review article supercritical algal extracts: a source of...

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]


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


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


(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


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


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


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


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


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


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


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


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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