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Review ArticleSupercritical Algal Extracts: A Source of Biologically ActiveCompounds from Nature
Izabela Michalak,1 Agnieszka Dmytryk,1 Piotr P. Wieczorek,2 Edward Rój,3
BogusBawa Awska,4 BogusBawa Górka,2 Beata Messyasz,5 Jacek Lipok,2
Marcin Mikulewicz,6 RadosBaw Wilk,1 Grzegorz Schroeder,4 and Katarzyna Chojnacka1
1Department of Advanced Material Technologies, Faculty of Chemistry, Wrocław University of Technology,Smoluchowskiego 25, 50-372 Wrocław, Poland2Faculty of Chemistry, Opole University, Plac Kopernika 11, 45-040 Opole, Poland3Supercritical Extraction Department, New Chemical Syntheses Institute, Aleja Tysiąclecia Panstwa Polskiego 13a,24-110 Puławy, Poland4Faculty of Chemistry, Adam Mickiewicz University in Poznan, Umultowska 89b, 61-614 Poznan, Poland5Department of Hydrobiology, Faculty of Biology, Adam Mickiewicz University in Poznan, Umultowska 89, 61-614 Poznan, Poland6Department of Dentofacial Orthopeadics and Orthodontics, Medical University of Wrocław, Krakowska 26, 50-425Wrocław, Poland
Correspondence should be addressed to Izabela Michalak; [email protected]
Received 9 May 2015; Accepted 28 June 2015
Academic Editor: Iciar Astiasaran
Copyright © 2015 Izabela Michalak et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.
The paper discusses the potential applicability of the process of supercritical fluid extraction (SFE) in the production of algal extractswith the consideration of the process conditions and yields. State of the art in the research on solvent-free isolation of biologicallyactive compounds from the biomass of algae was presented. Various aspects related with the properties of useful compoundsfound in cells of microalgae and macroalgae were discussed, including their potential applications as the natural components ofplant protection products (biostimulants and bioregulators), dietary feed and food supplements, and pharmaceuticals. Analyticalmethods of determination of the natural compounds derived from algae were discussed. Algal extracts produced by SFE processenable obtaining a solvent-free concentrate of biologically active compounds; however, detailed economic analysis, as well aselaboration of products standardization procedures, is required in order to implement the products in the market.
1. Introduction
The increase of public awareness, concerning potentiallyharmful ingredients present in commercial products, put thepressure on manufacturers to apply natural, environmentalfriendly materials of improved quality. Such requirementsare met by algal biomass, the capacity of which, despite longhistory in daily living products, still remains largely unex-plored [1]. Algae, both micro- and macrocellular (seaweeds),are known as a rich source of bioactive compounds, includingproteins, minerals, vitamins, polysaccharides, polyphenols,phlorotannins, pigments, unsaturated fatty acids, sterols, andphytohormones [2, 3]. The properties of these compoundswere used in various branches of industry, such as chemical,
pharmaceutical, human food, and animal feed productionand integrated systems of plant cultivation [1, 4].
Among possible ways of enriching a given product withalgal-derived material, application of extract is the mostfrequently reported. In order to extract bioactive substancesfrom raw biomass, the proper solvent (e.g., water and organicsolvents) should be chosen. The application of conventionalmethods of extraction (extraction in Soxhlet apparatus, solid-liquid extraction, and liquid-liquid extraction) has some dis-advantages, for example, the use of high volumes of solventsand difficult medium separation [5]. Therefore, in the recentyears, solvent-free methods of extraction have been devel-oped. Alternatively to conventional procedures, supercritical
Hindawi Publishing CorporationJournal of ChemistryVolume 2015, Article ID 597140, 14 pageshttp://dx.doi.org/10.1155/2015/597140
2 Journal of Chemistry
fluid extraction is usually proposed. This technique fulfillsthe market demand for both high quality and chemicallysafe products [6]. The term “supercritical” corresponds tosubstance behavior after exceeding the value of both criticaltemperature and pressure (the so-called “critical point”).Supercritical fluid (SCF) shows features bordering on char-acteristics of gaseous and liquid form of the compound andmight be classified somewhere between these two states.Thus, supercritical fluids have unusual capacity to extractselected constituents from complex material, the efficacy ofwhich is worth researching [7, 8].
In a present work, a review of SFE condition require-ments, technological aspects, and obstacles is presented, aswell as the application prospects of supercritical extracts fromalgae. Special attention was paid to the use of algae basedproducts in agriculture, as a rich source of natural plantgrowth stimulators. Another section was also devoted to thenovel analytical methods which are necessary to examinethe organic and inorganic composition of algal extracts. Onthe basis of the composition of the new product, potentialapplications are sought.
2. Supercritical Fluid Extraction asan Efficient Method of Isolation ofValuable Compounds
Solvation properties of supercritical fluids were reportedfor the first time in 1879 by Hannay and Hogarth [9]. Theidea of involving supercritical fluid extraction in industrialtechnologies was shown in public in 1969 by Zosel [10]. Dueto the global concern of environmental damage caused bylarge-scale use of organic solvents in classical extractions,the implementation of new technologies, using minimumvolumes of solvents, became the subject of great interest [11].Currently, a lot of attention is paid to optimize processesunder supercritical conditions, so that they can be used moreextensively. However, there are still economic and energyissues (e.g., high investment cost and labor-intensive step ofsample processing) that limit the use of SFE in commercialproductions [12].
2.1. Fundamentals of SFE. In the literature, supercritical fluidextraction is usually compared with conventional processesin order to present advantages and disadvantages of bothmethods. Absence of the harmful or toxic chemicals in thefinal product is the most evident advantage of SFE. Fluidsunder supercritical state surpass organic solvents in reducingprocess time and required amount of the sample by anorder of magnitude and in enhancing the yield of extraction[13]. Such differences resulting from the complex nature ofsupercritical fluids, lower viscosity and surface tension withhigher compressibility and diffusivity (gas- and liquid-likefeatures, alternately) enable more effectively penetrating thematerial and hence provide better mass transfer betweenphases [5, 14]. The high selectivity (ease of being modified bychanging temperature or pressure value only, e.g., tuneablesolvating power) and facility for fractionation of extractedcompounds are also emphasized as the major benefits of
using supercritical fluids. For industrial application of SFE,the exclusion of oxygen and low processing temperature(depended on the type of fluid) is worth mentioning, since itgives an opportunity to obtain volatile or labile constituentswithout their damage [12, 13, 15].
A variety of compounds, both inorganic (carbon dioxide,nitrous oxide, ammonia, sulphur hexafluoride, and water)and organic (ethane, propane, n-pentane, fluoroform, andchlorodifluoromethane (Freon-22)) was subjected to testsunder critical conditions [15]. Among examined fluids,supercritical CO
2(SC-CO
2) has been reported as the most
common choice. Benefits of using SC-CO2are well known
and include such features as relatively low critical parameters(𝑇 = 31∘C, 𝑝 = 73.8 bar), chemical inertness, no or lowtoxicity, nonflammability, noncorrosivity, and GRAS desig-nation (Generally Recognized as Safe) from both AmericanFood and Drug Administration and European Food SafetyAuthority [5, 15, 16]. Furthermore, under normal conditions,carbon dioxide is a gas, which can be easily separated fromthe extract and hence recovered, what reduces its cost [16].Despite several advantages, SC-CO
2is not a universal extrac-
tant, since it is nonpolar. Therefore, isolation of compoundswith high polarity needs to be supported with modifiers,cosolvents, which if added at low concentration increasesolvating power of the fluid towards the target compound [6].In case of supercritical carbon dioxide, methanol and ethanolare themost frequently reported.The former is more efficientand the latter is less toxic [6, 11, 13].
Extraction under supercritical fluid requires equipmentwhich involves a tank of the mobile phase (chosen solvent),a pump to pressurize the fluid, an oven comprising theextraction vessel with a matrix, a restrictor to maintainthe high pressure inside the system, and a trapping vessel.Extracts are trapped during decompression of the analyte-containing SCF into an empty vial, through a solvent, or ontoa solid or liquid material. There are three possible ways ofSFE: dynamic mode, static mode, or a combination mode. Inthe former, the fluid flows continuously through the sample(extraction vessel) and out of the restrictor to the trappingvessel. In static mode, the fluid circulates in a loop withinthe extraction vessel for some period before being releasedthrough the restrictor to the trapping vessel. In combinationmode, a static extraction is performed for some period oftime, followed by a dynamic extraction [17].
2.2. Parameters in SFE. Crampon et al. reviewed the param-eters that have an impact on the kinetics and efficiency ofextraction of microalgae and seaweeds carried out undersupercritical conditions (SC-CO
2) from dry biomass. Pres-
sure seems to be the most important parameter [7]. Atconstant temperature, the higher the pressure, the higher thedensity and thereby enhanced yields and/or faster extractionkinetics might be noted [7, 15]. In the case of temperature,its effect on the strength of supercritical fluid depends on thepressure (retrograde behavior). Correlation between thesetwo parameters varies and is determined by pressure valuecalled “crossover point,” above which increasing temperatureimproves solvating power [15].
Journal of Chemistry 3
Another important parameter in SFE is solvating power(selectivity) of supercritical fluids. It increases with density.Such correlation was not observed for conventional liquidsolvents. The density of extractant under supercritical con-ditions can be adjusted to the process needs by temperature,pressure, and/or composition (content of modifiers) [14, 18].Efficiency of the extraction is also clearly related tomolecularweight of analytes, their concentration in the sample, type andstrength of binding to the matrix, and solubility in specificSFC. Considering extraction with supercritical carbon diox-ide, it is advised to work with a high SC-CO
2/algaemass ratio
[19].Selection of the proper values of process variables is
crucial for obtaining high degree of extraction. Since thereare several variables to change, optimization of SFE might beperformed through various approaches, which are generallyclassified as phase equilibrium strategies and experimentaldesign with statistical modeling.The first approach considerslimitation of stages that influence the final effect of theprocess. The second approach complements this knowledgeby fitting statistical treatment to the results [6].
2.3. Preparation of the Biomass for SFE. Applying supercrit-ical fluids to treat biological materials, including algae, in aprofitable way is highly dependent on the proper pretreat-ment. In the first step, the biomass undergoes centrifugation,after which the concentrated algal suspension should besubjected to a drying process, freeze-drying or drying atlow temperatures. High sample moisture might lead to afew disadvantages, such as limitation of the matrix-SCFcontact [20] or, in case of applying supercritical CO
2, acidic
hydrolysis of the analytes due to carbonic acid formation[21]. Therefore, there is a common practice to removeexcess water during sample pretreatment. Finally, algae arecrushed to break the cellular wall and thus increase extractionefficiency. Concerning the effect of crushing, results obtainedby Crampon et al. indicated that the smaller the particle,the more rapid the kinetics of extraction and the higher theyields. Disintegration of cells is essential in the recovery ofintracellular products from algae [7]. According to literature,the SFE can be coupledwith cells disintegration techniques toobtain higher yields. Ultrasounds andmicrowaves are provedto facilitate extraction, hence the productivity, as well asreducing the time of the process [12]. Additionally, the fol-lowingmethodsmight be used: freezing, alkaline and organicsolvents, osmotic shocks, sonication, homogenization at highpressure, and bead milling [22, 23]. Moreover, in the study ofValderrama et al., cells of H. pluvialis and S. maxima werecrushed by cutting mills (coffee mill) and manually groundwith dry ice [2].
3. Production of Algal Extracts bySupercritical Fluid Extraction
3.1. Extraction of Biologically Active Compounds from Algaeby SFE. Algae form a diverse group of micro- and macroor-ganisms (seaweeds) containing a great amount of biologicallyactive compounds, which participate in processes of growth,
development, and protection and therefore are considered tobe capable of affecting other living organisms. The vast arrayof bioactive compounds in algae is the result of their adap-tation to unfavorable environmental conditions. Productionof these compounds increases when the environmental stressfactors are occurring, for example, changes of temperature,salinity, drought stress, tidal flows, lack of nutrients, orpresence of hostile organisms [24]. Algae are found in bothmarine and freshwater environments. Chemical compositionof algae has not been known as well as terrestrial plants. Onthe other hand, algae contain unique compounds that areabsent in higher plants [2, 3].
Supercritical fluidswere first used for treating algalmatrixto select biomolecules valuable in food processing industry[14, 17]. Currently, other functional compounds of provenactivity on human health, plant growth, or livestock produc-tivity and biofuels of new generation have been obtained fromalgae by using SFE [25–28].
Based on literature studies, microalgal cells are usedin extraction with supercritical fluids more frequently thanseaweeds. In the last 14 years, the words “supercritical fluidextraction andmicroalgae” appeared in the topic of the scien-tific papers 88 times, whereas “supercritical fluid extractionand seaweed” only 21 times (Web of Knowledge, Decem-ber 12, 2014; http://apps.webofknowledge.com/). Adequateexamples of applying supercritical conditions for microalgalbiomass processing are collected in Table 1.
In the presented examples, SC-CO2was chosen as a
solvent, occasionally supported by a modifier; ethanol andoperational conditions ranged within 40–85∘C and 78.6–500bar. In general, SFE was particularly used for the extractionof pigments, lipids including polyunsaturated fatty acids(PUFAs) (e.g., omega-3 fatty acids: eicosapentaenoic acid(EPA) and docosahexaenoic acid (DHA) and omega-6 fattyacid: 𝛾-linolenic acid (GLA) and arachidonic acid (AA)),polyphenols, and vitamins [23].
As it was mentioned above, there are fewer reportson the production of supercritical seaweed extracts. Mostresearch on producing supercritical extracts frommacroalgaefocuses on comparing solvating power of SCFs with organicextractants. Marine red macroalgae Hypnea charoides wereinvestigated as a nonconventional source of 𝜔-3 fatty acidsobtained by extraction with SC-CO
2. Tests were conducted
under mild conditions: temperature range 40–50∘C andpressure range 241–379 bar. Different conditions enabledobserving their influence on product yield: the higher thetemperature and pressure, the better the lipid recovery andratio of unsaturated fatty acids. Moreover, solubility, henceextractability, of 𝜔-3 fatty acids in supercritical CO
2was
proven to depend on the chain length [29]. SC-CO2was also
used to extract fucoxanthin from brown seaweed Undariapinnatifida [30]. A broad range of operational conditions wasapplied: temperature 25–60∘C, pressure 200–400 bar, andCO2flow rate 1.0–4.0mL/min, to investigate the variations
of process efficiency. It was shown that the product recoveryincreased with decreasing temperature and increasing pres-sure and the highest yield of fucoxanthin (almost 80%) wasachieved at 40∘C and 400 bar during 3-hour extraction.Moreexamples of the application of SFE of algal biomass (both
4 Journal of ChemistryTa
ble1:SFEextractio
nof
biom
asso
fmicroalgae:review
ofliteraturer
eports.
Extractio
nAlgae
Insta
llatio
nTemp.[∘ C
]Pressure
[bar]
Extract
Extractio
nyield
Reference
SFEwith
CO2
Botryococcus
braunii,
Chlorella
vulga
ris,
Dun
aliellasalin
a,and
Arthrospira
maxim
a,who
le,crushed,and
slightly
crushed
Flow
40–6
0125,200,and
300
B.braunii:alkadienes;
C.vulga
ris:carotenoids
(canthaxanthin,
astaxanthin);
D.salin
a:𝛽-carotene(trans-and
cis-iso
mer);A.
maxim
a:GLA
,C18:3𝜔6
(CO
2andCO
2+10mol%ethano
l)and
lipids
TotalG
LA:45%
:35.0M
Pa,333.1Kwith
them
ixture
(CO
2+10mol%ethano
l)[26]
SFEwith
CO2and
ethano
l(9.4
%mass)
Haematococcus
pluvialis
and
Arthrospira
maxim
a(Spirulin
a)
Colum
n;flo
wrate:
1mL/min
60300
H.pluvialis:
astaxanthin,
A.maxim
a:ph
ycocyanin
Asta
xanthin:
1.7%mass(no
effecto
fethano
l);ph
ycocyanin:
1.1%mass(CO
2),1.7%
mass(CO
2+ethano
l)[2]
SFEwith
CO2and
ethano
l(9.2
3mL/g)
Haematococcus
pluvialis
Biom
ass
6.5g
;CO
2flo
wrate:
6.0m
L/min;
time:20
min
50310
Pigm
ent(astaxanthin)
Asta
xanthin:
74%(11m
g/gdrycells),
8extractio
ncycle
s[5]
SFEwith
CO2
Chlorella
vulga
risFlow
type
40,55
350
Caroteno
ids,lip
ids
Carotenoids
andlip
ids:im
proved
for
crushedcells
andathigh
erp
[25]
SFEwith
CO2
Botryococcus
braunii
Flow
type
50–85
200–
250
Fatty
acids
Lipidyielddecreasedwith
temperature
andincreasedwith
pressure
[36]
SFEwith
CO2
Botryococcus
braunii
andCh
lorella
vulga
risFlow
type
40300(B.
braunii)and
350(C.
vulga
ris)
B.braunii:hydrocarbo
nsC.
vulga
ris:carotenoids
(canthaxanthin
andastaxanthin)
Thee
xtractionyieldof
caroteno
ids
increasedwith
thed
egreeo
fcrushingof
them
icroalga
[37]
SFEwith
CO2
Arthrospira
platensis
(Spirulin
a)Time:4h
48200
85g/kg
offlavono
ids;
78g/kg
of𝛽-carotene;113g
/kgof
vitamin
A;3.4g/kg
of𝛼-to
coph
erol;fattyacids:
palm
itic(35%),lin
olenic(22%
),and
linoleic(21%)
Yieldof
thee
xtractsfrom
S.platensis–10g
/kg
[38]
SFEwith
CO2
(soybean
oiland
ethano
lasm
odifier)
Chlorella
vulga
risFlow
type
40300
Carotenoids:69%
,crushingstr
ongly
improved
extractio
nrecovery
[39]
SFEwith
CO2
Haematococcus
pluvialis
Time:4h
70500
Astaxanthin
Thep
redicted
amou
ntof
astaxanthin
extractedwas
23mg/g
[40]
SFEwith
CO2and
ethano
l(0.856m
L/g
ofbiom
ass)
Arthrospira
platensis
(Spirulin
a)Time:1h
4040
0𝛾-lino
lenica
cid
Arecovery
of102%
GLA
[41]
SFEwith
CO2and
ethano
lAr
throspira
maxim
a(fr
eeze-drie
d)Flow
type
50–6
0250𝛾-lino
lenica
cidandlip
ids
GLA
andlip
ids:up
to45%
[27,28]
SFEwith
CO2
Synechococcussp.
Flow
type
50(fo
rcaroteno
ids)
and
60(fo
rchloroph
yll)
300(fo
rcaroteno
ids)
and
500(fo
rchloroph
yll)
Caroteno
idsa
ndchloroph
ylls
Carotenoids
(1.5𝜇g/mgdryweighto
fmicroalga);chloroph
ylla
(0.71𝜇
g/mg
dryweighto
fmicroalga)
[42]
SFEwith
CO2and
CO2:ethanol
Arthrospira
platensis
(Spirulin
a)Pilot-s
cale
plant
75(C
O2)
55and
(CO
2:EtO
H)
320(C
O2)
and
78.6
(CO
2:EtO
H)Vitamin
ECO
2:yield0.85%;16m
gof
vitamin
E/g
ofextract;
CO2:ethanol:yield
8.1%
;0.49m
gof
vitamin
E/gof
extract;CO
2(w
/w)6
9%[43]
PLEusingmixture
ofethano
l:ethyllactate
Arthrospira
platensis
(Spirulin
a)Time:15min
180
207𝛾-lino
lenica
cid
Totalyieldsu
pto
21%(w
/w),fora
solventcom
positionof
ethano
l:ethyl
lactate(50
:50,v/v),G
LArecovery
of68%
[44]
Journal of Chemistry 5
micro- and macroalgae) are presented in a review paper ofMichalak and Chojnacka [23].
3.2. Comparison SFE with Other Extraction Techniques fromBiomass of Algae. Despite regulations limiting the use ofchemicals and proven efficacy of using supercritical fluids asextractants, conventional solvent extraction still remains theleading technique to provide algae derived compounds forcosmetic or food industry [5, 26]. Applying organic solventsrequires additional step of their recovery and posttreatmentand might change physicochemical properties and func-tionality of isolated compounds [5]. There is an increasingnumber of reports focusing on comparison of conventionalmethods with SFE and some are summed in Table 2.
Halim et al. investigated a lab-scale biodiesel productionby extracting lipids from green microalgae Chlorococcumsp. with the use of two different solvents: SC-CO
2and n-
hexane. Obtained results confirmed usability of supercriticalconditions to algal lipid recovery. It was concluded thatSFE generated comparable yield to Soxhlet extraction andshortened process time by over five times. Nevertheless,the time required to complete the extraction might beinsufficient criterion [31]. In research of Crespo and Yusty,isolation of n-alkanes, C18, C19, C20, C22, C24, C28, C32,and C36, and the acyclic isoprenoid Pristane from brownseaweed Undaria pinnatifida was conducted by the use ofn-hexane:dichloromethane mixture (Soxhlet mode) and SC-CO2with a modifier. Although SFE enabled hastening the
process from days (conventional extraction) to 1 hour, theauthors concluded that both methods are comparable, sincethe obtained yields of hydrocarbons were not significantlydifferent. It was also noted that solvating power of SC-CO
2
is higher than organic solvent in case of longer-chain n-alkanes [32]. As opposed to investigation of Crespo andYusty,supercritical CO
2(with and without cosolvent) in experi-
ments on isolation of astaxanthin (AXA) and chlorophyllfrom microalgae Monoraphidium sp. GK12 was proved tosurpass ethanol. Efficacy of using chosen solvents was verifiedby performing bead beater extraction (BBE), the results ofwhich were established as 100%. The yield of astaxanthinobtained by SFE was twice as high as in EtOH-extractsand this advantage increased with higher concentration ofmodifier to finally achieve similar level to the result of BBE.In case of extraction of chlorophyll, applying supercriticalconditions was slightly more effective than both of the othermethods [33]. Supercritical fluid extraction was also shownto be an efficient pretreatment method in the productionof polysaccharides (fucoidan) from biomass of brown sea-weeds Fucus evanescens, Saccharina japonica, and Sargassumoligocystum. It provided the equivalent yield as conventional(organic solvent) method [34].
3.3. General Application of Algae Derived Compounds.Extracted algal compounds are characterized by anticoagu-lant, anticancer, antiallergic, antiviral, antifungal, antioxida-tive, and immunomodulating activities [35]. These proper-ties make that algal extracts have broad potential applica-tions, for example, as components in cosmetics, medicines,
pharmaceuticals, nutraceuticals, feed additives, nutrition(feed) and food additives, aquaculture, plant growth biostim-ulants and bioregulators, biofuels, and pollution prevention[23].
It should be underlined that some algal-origin moleculesare assigned for specific species or taxonomic groups. Forexample, in cyanobacteria typical bioactive compounds aremalyngolide (Lyngbya majuscula (Dillwyn) Harvey), nosto-dione (Nostoc communeVaucher), cyanobacterin (Scytonemahofmanni Kutz., and Nostoc linckia (Roth) Bornet & Fla-hault), aponin (Gomphosphaeria aponina Kutz.), fischerellin(Fischerella muscicola (Thuret) Gom.), and scytophycins(Scytonema pseudohofmanni Bharad.). It was shown thatthey demonstrate antibacterial, antifungal, and even antialgalproperties that can be used in pharmaceutical industry [45].In the work of Ramesh et al., the main attention was paidto active substances isolated from freshwater algae withpharmaceutical applications. This group of algae producescompounds with a vast array of properties: from antimi-crobial and antiviral to cytotoxicity and immunomodulatoryactivity. Freshwater algae provide a diverse and unique sourceof bioactive compounds that can be used for the discoveryof modern drugs (antibiotics, mycotoxins, alkaloids, andphenolic compounds) [46].
Another group of bioactive compounds constitutecarotenoids (𝛽-carotene, astaxanthin, and canthaxanthin)and phycocyanin (water-soluble phycobiliprotein) isolatedfrom algal biomass. They can be used as natural pigmentsin nutrition of animals and humans [2]. Algae are apromising commercial source of carotenoids due to therelative fast growth rate (especially microalgae). Somespecies, such as unicellular Dunaliella salina (Dunal) Teod.or Dunaliella bardawil Ben-Amotz & Avron, demonstratetheir capability to accumulate large amount of 𝛽-carotenein chloroplasts in the form of lipid globules. Adverseenvironmental conditions, such as high salinity, rapid changein temperature, and nutrient limitation acting as a stressor,may increase this capacity [47].
Other important compounds extracted from algae areunsaturated fatty acids [31, 48]. Fatty acid compositionof marine algae species differs totally from higher plants.For example, cells of Arthrospira (Spirulina) maxima con-tain polyunsaturated 𝛾-linolenic acid and 𝛼-linolenic acid(ALA), which can be the component of pharmaceuticals(schizophrenia, multiple sclerosis, diabetes, and rheumatoidarthritis) [26]. Fatty acids produced from algae, linolenicacid (from Arthrospira sp.), arachidonic acid (Porphyridiumsp.), eicosapentaenoic acid (Chlorella vulgaris Beij.), anddocosahexaenoic acid (C. vulgaris), have high biologicalactivity and are mainly used in nutritional supplements [49,50].
The extraction of the mentioned biologically activecompounds from algae by SFE is especially recommendedbecause this technique protects them from thermal or chem-ical degradation. Active ingredients in solvent-free environ-ments are particularly important for their applications inmedicines and nutraceuticals [26].The industrial importanceis due to not only the algal extract but also the postextractionresidue which can be used as animal feedstock [12]. Algal
6 Journal of Chemistry
Table2:Com
paris
onof
SFEwith
conventio
nalextractionmetho
ds,o
ntheexam
pleof
algaeprocessin
g;theresults
ofindepend
ente
xperim
entsareshow
nseparately
andmarkedwith
(a)–(d).
(a)
Extractio
nBiom
assp
retre
atment
Samplea
ndsolvent
Con
ditio
nsEx
tractio
nyield
Reference
Solventextractionwith
𝑛-hexanea
tstatic
and
dynamic(Soxhlet)m
ode
Microalgalp
owder:
Oven-drying
at85∘Cfor16ho
urs
andgrinding
inar
ingmill
Microalgalp
aste(solid
conc.30%
,by
mass):centrifu
ging
wetalgaein
benchtop
centrifuge
Staticmode:
(i)Microalgalp
owder:4g
(ii)M
icroalgalp
aste:13.3g
(iii)Solventsfor
perfo
rmance
onpo
wder:
pure𝑛-hexanea
ndmixture
of𝑛-hexanea
ndiso
prop
anol(3:2),
separately,
both
300m
L(iv
)Solvent
for
perfo
rmance
onpaste
:pure
𝑛-hexane,300m
LDynam
icmode(Soxhlet
extra
ction):
(i)Microalgalp
owder:4g
(ii)S
olvent:pure𝑛
-hexane,
300m
L
Staticmode:
Ambientcon
ditio
ns,
agitatio
n:800r
pm,
𝑡:7.5h
Dynam
icmode:
Rateof
reflu
xes:10
per
hour,𝑡:7.5h
Posttreatment:
Removalof
solid
resid
ues
from
extractb
yseparatio
non
afiltrationpaper
Results
form
icroalga
lpow
der:
Lipidyield[g
lipid
extract/g
d.m.]:
(i)Staticmod
e:0.015and0.04
8,with
andwith
outcosolvent,respectively
(ii)D
ynam
icmod
e:0.057
Results
forw
etmicroalga
lpaste:
Lipidyieldaft
er80
min
[glip
idextract/g
d.m.]:
Staticmod
e:0.010
[31]
SFEwith
CO2
(i)Microalgalpow
der:20
g(ii)M
icroalgalp
aste:8
gBo
thpo
wdera
ndpaste
wereformerlymixed
with
inertd
iatomaceous
earth
(d.e.)atratios2
:1w/w
and
1:2w
/w,respectively
Solventf.r.:400
mL/min
𝑇:60or
80∘C,𝑝:100–300
or300–
500b
ar(lo
wer-a
ndhigh
er-pressure
experim
ents,
resp.),𝑡:
80–120
min
Results
form
icroalga
lpow
der:
Lipidyieldaft
er80
min
[glip
idextract/g
d.m.]:
(i)60∘C,
300–
500b
ar:0.058
(ii)8
0∘C,
300–
500b
ar:0.048
Effecto
fpressure:increase
ofthelipid
yieldalmosttwiceincase
ofhigh
er-pressuree
xtractions,com
pared
tolower-pressuree
xperim
ents
FAMEcontentvariatio
nsdu
ring
80min
ofthep
rocess[g
FAME/gd
.m.]:
(i)60∘C,
300–
400b
ar:0.23–0.29
(ii)8
0∘C,
300–
400b
ar:0.31–0
.44
Effecto
fpressure:no
tsignificant
Results
forw
etmicroalga
lpaste
Lipidyieldaft
er120m
in[g
lipid
extract/g
d.m.]:
60∘C:
0.071
Journal of Chemistry 7(b)
Extractio
nBiom
ass
pretreatment
Samplea
ndsolvent
Con
ditio
nsEx
tractio
nyield
Reference
Soxh
letextraction
with
mixture
of𝑛-hexanea
nddichloromethane
(1)D
ryingby
lyop
hilization
(sam
ples
number1
and2)o
rdehydration
(sam
plen
umber
3) (2)P
ulveriz
ing
(3)
Hom
ogenization
(i)Samples:1gof
each
of3different
matrix
escollected
from
two
locatio
nsin
North-W
est
Spaindu
ringtwo
consecutives
pring
season
s)Ea
chsamplew
asmixed
with
15gof
seas
and
(ii)S
olvent:
𝑛-hexane:dichlo-
romethane
(50:
50),
250m
L
𝑡:7
hEx
tractp
osttreatm
ent:
Removalof
solvento
nar
otatory
evaporator
at40∘C,
oven-drying
obtained
extractat75∘Cfor9
0min
andthen
coolingto
room
temperature
(20∘C)
Providingfractio
nof
hydrocarbo
ns:
Redissolving
thee
xtractin
𝑛-hexane(5m
L)andnext
passing
throug
hSep-Paksilicas
olid-phase
extractio
n(SPE
)colum
n,previously
activ
ated
with𝑛-hexane(4m
L);
eluatinghydrocarbo
nsfro
mSP
Ecartrid
gewith
10mLof𝑛-hexane
andthen
evaporatingthee
luent
undera
stream
ofair,redissolving
ther
esidue
in𝑛-hexane(1m
L)and
subjectin
gitto
analysis(G
C-FID)
Results
forsam
ple1
(𝑛=6):
Recoverie
s[%],RS
D[%
]:Pristane,samplea
t5mg/L:74.1,
5.81;
Pristane,samplea
t40m
g/L:90.5,6.59;C 1
8,samplea
t5mg/L:87.9,
5.36;C
18,sam
plea
t40m
g/L:97.8,6.88;C 1
9,samplea
t5mg/L:86.0,
1.52;C 1
9,samplea
t40m
g/L:96.7,
3.20;C
20,sam
plea
t5mg/L:69.1,
4.90;C
20,sam
plea
t40m
g/L:96.5,3.31
;C22,sam
plea
t5mg/L:69.6,
4.98;C
22,sam
plea
t40m
g/L:99.4,4.69;C 2
4,samplea
t5mg/L:78.4,
4.42;C
24,sam
plea
t40m
g/L:99.8,3.39
;C28,sam
plea
t5mg/L:90.4,
7.58;C 2
8,samplea
t40m
g/L:101,1.6
5;C 3
2,samplea
t5mg/L:98.8,1.05;
C 32,samplea
t40m
g/L:99.7,
1.74;C 3
6,samplea
t5mg/L:97.9,
0.58;
C 36,samplea
t40m
g/L:97.4,3.10
Alip
hatic
hydrocarbo
nyield
[𝜇g/gd
.m.]:
C 18:4.32±0.08,C
20:5.35±
0.34,C
22:3.99±0.20,C
24:2.65±0.06,C
28:1.06±0.06,total:14.9±
0.5
Results
forsam
ple2
(𝑛=2):
Alip
hatic
hydrocarbo
nyield
[𝜇g/gd
.m.]:
C 18:5.14±0.19,C
20:5.88±
0.27,C
22:5.03±0.14,C
24:2.50±0.11,C
28:0.64±0.00,and
total:17.9±
0.3,
Results
forsam
ple3
(𝑛=2):
Alip
hatic
hydrocarbo
nyield
[𝜇g/gd
.m.]:
C 18:4.25±0.25,C
20:5.10±
0.30,C
22:5.32±0.44
,C24:2.88±0.25,C
28:1.03±0.15,and
total:17.0±
1.0,
[32]
SFEwith
CO2
(+methano
las
mod
ifier)
(i)Samples:0.5gof
each
of3matrix
esmentio
ned
above;
each
samplew
aspreadsorbedon
to10%
deactiv
ated
alum
ina
(3g)
byadmixingto
obtain
homogenou
smixture,w
hich
was
then
complem
entedwith
alum
ina(
1g)
(ii)S
olvent:SC-
CO2
with
methano
l(200m
L)
Initialsta
ticequilib
ratio
nperio
d:𝑇:100∘C,𝑝:229
bar,𝑡:10m
inEx
tractio
n:SC
-CO
2density
:0.55g
/mL,f.r.:
1mL/min
(dyn
amicor
continuo
usflo
wmod
e),𝑡:50m
in,𝑇
ofno
zzle:
45∘C;𝑇of
analyte-collectingtrap:
40∘C
Providingfractio
nof
hydrocarbo
ns:
eluatingaliphatic
hydrocarbo
nsfro
manalytes
containedin
thetrap
were
with
5po
rtions
of𝑛-hexane
(1.5mLpere
achfractio
n);
concentratingob
tained
extractto
1mLun
dera
stream
ofaira
ndthen
purifying
(SPE
column)
and
analyzingas
itwas
describ
edin
case
ofSoxh
letextraction
Results
forsam
ple1
(𝑛=6):
Recoverie
s[%],RS
D[%
]:Pristane,samplea
t5mg/L:36.8,7.53
;Pristane,samplea
t40m
g/L:52.3,8.11;C
18,sam
plea
t5mg/L:66.4,
9.28;C 1
8,samplea
t40m
g/L:76.0,3.49;C 1
9,samplea
t5mg/L:77.6,
4.69;C
19,sam
plea
t40m
g/L:89.1,
4.71;C
20,sam
plea
t5mg/L:84.6,
4.01;C
20,sam
plea
t40m
g/L:94.3,4.42;C 2
2,samplea
t5mg/L:87.9,
3.04;C
22,sam
plea
t40m
g/L:98.4,5.18
;C24,sam
plea
t5mg/L:90.8,
2.46
;C24,sam
plea
t40m
g/L:100,3.85;C
28,sam
plea
t5mg/L:88.6,
5.75;C
28,sam
plea
t40m
g/L:100,3.35;C
32,sam
plea
t5mg/L:87.8,
8.40
;C32,sam
plea
t40m
g/L:98.6,3.86;C 3
6,samplea
t5mg/L:84.8,
5.76;C
36,sam
plea
t40m
g/L:98.1,
4.09
Alip
hatic
hydrocarbo
nyield
[𝜇g/gd
.m.]:
C 18:6.28±0.66,C
20:6.66±
0.79,C
22:4.21±
0.44
,C24:2.56±0.28,C
28:0.71±
0.07,and
total:19.1±
2.0
Results
forsam
ple2
(𝑛=2):
Alip
hatic
hydrocarbo
nyield
[𝜇g/gd
.m.]:
C 18:4.54±0.23,C
20:4.25±
0.10,C
22:2.99±0.04,C
24:2.30±0.18,C
28:0.59±0.05,and
total:13.6
±0.6,
Results
forsam
ple3
(𝑛=2):
Alip
hatic
hydrocarbo
nyield
[𝜇g/gd
.m.]:
C 18:7.9
0±1.0
7,C 2
0:6.73±
0.63,C
22:4.49±1.2
6,C 2
4:2.99±3.55,C
28:1.15±5.17,and
total:21.7±
0.2
8 Journal of Chemistry
(c)
Extractio
nBiom
ass
pretreatment
Samplea
ndsolvent
Con
ditio
nsEx
tractio
nyield
Reference
Solvent
extractio
nwith
ethano
l
Freeze-
drying
(i)Sample:1g
(ii)S
olvent:20m
L
𝑡:30m
in(soaking
matrix
inethano
l)Po
sttreatment:
Separatio
nof
extractfrom
solid
resid
uesb
ycentrifuging(400
0×g,
20∘C,
10min,extract=
supernatant)
Results
ofAX
Aextra
ction:
Yield±𝛿[m
gAXA/gd.m]:1.16±0.18
Recovery
[%of
yield
obtained
with
BBM
metho
d]:48
Results
ofchlorophyll
extra
ction:
Yield±SD
[mgchloroph
yll/g
d.m]:16.1±1.9
1Re
covery
[%of
yield
obtained
with
BBM
metho
d]:56
[33]
SFEwith
CO2
(+ethano
las
mod
ifier)
(i)Sample:1g
(ii)S
olvent:
(a)P
ures
olvent:
SC-C
O2
(b)C
osolvent:EtO
H,0.1,
0.2,0.5,1.0
,2.0,or2
0mL
𝑇:60∘C,𝑝:200
bar,𝑡:
60min
Providingextractafte
rSFE
:soakingob
tained
biom
ass
in20
mLof
ethano
lfor
30min
andthen
separatio
nof
supernatantb
ycentrifugation(400
0×g,
20∘C,
10min);in
case
ofsamples
treated
with
0.1–2.0m
Lof
cosolvent,
prop
ervolumeo
fethanol
hadto
beaddedto
complem
entitto20
mL
Extractp
osttreatm
ent:
Removalof
chloroph
yllto
redu
cethes
aturationof
greencolorb
ytre
ating
extractw
ithseveralacids,
such
asH
2SO
4,HCl,
H3PO
4,andCH
3COOH,at
thec
oncentratio
nrangeo
f0.002–0.1N
Results
ofAX
Aextra
ction:
Yield±𝛿[m
gAXA/gd.m];recovery
[%of
yieldob
tained
with
BBM
metho
d]:
Pure
SC-C
O2:2.02±0.20,83;SC
-CO
2+0.1m
Lof
EtOH:2.13±0.36;
87;SC-
CO2+0.2m
Lof
EtOH:2.33±0.62,96;SC
-CO
2+0.5m
Lof
EtOH:2.40±0.37,98;SC
-CO
2+1.0
mLof
EtOH:2.32±0.43,95;
SC-C
O2+2.0m
Lof
EtOH:2.41±
0.49,99;SC
-CO
2+20
mLof
EtOH:
2.46±0.23,101
Results
forsam
ples
treated
with
pure
SC-C
O2andSC
-CO
2+
cosolventatthe
high
estcon
centratio
nshow
edsta
tisticallysig
nificant
difference(𝑝<0.05by
Stud
ent’s𝑡-te
st)with
AXAyieldof
both:
conventio
nalm
etho
dandther
esto
fsup
ercriticalextracts
Results
ofchlorophyll
extra
ction:
Yield±SD
[mgchloroph
yll/g
d.m],recovery
[%of
yield
obtained
with
BBM
metho
d]:
Pure
SC-C
O2:29.4±1.7
0,103;SC
-CO
2+0.1m
Lof
EtOH:28.8±0.91,
104;SC
-CO
2+0.2m
Lof
EtOH:30.8±2.31,108;SC-
CO2+0.5m
Lof
EtOH:28.7±1.2
4,104;SC
-CO
2+1.0
mLof
EtOH:28.4±0.91,103;
SC-C
O2+2.0m
Lof
EtOH:29.1±0.36,102;SC-
CO2+20
mLof
EtOH:29.5±1.0
4;103
Results
fora
llsamples
show
edstatisticallysig
nificantd
ifferences
(s.s.d.,𝑝<0.05)w
ithchloroph
yllyield
obtained
usingconventio
nal
metho
dandno
s.s.d.betweeneach
other
Journal of Chemistry 9(d)
Extractio
nBiom
ass
pretreatment
Samplea
ndsolvent
Con
ditio
nsEx
tractio
nyield
Reference
Solvent
extractio
nwith
aqueou
sethano
l
(1)G
rinding
beforehand
usinga
labo
ratory
mill
inequal
shorttim
eintervalsin
orderto
avoid
overheating
(2)P
assin
gthem
aterial
throug
hlabo
ratory
sieves
(diameter
3mm),
perio
dically,
and
collectingthe
finefraction
after
each
sieving
(i)Sample:30
g(ii)S
olvent:aqu
eous
ethano
latthe
concentrationof
70%
Solvent:sampler
atio
(w/w
):1:1
𝑡:10days
Providingextract(fractio
n)of
polysaccharid
es:
DryingEtOHextractinaira
ndthen
treatingittwice
with
0.1M
HCl,atratio
(w/w
)1:20,at60∘Cfor
120m
in;n
eutralizingnewlyob
tained
extractand
centrifugingto
separatesupernatant,fro
mwhich
WSP
Sfractio
nswereisolated
(con
centratin
gsupernatantinar
otaryevaporator→
dialyzing
againstd
istilled
H2O→
lyop
hilization)
HCl
extractp
osttreatm
ent:
Separatio
nof
fucoidansfrom
thep
olysaccharide
fractio
nsby
anion-exchange
chromatograph
y(elutio
nby
alinearg
radiento
fH2O
and2M
NaC
lsolutio
n),w
hich
resultedin
providingon
e,two,or
threefucoidansubfractions
ofdifferent
degree
ofsulfatio
n(m
arkedas𝐹1–𝐹3,according
toan
increasin
gordero
fsulfatedgrou
pcontent)fractio
ns
Results
forF
.evanescensextracts:
Yieldof
fucoidans[%,byd.m.];
contento
ftotalsugar[%,
bymass]:5.11;48.0
Con
tent
ofSO
3Naa
ndpo
lyph
enols[%,bymass]:34.6and
0.5
Results
forS
.japonica
extra
cts:
Yieldof
fucoidans[%,byd.m.];
contento
ftotalsugar[%,
bymass]:𝐹1:0.38;46
.4;𝐹2:1.28;45.2
Con
tent
ofSO
3Naa
ndpo
lyph
enols[%,bymass]:𝐹1:14.0
and0;𝐹2:26.3and0.1
Results
forS
.oligocystum
extra
cts:
Yieldof
fucoidans[%,byd.m.];
contento
ftotalsugar[%,
bymass]:𝐹1:0.34;46
.0;𝐹2:0.65;48.1;𝐹3:0.55;44
.0Con
tent
ofSO
3Naa
ndpo
lyph
enols[%,bymass]:𝐹1:17.4
and0.4;𝐹2:24.0and1.1;𝐹3:32.0and0.1
[34]
SFEwith
CO2(+
ethano
las
mod
ifier)
(i)Sample:30
g(ii)S
olvent:
Treatin
gF.evanescens:pure
SC-C
O2
Treatin
gS.japonica
andS.
oligocystum:bothpu
reSC
-CO
2andSC
-CO
2+
EtOH(5%)
Teste
dflu
id:sam
pler
atios
(w/w
):fro
m10:1to>30
:1;
fluid:sam
pler
atio
(w/w
)chosen
fore
xperim
ents:
30:1
𝑇=60∘C,𝑝:550
bar,𝑡:60m
in
Providingfractio
nof
polysaccharid
esand
posttreatmento
fHCl
extractw
erea
sdescribed
incase
ofsolventextraction
Results
forF
.evanescensextracts:
Yieldof
fucoidans[%,byd.m.];
contento
ftotalsugar[%,
bymass]:P
ureS
C-CO
2:3.02;49.2
Con
tent
ofSO
3Naa
ndpo
lyph
enols[%,bymass]:P
ure
SC-C
O2:39.2and1.5
Results
forS
.japonica
extra
cts:
Yieldof
fucoidans[%,byd.m.];
contento
ftotalsugar[%,
bymass]:P
ureS
C-CO
2,𝐹1:0.35;48.5;pureS
C-CO
2,𝐹2:
1.26;44
.2;SC-
CO2+EtOH(5%),𝐹2:1.35
;45.1
Con
tent
ofSO
3Naa
ndpo
lyph
enols[%,bymass]:P
ure
SC-C
O2,𝐹1:11.8
and0.1;pu
reSC
-CO
2,𝐹2:27.0
and0.1;
SC-C
O2+EtOH(5%),𝐹2:27.3
and0.1
Results
forS
.oligocystum
extra
cts:
Yieldof
fucoidans[%,byd.m.];
contento
ftotalsugar[%,
bymass]:P
ureS
C-CO
2,𝐹1:0.38;47.4;pureS
C-CO
2,𝐹2:
0.57;45.2;pu
reSC
-CO
2,𝐹3:0.27;42.0;SC-
CO2+EtOH
(5%),𝐹2:0.55;49.0;SC-
CO2+EtOH(5%),𝐹3:0.32
;34.9
Con
tent
ofSO
3Naa
ndpo
lyph
enols[%,bymass]:pure
SC-C
O2,𝐹1:16.4and2.1;pu
reSC
-CO
2,𝐹2:23.4and1.7
;pu
reSC
-CO
2,𝐹3:34.0and0.1;SC
-CO
2+EtOH(5%),𝐹2:
28.8and1.5
;SC-
CO2+EtOH(5%),𝐹3:32.0and0.5
Effecto
fdifferentfl
uid:sam
pler
atios:10:1ratio
enabled
toprovidea
bout
95%,bymass,of
allextractable
substances,w
hileincreasin
gratio
over
30:1didno
tresult
insig
nificantenh
ancemento
fSFE
efficacy
10 Journal of Chemistry
proteins have significant nutritional value to the animalorganism [51].
3.4. Algae Based Products in Agriculture. Nowadays, due tofuture changes in European Union legislations, there is agrowing interest in the use of supercritical algal extracts asnatural foliar biostimulants for crop production. Plant growthstimulators which are known as phytohormones are the nextimportant group of compounds, which can be extracted fromalgal biomass by SFE. From a chemical point of view, planthormones are structurally diverse groups of compounds,which include auxins, gibberellins, cytokinins, salicylic acid,jasmonates, and brassinosteroids [52]. Plant hormones arethe promoters of many essential physiological processes suchas cell division, growth, and differentiation, organogenesis,sleep and seed germination, aging, and leaves pigments andfor the response to biotic stress and abiotic factors [53, 54].
The effect induced on plants, by the treatment withproducts of algal origin, is mainly determined by the contentof different types of plant hormones and their concentrations[55].The functional importance is that these products shouldbe applied in high dilutions. In many bioassays, researchersproved that products made from seaweeds stimulate thegrowth of many plants. The concentration of used extractand the method of application play an important role in suchphenomena. As far as plant hormones and other biologicallyactive compounds affect positively the plants in small con-centrations, in higher doses they may cause inhibitory effecton some processes [56].
Algae are also rich in mineral compounds and traceelements.Their role in enhancing the plant growth should beunderlined [57]. Moller and Smith investigated the impor-tance of mineral components in suspensions made fromseaweeds. Two brown algae extracts were tested on lettuceseedlings. The results showed that extracts were promotingthe growth of cotyledon of lettuce. The experiment has led tothe conclusion thatmineral components weremainly respon-sible for this effect. Additionally, it was noticed that seaweedsuspension was less effective than ashed extract. There isa possibility that suspensions contained some inhibitingorganic compounds [58]. Cyanobacteria and eukaryotic algaehave also the ability of phosphorus accumulation in the formof polyphosphates which as the reserve of phosphorus cansignificantly enrich the algal biomass used for the purposes ofsoil fertility and better plant growth. Seaweed extracts testedon Vigna sinensis stimulated the growth of this plant but onlyat concentration smaller than 20%. At higher concentration,the effect was the opposite [59].The use of algal biostimulantsmay improve seedling growth, shoot and root length andweight, chlorophyll content, and in consequence total proteincontent. In another bioassay, the information about theinfluence of seaweed extract on spinach (Spinacia oleraceaL.) was presented. Spinach seeds were irrigated with differentconcentrations of extract from Ascophyllum nodosum. Totalflavonoids and phenolic compounds content and antioxidantactivity were measured at a certain time after applicationwhich confirmed that the use of seaweed extract enhanced allof the tested parameters. Total flavonoids content increased1.2 and 1.5 times compared to control and the upswing
depended on concentration of seaweed extract. Since, totalcontent of phenolic compounds increased, the antioxidantactivity also has been improved. The optimal concentrationof extract, which showed the desired activity, was determinedas 1 g/L [60].
Plants treated with algal extracts showed more intensegrowth of their roots, which significantly improved theuptake of the nutrients from soil. This phenomenon seems tobe crucial, especially if regarding the habitats poor in mineralcompounds [1]. Field experiment on soybean showed thatapplication of seaweed extract form Kappaphycus alvareziienhanced yield parameters. Researchers observed also betternutrient uptake by this crop after foliar spraying. The max-imum straw yield was obtained after using the extract at aconcentration of 15% [61].
Besides growth promoting effect, seaweed extracts alsoshow antibacterial and antifungal properties. Carrot plantswere treated with seaweed extract (0.2%) from Ascophyllumnodosum 6 h after the conidial suspension ofA. radicina or B.cinerea was inoculated. After 25 days, the results were mea-sured and the plants treated with seaweed extract exhibitedreduced infection by around 50%.Molecular analysis showedthe accumulation of defense gene transcripts, phenolics, andphytoalexins [62].
Due to a wide spectrum of positive influence on manyaspects of plant growth, several commercially available prod-ucts derived from algae are being usedworldwide. Brown andred algae are the most popular in biofertilizers production,because of their availability throughout all the seasons ofthe year and high content of bioactive substances. The mostknown plant growth stimulants manufactured by BASF areKelpak and Profert, manufactured from Ecklonia maximaand Durvillaea antarctica, respectively. Many manufacturersutilize also brown algae Ascophyllum nodosum for theirbiostimulating properties [56].
4. Composition of Algal Extracts:Analytical Methods
Determination of the full chemical profile of algae is a compli-cated task, even under unfavorable growth conditions, whichsignificantly enhance the synthesis of active compounds inalgal cells. Complexity of the matrix is the major obstacleneeded to be overcome.
Furthermore, there are problems in the preparation ofbiomass samples suitable for the analysis, whereas this pre-treatment is the key step in the whole test. This procedureis time consuming and requires several steps includingtissue fragmentation (mechanical, using radiation or ultra-sounds) and extraction (different types of solvent techniques,supercritical fluid extraction). Algal extract obtained usingthe above methods needs the specific sample preparationbefore qualitative and quantitative analysis.Themost populartechniques used for this purpose are solid-phase extrac-tion,membranemicroextraction, immunoaffinity extraction,vapor-phase extraction, extract filtration, evaporation, and inmany cases sample fractionation and derivatization.Workingwith plants is also hindered considering vulnerability. In
Journal of Chemistry 11
some conditions, content of chemical compounds mightbe changing during the extraction and sample preparation.Consequently, the total concentrations of desired compoundsmight be different compared to thewhole plant [63]. A varietyof analytical methods are available to determine chemicalcomposition of biological material, depending however onthe chemical properties of desired compounds: analytes.
Instrumental methods, combined with various detectors,made it possible to determine several hormones simultane-ously in fresh plant material, as well as in products (mostlyfood) that are used in many bioassays [3, 64–66]. Amongthese methods, chromatographic techniques seem to be thebest and the most accurate for measuring trace amounts ofphytohormones in seaweeds and extracts from plants.
Since the early 1970s of the last century, liquid chromatog-raphy and high pressure liquid chromatography have becomemore popular in plant hormones analysis. Good resolutionand relatively low limit of detection allowed for simultaneousqualitative and quantitative determination of various classesof plant hormones [63]. Liquid chromatography used forplant hormone analysis does not require sample derivatiza-tion. Popular methods of detection connected with LC orHPLC are UV–V is detectors, diode array and fluorescencedetection, and especially MS that allow determining thechemical structure of the analytes [67]. The potential ofthis method is being multiplied when tandem mass detectoris used [68]. Other instrumental methods like capillaryelectrophoresis or spectral and electrochemical methods andespecially biosensors are rather used for the analysis ofphytohormones.
For the analysis of nonpolar or volatile compoundsfrom algae extracts, gas chromatography is widely used.This technique, especially combined with mass detectors, isefficient for the structural identification and accurate quan-tification in multiple phytohormone analysis. Nevertheless,the requirement for sample volatility limits its application toonly few plant hormones and potential volatile biostimulants.Sometimes derivatization is needed in case of obtaining betterand more reliable results; nevertheless, this step significantlyextends the time of analytical procedure [69].
Chromatography assays might be combined with super-critical fluid extraction, resulting in analysis defined as super-critical fluid chromatography (SFC). This method enablesperformingmeasurements in automated systemunder onlinecontrol, providing shorter time of the assay and decreasedlevel of contamination [70]. Depending on particular need,different ways of collecting SF-extracted analytes might besuitable: (1) solvent collection in a solvent containing vessel,(2) solid-phase collection: highly selective separating analyteson packed-bed column, filled with inert or adsorbing mate-rial, from which they are eluted by the use of appropriatesolvent, (3) online collection: using a connection of collecteddevice with chromatograph, and (4) alternative collection:(4a) solid-liquid phase collection: recommended for highlyvolatile analytes, which are trapped in a system of a solid-phase and solvent containing vessel (catching losses), (4b)collection inside fused-silica capillaries, and (4c) empty vesseltrap collection: a way excluding solvent-sample separationstep [71, 72]. Besides the mentioned volatile compounds,
SFC is suitable for testing wide spectrum of molecules withdiverse characteristic, for example, polarity and molecularmass, including fat-soluble vitamins (without necessity offormer derivatization). In case of algae derived constituents,supercritical fluid chromatography was applied to determinecontent of isoflavones from seaweeds (brown algae: Sargas-sum muticum, Sargassum vulgare, and Undaria pinnatifida;red algae: Hypnea spinella, Porphyra sp., Chondrus crispus,andHalopytis incurvus), freshwater green algae (Spongiochlo-ris spongiosa), and cyanobacteria (Scenedesmus and Nostoc17). The whole experiment involved biomass pretreatment(sonication) and dynamic extraction with SC-CO
2(modified
with aqueous methanol) at 40∘C and 350 bar for 60 minutes,followed by fast chromatography analysis and tandem massspectrometry detection [73]. Abrahamsson et al. investigatedsupercritical fluid chromatography for quantitative deter-mination of carotenoids from microalgae Scenedesmus sp.SFE was performed on pretreated sample (freeze-drying andgrinding with liquid nitrogen) using CO
2(with or without
co-solvent – ethanol) at flow rate 2mL/min, at 60∘C and 300bar for 60 minutes, and the obtained extract was analyzedwith a series of two columns: C18 and 2-ethyl pyridine.Research proved validation of the method to separate andquantify carotenoids to be comparable to standard approach[74].
5. Conclusions
Supercritical fluid extraction gives the possibility to isolatebiologically active compounds from the biomass withouttheir degradation. Solvent-free extracts can be used in manybranches of industry: as active ingredients in cosmetic prod-ucts, as components of biostimulant formulations in order toincrease crop production, or as feed additive allowing for theproduction of healthy animal dietary feed supplement. Theimplementation of new algal-derived products in the marketcoincides with the public demand for natural products.Thereis also a need to replace classical extraction methods withinnovative technologies based on bioresources. Limited useof environmental friendly CO
2solvent and the possibility of
the reuse of waste byproducts produced by SFE are the mainadvantages of this process, instead of the high costs of the SFEinstallation. Ingredients derived from raw algal material inSFE process ensure no residues of organic solvents.Therefore,algal extracts have promising future prospects as products forhumans, animals, and plants. In this review, special attentionwas paid to the application of algal extracts in plant cultiva-tion, since this issue is rarely studied in the literature. In orderto properly apply algal extract, detailed characteristics shouldbe provided by the use of novel analytical methods.
The potential of algae as the source of many specificsubstances of biological activity, as well as growing interestand possibilities of using these organisms, creates favorableconditions in many areas of research and development.
Abbreviations
AA: Arachidonic acidGLA: 𝛾-Linolenic acid
12 Journal of Chemistry
ALA: 𝛼-Linolenic acidPLE: Pressurized liquid extractionAXA: AstaxanthinPUFA: Polyunsaturated fatty acidsBBE: Bead beater extractionSCF: Supercritical fluidsDHA: Docosahexaenoic acidSC-CO
2: Supercritical carbon dioxide
EPA: Eicosapentaenoic acidSFC: Supercritical fluid chromatographyFAME: Fatty acid methyl estersSFE: Supercritical fluid extraction.
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper.
Acknowledgment
This project is financed in the framework of the grant entitledInnovative Technology of Seaweed Extracts—Components ofFertilizers, Feed, and Cosmetics (PBS/1/A1/2/2012) attributedby The National Centre for Research and Development inPoland.
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