 1.0 L/ha (statistically significant differences). Number of
grains in ear (statistically significant differences) and shank length was the highest in the
group treated with Spirulina at the dose 1.5 L/ha. In the group with Ascophyllum at the
dose 1.0 L/ha, the highest length of ear was observed. The yield was comparable in all
the experimental groups (lack of statistically significant differences). Among the tested
supercritical extracts, the best results were obtained for Spirulina (1.5 L/ha). The mass
of 1000 grains was the highest for extract from Baltic macroalgae and was 3.5% higher
than for Asahi, 4.0% higher than for Forthial and 18.5% higher than for the control
group (statistically significant differences). Future work is needed to fully characterize
the chemical composition of the applied algal extracts. A special attention should be
paid to the extracts obtained from Baltic algae because they are inexpensive source of
naturally occurring bioactive compounds, which can be used in sustainable agriculture
and horticulture.
Keywords: algae, supercritical fluid extraction, biostimulant, field trials, winter wheat
INTRODUCTION
Recently, there is increased interest in natural products that stimulate the growth of plants.
A special attention has been paid to the raw material – the biomass of algae that is useful in the
production of plant growth biostimulants (
Calvo et al., 2014). The natural products obtained from
algae constitute the subject of interest in agriculture with emphasis on its application in sustainable
agriculture (Khan et al., 2009).
Algae have long been viewed a valuable source of food and traditional remedies. Over the
centuries, various types of macroalgae, such as Undaria and Laminaria were grown and harvested
in coastal areas. Another import ant commercial application of algae is the production of healthcare
Frontiers in Plant Science | www.frontiersin.org 1 October 2016 | Volume 7 | Article 1591
Michalak et al. Algal Extracts in Plant Cultivation
products and cosmetics, as well as the biochemical industry.
Dietary supplements based on Chlorella and Spirulina are the
most popular and successful commercial products from algae
(Fradique et al., 2010; Moorhead et al., 2011; Enzing et al., 2014).
The beneficial, from the viewpoint of agricultural applications,
properties of algae result from their living conditions –
permanent abiotic and biotic stress. This caused that these
organisms developed mechanisms that protect them from
drought, salinity, changing light intensity, frost, colonization
by bacteria or fungi. Resulting, algal cells contain bioactive
compounds that are prospective in the protection of plants –
carbohydrates, minerals, and trace elements, growth hormones
(cytokinins, auxins and auxin-like compounds), betaines, sterols
(
Khan et al., 2009).
Since chemical synthesis of biologically active compounds is
not profitable or difficult, the best source of these compounds
are extracts obtained from algae. Algal extracts containing
natural active compounds comprise a wide variety of structures
and functions that provide excellent pool of molecules for the
preparation of nutraceuticals, functional foods, food additives,
and biological agrochemicals (
Gil-Chávez et al., 2013; Jacob-
Lopes et al., 2015).
By using different extraction techniques, it is possible to
isolate these compounds in the process of extraction, to
formulate products and find their direct applications. Among
many extraction techniques (conventional liquid-liquid or solid–
liquid extraction, pressurized-liquid extraction, subcritical and
supercritical extractions, microwave- and ultrasound-assisted
extractions), supercritical fluid extraction (SFE) is gaining
increasing interest because the obtained extract is t he concentrate
of biologically active compounds in a solvent-free environment
and is safe to plants (
Gil-Chávez et al., 2013; Michalak and
Chojnacka, 2014
). These extraction techniques can be improved
with biomass pretreatment steps (enzyme-and instant controlled
pressure drop-assisted extractions) which improve the efficiency
of extraction process (
Gil-Chávez et al., 2013).
Upstream and downstream processing of supercritical algal
extracts production was described by (Wilk and Chojnacka,
2015a,b). Supercritical carbon dioxide extraction was pointed
out as a promising method for the production of algal-
based biostimulants. High efficiency, full biodegradability,
no phytotoxicity make biostimulants based on algal extracts
promising plant protection products (
Kim and Chojnacka, 2015).
Algae and their extracts can be used in crop management
to reduce abiotic and biotic stresses (Sharma et al., 2014). They
can act as chelators by the improvement of the utilization of
mineral nutrients by plants and improvement of soil structure
and aeration, which may stimulate root growth (
Milton, 1964).
As biostimulants of plant growth, algal extracts enhance seed
germination, improve plant growth, yield, flower set and fruit
production, as well as a post-harvest shelf life (Khan et al., 2009;
Calvo et al., 2014). Because seaweed extracts are the inexpensive
source of naturally occurring plant growth regulators they can
be successfully used in sustainable agriculture and horticulture
(
Panda et al., 2012).
It is important to emphasize that algal extracts obtained by
SFE have not been studied in the field experiments, so far.
The results presented in the present paper are a continuation
of the field trials conducted in the growing season 2013/2014
on winter wheat using supercritical Spirulina sp. extract in two
rates: 1.20 and 1.8 L/ha (
Chojnacka et al., 2014). The basis to
conduct field trials were germination tests c arried out on different
species of plants using supercritical extracts: from the mixture of
marine macroalgae from the Baltic Sea (species of Polysiphonia,
Ulva, and Cladophora) and Spirulina sp. on cress (Lepidium
sativum) (
Dmytryk et al., 2014a,b) and from the mixture of Baltic
macroalgae on garden cress and wheat (Michalak et al., 2016).
The objective of the present work was to evaluate the efficacy
of biostimulants (different types of supercritical algae extracts)
and their impact on the yield quantity and quality of winter
wheat. As a raw material for the production of extracts was used
the biomass of microalga Spirulina platensis and two macroalgae:
the mixture of Baltic macroalgae (Poland, Sopot city) and
Ascophyllum nodosum from Atlantic Ocean (France, Brittany).
MATERIALS AND METHODS
Feedstock
The characteristics of commercially available S. platensis provided
by WB Im-und Export W. Beringer & Co. GmbH. was described
in the work of
Chojnacka et al. (2014). Marine macroalgae
were collected from the Baltic Sea directly from t he water
near Sopot beach (Poland). The detailed description of the
harvesting of the biomass and pre-treatment before extraction
process was described in the work of Michalak et al. (2016).
Brown macroalga – A. nodosum was supplied by Laboratoires
Goëmar SAS
1
. It was collected by hand from the Brittany
coast (France) in June 2014 and stored in the frozen state.
Before SFE, the biomass was defrosted in a fridge in the
temperature ∼5
◦
C. Then it was dried to the moisture 15%
in the temperature 40
◦
C. Finally, it was ground in the mill
(Retsch SM 300) in order to obtain the fraction of the size
500 µm.
Supercritical Extraction of the Biomass
with CO
2
Supercritical fluid extraction of all examined algal biomasses was
performed in the New Chemical Syntheses Institute in Puławy –
Supercritical Extraction Department (Poland). SFE of microalga
Spirulina sp. was described in the work of
Chojnacka et al. (2014)
and of Baltic green macroalgae in the work of Michalak et al.
(2016). In the case of brown macroalga A. nodosum, SFE was
performed in the laboratory extractor with the volume – 1L. See
Rój (2014) for further details. The following conditions: pressure
500 bar; temperature 50
◦
C; load mass 257 g; CO
2
passed through
a bed of algal biomass 23.8 kg; CO
2
consumption in relation
to the initial mass of t he plant sample was 100 kg CO
2
/1 kg
of the load mass were used. The mass of the obtained liquid,
dark brown extract and water was 30.9 g; extraction yield was
5.77%.
1
www.goemar.com
Frontiers in Plant Science | www.frontiersin.org 2 October 2016 | Volume 7 | Article 1591
Michalak et al. Algal Extracts in Plant Cultivation
Algal Extracts Tested in the Field
Experiments
The following products were tested in field experiments on winter
wheat, variety Akteur (Table 1) – growing season 2014/205.
The choice of t he application rates was based on the previous
field experiments described by Chojnacka et al. (2014). The
dose of the formulation containing supercritical algal extract was
selected on the basis of the content of polyphenols in the applied
reference product – Asahi SL. The concentration of polyphenols
in the supercritical extract from Spirulina sp. was 3.0%. The
prepared formulation in one liter contained 100 g of extract –
the concentration of polyphenols was then equal to 0.3%. The
content of polyphenols in this preparation (dose 1.20 L/ha) was
the same as the content in the commercial product – Asahi SL
(dose 0.60 L/ha) (
Chojnacka et al., 2014).
In the present study, Spirulina extract was applied in three
different rates: 1.0, 1.5, and 1.8 L/ha in order to examine if the
extract dose stimulates the plant growth and development. It was
the continuation of field experiments conducted in the growing
season 2012/2013 on the winter wheat, variety Tacitus, when
supercritical Spirulina extract was applied in two rates: 1.2 and
1.8 L/ha (Chojnacka et al., 2014).
The formulations for the field trials were prepared according
to the data collected in Table 2. One liter of the final formulation
of algal extract was diluted in 200 L of water and applied on one
hectare (spray volume – 200 L/ha).
Field Experiments
The experiments were performed in accordance to the EPPO PP
1/144 (3), EPPO PP 1/135(4), EPPO PP 1/152 (4), and EPPO
PP 1/181(4) guidelines. The trial site was Miechowice Oławskie,
Lower Silesia, South-western Poland, GPS coordinates (N 50
◦
49
′
00,9
′′
; E 17
◦
13
′
58,2
′′
). The experiments were performed in
randomized complete blocks in four replications (N = 4) for each
tested product. The plot size was 20.0 m
2
(2.0 m × 10.0 m). The
soil had t he following characteristics – soil type: loam, soil quality
class: IIb, organic matter: 2.9%, soil pH: 6.4.
The sowing of winter wheat was on 3/10/2014 (the sowing
density of winter wheat was 200 kg/ha), the harvesting on
30/07/2015 (BBCH 89: fully ripe – grain hard, difficult to divide
with thumbnail). The tested products were applied twice: on
22/04/2015 (crop growth stage BBCH 31–32: 31 – first node at
least 1 cm above tillering node; 32 – node 2 at least 2 cm above
node 1) and 3/06/2015 (crop growth stage BBCH 59–61: 59 –
end of heading: inflorescence fully emerged; 61 – beginning of
flowering: first anthers v isible). The average temperature and the
total rainfall (mm) during the field experiments was as follows:
April 9.9
◦
C and 11.6 mm, May 13.8
◦
C and 28.4 mm, June
17.4
◦
C and 42.0 mm, July 21.1
◦
C and 59.2 mm. The tested
products were dosed to plants with t he use of sprayer with a
boom UP-02 (sprayer volume 6.0 L, nozzle ID: 8 nozzles TeeJet
XR11002VS).
The trials were carried out with standard fertilization:
26/08/2014: CaCO
3
(liming, CaO 50%, 2.2 t/ha), 02/10/2014:
Polifoska 7 [NPK(S) 7-18-28(11), 150 kg/ha], 04/03/2015:
ammonium nitrate (N 32%, 140 kg/ha), 14/03/2105: ammonium
nitrate (N 32%, 140 kg/ha), 10/04/2015: ADOB (N-NO
3
2.7%,
Cu 6.0%, 1.0 L/ha), 10/04/2015: magnesium sulfate (MgS 21–
30, 5.0 kg/ha), 10/04/2015: OSD Mineral N 19.5%, 2.0 kg/ha,
22/04/2015: ammonium nitrate (N 32%, 140 kg/ha), 22/05/2015:
TABLE 2 | The composition of formulations designed for field trial
containing supercritical extracts of S. platensis, Baltic macroalgae, and
A. nodosum.
Active substance (% mas.) Remarks
Supercritical algal extract 10.0
Amphoteric emulsifier (Atlox 4915) 2.50 Croda Europe Ltd.
Non-ionic emulsifier (Atlas G-5000) 2.50 Croda Europe Ltd.
Potassium sorbate 0.010 C
6
H
7
KO
2
, POCH
Polyethylene glycol 2.50 POCH
B(III) 0.020 H
3
BO
3
, POCH
Cu(II) 0.050 CuSO
4
·5H
2
O, Acros Organics
Fe(II) 0.100 FeSO
4
·7H
2
O, Acros Organics
Mn(II) 0.050 MnSO
4
·H
2
O, POCH
Mo(VI) 1.00·10
−3
(NH
4
)
6
Mo
7
O
24
·4H
2
O, POCH
Zn(II) 0.050 ZnSO
4
·7H
2
O, POCH
Demineralised water fulfill to 100%
TABLE 1 | Products tested in field experiments on winter wheat.
Name Rate, L/ha Active substance
Tested product – supercritical algal extracts
Baltic Sea algal extract 1.0 10% of Baltic Sea algal extract by weight
Ascophyllum nodosum extract 1.0 10% of A. nodosum extract by weight
Spirulina platensis extract 1.0 10% of Spirulina extract by weight
S. platensis extract 1.5 10% of Spirulina extract by weight
S. platensis extract 1.8 10% of Spirulina extract by weight
Reference product
Control Untreated –
Forthial 1.0 N-NO
3
– 6.2%; MgO – 9.0% Biologically active GA 142
filtrate from A. nodosum marine algae
Asahi SL 0.6 Bioactive compounds: sodium para-nitrophenolate – 0.3%,
sodium ortho-nitrophenolate – 0.2%, sodium
5-nitroguaiacolate – 0.1%
Frontiers in Plant Science | www.frontiersin.org 3 October 2016 | Volume 7 | Article 1591
Michalak et al. Algal Extracts in Plant Cultivation
ammonium nitrate (N 32%, 110 kg/ha). Full protection of plants
was maint ained (application of pesticides).
Assessments Methods
The following parameters of plant growth were assessed. Before
the first application of the tested products (22/04/2014; BBCH
31–32): crop height (cm) of 25 plants/plot and crop vigor –
visual assessment on a 0–10 scale (5: control – optimal vigor,
<5: worse vigor, >5: better vigor) were measured. Before the
second applic ation of the tested products (02/06/2015; BBCH
59–61) additionally phytotoxicity – visual assessment (0% – no
phytotoxicity, 100% – plants totally destroyed) was performed.
Eight (30/04/2015; BBCH 8) and 21 (13/05/2015; BBCH 35–
37) days after the first application of the tested products and 9
(12/06/2015; BBCH 64–67) and 22 (25/06/2015; BBCH 69–71)
days after the second application: phytotoxicity and crop vigor
were determined.
Before harvest (16/07/2015; BBCH 87: hard doug h –
grain content solid. Fingernail impression held) the following
parameters were determined: crop height (cm), ear-bearing
culms and barren culms number – 25 plants/plot, ear number per
m
2
, grains in ear number – 25 ears/plot, length of ear (cm) – 25
ears/ plot, shank length (cm) – 25 ears/plot. Lodging assessment
concerned the following observations: area lodged (%), intensity
of lodging, phytotoxicity and crop vigor.
After the harvest (30/07/2015, BBCH 89), grain yield quantity
based on a standard 15% moisture (t/ha) and mass of 1000 grains
(g) were assessed.
Statistical Methods
The results were elaborated statistically by Statistica ver. 10.
Normality of distribution of experimental results was assessed
by the Shapiro–Wilk test. On this basis, a statistical test was
selected which was used to investigate the significance of the
differences between the groups. For normal distribution, the
differences between the groups were investigated with a one-
way analysis of variance (ANOVA) using the Tukey test. If
the distribution was not normal, the Kruskal–Wallis test was
applied. Results were considered significantly different when
p < 0.05.
RESULTS AND DISCUSSION
In the present paper, the effect of three supercritical algal extracts
(obtained from S. platensis, A. nodosum, and Baltic macroalgae)
and two reference materials (Forthial, Asahi SL) on the growth
and development of winter wheat (variety Akteur) in the field
trials was examined.
Phytotoxicity, Plant Vigor, and Lodging
Assessment
During the field experiments no phytotoxicity symptoms (0% –
no phytotoxicity) were observed in the c ase of the application of
all the tested products. Plant vigor was equal five for all products
throughout the entire experimental period – till BBCH 87. The
lodging assessment was performed in the crop growth stage
BBCH 87. Area lodging (%) and lodging intensity was equal zero
for all the preparations.
Crop Height and the Number of
Ear-Bearing Culm and Barren Culm
Figure 1 presents crop height at different BBCH crop growth
stages. For all stages, the differences were not statistically
different. The average height in the experimental and reference
groups is presented in Table 3. The height of the winter wheat
was comparable in all the groups. The examined algal extracts
and reference products have no significant effect on plant growth.
These results are in agreement with the results obtained by
other authors.
Matysiak et al. (2012) evaluated the effect of
seaweed extracts from Ecklonia maxima (Kelpak SL) on the
height of winter wheat, variety Tonacja in the field trials. It
was found that the extract applied in autumn (BBCH 20) or
in spring (BBCH 39) and twice application in the both terms
in the rate 2 L/ha (soil irrigation) did not affect significantly
the height of plants. However, the method of the application
of the tested product – for example: foliar, soil application, or
seed treatment can influence the plant growth (
Khan et al.,
2009; Michalak et al., 2016). In the work of Carvalho et al.
(2014) it was found that the height of the wheat (Triticum
aestivum cv. IAC 364) in a greenhouse depended on the met hod
of the applic ation of Acadian
R
Marine Plant Extract produced
from A. nodosum. Plants irrigated with A. nodosum extract
(5 mL/L) were higher than the control (water) and when the
seeds were previously treated with the extracts (0.1 mL of
the A. nodosum extract on the 100 g of seeds), regardless
of the evaluation period – at 14
th
, 28
th
, and 42
nd
days after
sowing.
In the present study it was shown that the average ear-bearing
culms number (25 plants/plot) in BBCH 87 was 4.1 in the tested
groups with the exception of Baltic macroalgae and Ascophyllum
for which it was 4.0. The average barren culms number was 0.1
for the tested groups with the exception of Baltic macroalgae and
Spirulina – 1.5 for which it was 0. The effect of algal extracts
on these parameters of wheat was not studied in the literature.
Therefore the comparison is not possible.
Effect of Biostimulants on the Ear
Number of Winter Wheat and the
Number of Grains in Ear
The effect of the tested biostimulants on the ear number per m
2
and number of winter wheat grains in ear is presented in Table 4.
Statistically significant differences between tested products were
observed for both measured parameters (Figure 2).
For ear number per m
2
, the best results were obtained
for the extract from Baltic macroalgae and the weakest for
Spirulina extract – 1.0. The average ear number for group with
Baltic macroalgae was 13.8% higher than for Spirulina extract –
1.0 (statistically significant difference). Statistically significant
differences were also noted between this group and Spirulina –
1.5, Spirulina – 1.8, Forthial and Asahi SL. Only for Spirulina –
1.0, the ear number per m
2
was lower than in the control group –
untreated.
Frontiers in Plant Science | www.frontiersin.org 4 October 2016 | Volume 7 | Article 1591
Michalak et al. Algal Extracts in Plant Cultivation
FIGURE 1 | Crop height (cm) at different BBCH crop growth stages: (A) 31–32, (B) 59–61, and (C) 87.
This result is in agreement with the data obtained in the
growing season 2012/2013 when Spirulina extract was applied on
wheat at two doses: 1.2 and 1.8 L/ha (
Chojnacka et al., 2014).
The ear number per m
2
for 1.2 L/ha was 3.0% higher than for
1.8 L/ha and 11% higher than for the control group (untreated,
without fertilization). In this growing season, this parameter was
11% higher for Spirulina – 1.5 than for Spirulina – 1.0, 2.3% than
for Spirulina – 1.8 and 4.3 than for the control group (untreated,
Frontiers in Plant Science | www.frontiersin.org 5 October 2016 | Volume 7 | Article 1591