High-Voltage Pulsed Electric Field Preprocessing EnhancesExtraction of Starch, Proteins, and Ash from Marine Macroalgae UlvaohnoiMeghanath S. Prabhu,*,†,⊥ Klimentiy Levkov,† Yoav D. Livney,‡ Alvaro Israel,§

and Alexander Golberg*,†

†Porter School of the Environment and Earth Sciences, Tel Aviv University, Dr. George Wise Street 20, Tel Aviv 6997801, Israel‡Faculty of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa 3200000 Israel§Israel Oceanographic and Limnological Research Ltd., The National Institute of Oceanography, Tel-Shikmona, P.O. Box 8030,Haifa 3108000 Israel

*S Supporting Information

ABSTRACT: Marine macroalgae are an attractive source forbiorefineries as an alternative to terrestrial crops, and new,sustainable macroalgae biomass fractionation methods areneeded. One of the least investigated macroalgae-derivedproducts is starch. In this work, we report on a device and aprotocol for pretreatment for starch extraction from a greenmacroalga Ulva ohnoi (U. ohnoi) with an emerging, non-thermal, and environmental friendly technologypulsedelectric field (PEF). Using the custom-made insulated gatebipolar transistor-pulsed generator combined with a grav-itation press-electrode device, we show that 200 pulses with afield strength of 1 kV cm−1, pulse duration of 50 μs, and pulserepetition rate of 3 Hz concentrate the starch in the U. ohnoi biomass by 59.38% by removing the salts, proteins, and other smallmolecules. The starch extraction yield from the PEF-pretreated biomass is 59.54 ± 1.34%, compared to 52.31 ± 3.21% fromuntreated biomass. In addition, PEF combined with pressing increased the coextracted macroalgae protein by more than 4 timesand ash by 1.5 times in comparison with pressing alone. These results indicate the potential of PEF pretreatment for challengingmacroalgae biomass fractionation in the sustainable marine biorefinery.

KEYWORDS: pulsed electric fields, macroalgae, seaweeds, Ulva ohnoi, starch, biorefinery, marine proteins, deashing

■ INTRODUCTION

Increasing population and food consumption rate will demand70−100% more food by 2050.1 Starch is the maincarbohydrate ingredient of human food in addition to theprotein. Starches, including modified starches, are importantingredients of various industries and are used for numerousapplications including food, fermentation, textile, cosmetics,pharmaceuticals, packaging, synthetic polymer industries, andmore.2,3 Modified starches and different native starch-basedblends are an attractive bio-based alternative to biodegradablesynthetic polymers.2,4 Starch-based bioplastics represent 80−85% of marketed bioplastic.4 In addition, starch from cereals isused for biofuel production for the past 10 years in the UnitedStates and has also become prominent in other parts of theworld.5 These competing applications cause shortage and priceincrease of cereal-based food starch6 and also put pressure onworld food supplies.1 The shortage of arable land for terrestrialagriculture and scarcity of water raise the question on thesustainability of starch supply. To meet these challenges,offshore macroalgae biomass production, known as seagricul-

ture, can provide a sustainable solution for the feedstockrequired for starch production. Furthermore, starch canprovide an additional revenue stream for the emergingmacroalgae biorefineries.7−10

Starch is a major storage carbohydrate in various species ofseaweeds such as Cladophora, Gracilariopsis, Rhodophyta, Ulva,and other species.10−16 In Ulva sp., starch granules are situatedinside the chloroplast of the cell.13,15 Starch content forinstance in Ulva rigida and Ulva ohnoi (U. ohnoi) fluctuatesseasonally and reaches 32 and 21.4% of the dry matter (DM),respectively.10,17 Although seaweed starch can serve as asustainable alternative to conventional starch for food andother industrial applications, it does not require arable landand fresh water for biomass production, and not muchattention has yet been given to its extraction. We demonstratedextraction of these starch granules from the macroalga U. ohnoi

Received: August 9, 2019Revised: September 5, 2019Published: September 9, 2019

Research Article

pubs.acs.org/journal/ascecgCite This: ACS Sustainable Chem. Eng. 2019, 7, 17453−17463

© 2019 American Chemical Society 17453 DOI: 10.1021/acssuschemeng.9b04669ACS Sustainable Chem. Eng. 2019, 7, 17453−17463

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and obtained an extraction yield of 51%.10 Previously, Yu et al.reported extraction and characterization of Floridean starchfrom red alga Gracilariopsis lemaneiformis.16 These two studiessuggest that the starch from marine macroalgae is similar toseveral terrestrial starches; however, it can be producedoffshore.New and sustainable green methods for ocean-based

biomass fractionation are still not mature. The macroalgaebiomass structure, electrical conductivity, and chemicalcomposition are very different from crops and other terrestrialplants. Therefore, direct translation of processing technologyfrom land crops to marine biomass is challenging and requiresnew devices and processes.18,19 In this work, we focus onpulsed electric fields (PEF)an emerging sustainable biomassprocessing technology.Pulse electric fields (PEFs) is an emerging nonthermal,

nonchemical, energy efficient method, used in the foodindustry for disinfection, enzyme activity modification, andextraction of various compounds from biomass.20−22 PEFtreatment is less energy-consuming process compared to theconventional thermal extraction and dehydration operations.22

PEF has been already shown as a potential pretreatmentmethod for potato starch extraction at the industrial scale inGermany.23−25 However, compared to the starch extractionprocess from terrestrial biomass sources such as potato, wheat,maize, and so on, which are rich in starch (around 80%),3 thestarch extraction from leaf-like biomass such as Ulva is muchmore difficult to process as the starch granules are presentinside the chloroplast. In addition, the starch content inside theseaweed biomass is low,13,15 between 2 and 21%,10 and itsconcentration could enable more efficient storage andextraction. Starch granules gelatinize and lose their granularand crystalline structure low temperatures between 50 and 70°C;10,26,27 therefore, the extraction or concentration of starchgranules in their native form seaweeds requires use ofnonthermal processes, such as PEF.10,28,29 However, currentlyused PEF systems at the industrial and laboratory scale do notfit for macroalgae processing because of multiple difficultiesrelated to the application of PEF on seawater and highlyconductive biomass.Indeed, our recent work at the laboratory scale showed that

PEF can be used for selective extraction of proteins and ash(minerals) from macroalgae such as Ulva sp.20,21,30 Extractionof water-soluble protein and carbohydrates from macroalgaeusing PEF was also demonstrated in refs 19 21 30, and 31indicating that PEF could be used in the processing ofmacroalgae biomass. However, to the best of our knowledge,there are no works mentioning the use of PEF as apretreatment method for starch concentration and extractionfrom seaweed or even from any green biomass. Such a use canprovide an opportunity to the PEF for the simultaneousfractionation and extraction of soluble molecules, such asproteins, and enhancement of starch extraction in thedownstream processing, providing a new process forbiorefinery.The objective of this work was to evaluate the use of PEF for

nonthermal preprocessing of the extraction of native starchgranules from macroalga U. ohnoi coupled with thefractionation of biomass into ash and proteins. To attain thistask, we developed a PEF system that consists of an insulatedgate bipolar transistor (IGBT)-based pulse generator, coupledto a press-electrode device for simultaneous electric fielddelivery and intracellular juice extraction. We show that PEF

coupled to pressing pretreatment concentrates starch at theresidual macroalgae biomass and enhances the starch yield inthe downstream processing by extraction of intracellular water,ash, proteins, and other water-soluble molecules. Theapplication of PEF as reported in this paper is innovativeand critically important for future production of starch frommarine sourcesa truly sustainable approach for theproduction of this important commodity chemical in a marinebiorefinery.

■ EXPERIMENTAL SECTIONU. ohnoi Biomass Cultivation. A culture of U. ohnoi was

collected from an outdoors seaweed culture cultivation system atIsrael Oceanography and Limnological Research, Ltd. (IOLR), Haifa,Israel. U. ohnoi originated from random collections conducted overthe past years along the Israeli Mediterranean Sea.32 The cultivationunits were made up of 1000 L fiberglass tanks equipped with aerationand running seawater pumped from a Mediterranean shore nearbyIOLR, Haifa. U. ohnoi cultivated in 1000 L tanks got fertilized once aweek with sodium dihydrogen phosphate (NaH2PO4, added to 0.057mM in the tank) and ammonium chloride (NH4Cl, 0.59 mM).

Initial Characterization of U. ohnoi Biomass Composition.At the end of a 4 week growth period, U. ohnoi was harvested from thecultivation tank and transported to our laboratory in a cool and darkcondition within 2 h post-harvesting. The surface water from thebiomass was removed at 3200 rpm, using a portable spin dryer(Beswin Electric Co. Ltd., Zhejiang Province, China). This surfacedewatered fresh biomass, defined as wet mass (WM), was used for theestimation of moisture content, DM content, and starch concen-tration.

Determination of Moisture, Dry Mass, and Ash Content.Moisture content and DM were determined by drying U. ohnoi at 105°C to a constant mass using a moisture analyzer (BM-50-5, BiobaseBiodustry (Shandong) Co. Ltd., China). Ash content was measured at550 °C for 3 h based on ISO 5984,33 and calculations were madeusing eq 1

= ×−−

M MM M

ash content(%) 100( )( )

4 1

3 1 (1)

where M1 is the mass of the empty crucible at 105 °C, M3 is the massof the crucible and sample dried at 105 °C, M4 is the mass of thesample and crucible at 105 °C, after treating at 550 °C.

Determination of Starch Content. Starch concentration wasmeasured by using the total starch assay kit (K-TSTA-100A,Megazyme, Ireland).10 Briefly, harvested U. ohnoi was dried to aconstant mass at 40 °C in a conventional oven and ground to aparticle size of less than 1 mm using a mortar and pestle with the helpof liquid N2. From this powder, 10 mg of sample (n = 3) was weighedin 2 mL tubes and washed twice in 500 μL of 80% (v/v) ethanol toremove any glucose present. Two hundred microliters of 2 Mpotassium hydroxide (KOH) was then added, and the tubes wereshaken horizontally for 30 min at 37 °C and 150 rpm. At the end of30 min, the mixture was further incubated at 100 °C for 1 min tocompletely dissolve the starch. Tubes were short spun for 1 min at 23°C, and sodium acetate buffer (800 μL, 1.2 M, pH 3.8) was added.Immediately, 10 μL of α-amylase and 10 μL of amyloglucosidase wereadded and mixed using a vortex mixer. The mixture was shaken for 2h at 50 °C and 150 rpm. The tubes were then centrifuged at 1800g for10 min (Eppendorf centrifuge 5424, Hamburg). The glucose releasedwas measured at 510 nm by reacting 0.01 mL of supernatant with 0.3mL of glucose oxidase−peroxidase (GODPOD) enzyme mixture for20 min against the glucose standard. Starch concentration, as thepercentage of the DM, was calculated with a molar mass conversionfrom glucose to anyhydroglucose (the starch monomer unit) of 0.9.

IGBT-Based PEF Generator and Gravitation-Based Electro-des for Biomass Pretreatment and Fractionation. A custom-made pulsed electric field generator was developed. The generatorprovides at a maximum voltage of 1000 V and a current of 160 A at a

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5 ohm load. The maximal pulse duration, the number of pulses, andpulse frequencies are limited by the permissible heating of the IGBTtransistors. In our system, as described below, for a 5 ohm load and 1Hz pulse repetition rate, the maximum pulse duration is 100 μs.The functional circuit diagram of the pulsed generator developed is

shown in Figure 1a. The main functional nodes of the system include(1) an energy storage capacitor (ESC) with a capacity of 50 μF for avoltage of 1.25 kV, (2) a high-voltage source of charge of energystorage capacitors (CCM1KW, Spellman, NY), (3) parallel-connectedhigh-voltage switches for pulsed discharge of ESCs (IX-YN120N120C3 (IXYS, CA) with parameters of 1200 volts, 120 A),(4) a driver of high-voltage switch with electrical circuits of control oftransistor gates and own power supply (Gate Driver OptocouplerFOD3184, Fairchild, CA), (5) high-power current-limiting resistors(RR02−3 OHM-2 W), (6) a circuit node for manual control of high-voltage switch and high-voltage power supply in the testing mode, (7)a microcontroller for controlling PEF treatment, calculating thecurrent through the treated biomass, and transferring the results tothe computer recorder, and (8) a low-voltage power supply forcontrol circuits and fans of the device. The device is connected by aUSB interface to a computer for inputting the experiment parametersin the microcontroller, displaying the current state of the process andrecording the received data in the experiment file. Currents weremeasured using a PicoScope 4224 oscilloscope with a pico currentclamp (60A AC/DC). The voltage was measured with a PicoScopeTA044 70 MHz 7000 V differential oscilloscope probe 100:1/1000:1.Voltages and currents were analyzed with Pico Scope 6 software (PicoTechnology Inc., U.K.).The electroporation extraction cell holds the treated biomass

between the electrodes during the application of PEF. It also allowsseparation of the seaweed biomass into liquid and solid phasessimultaneously with the application of electric fields and mechanicalpressure. The electroporation cell consists of a cylindrical Teflonchamber (2.5 cm diameter), and the positive electrode is located atthe bottom of the chamber. In the lateral part of the electroporationcell, there are narrow slit-like openings for the outlet of the liquidfraction during the electroporation of the biomass. The extractedliquid is collected and discharged through a groove at the base of the

cell. The gravitational press-electrode device is shown in Figure 1b.The load-receiving platform is fixed at the upper end of the rod, and atits lower end, there is a negative electrode, which can freely slideinside the electroporation cell. The complete developed experimentalsetup is shown in Figure 1c. A load weighing up to 10 kg can beplaced on the load-receiving platform to exert the necessaryinterelectrode pressure on the biomass (Figure 1c). A displacementsensor (optoNCDT, Micro-Epsilon, Germany) is installed on thesliding rod to monitor the volume change of the biomass duringelectroporation (Figure 1b).

PEF Treatment Coupled with Mechanical Press. A custom-made PEF generator and gravitation-based electrodes were used forthe electroporation of the U. ohnoi thalli. Ten grams (WM) of U.ohnoi was suspended in 100 mL of distilled water. The biomass (10 gfresh, corresponding to 2.61 ± 0.08 g dry) was then loaded in theelectroporation cell in batches of 2 g, and PEF was applied with thefollowing conditions: electric field strength of 1 kV cm−1, pulserepetition rate of 3 Hz, number of pulses, 200 with 50 μs pulse widthduration.

These parameters (field strengths E ≥ 1 kV cm−1) were reported tobe sufficient to result in permanent pore formation of plant cellmembranes and were recommended in refs 20 and 36. Thus,parameters were chosen to have conditions that enable the breakingof covalent bonds within the cell wall and allow the investigation ofthe PEF effect on Ulva biomass fractionation and starch extraction.The distance between the fixed electrode inside the electroporationcell (positive electrode) and the free slide gravitational press electrode(negative electrode) was adjusted before applying the first pulse. Thedistance between the two electrodes was measured continuously withthe displacement sensor (opto NCDT 1302, Micro-Epsilon,Germany). An additional pressure of 107,682 N/m2 was appliedduring the PEF. Internal resistance, Ri, for the device with 1000 V and160 A was 0.75 ohm. The initial (I0) and residual (I) currents weredetermined from the oscillogram. VESC is the voltage applied on theenergy storage capacitor (ESC), CESC is the capacitance of the energystorage capacitor (1 × 10−6), and ti is the width of the pulse inmicroseconds. The electroporation cell (EPC) resistance wascalculated using eq 2

Figure 1. (a) Functional circuit diagram of the electroporation device 1 kV, 160A. (b) Electroporation cell. (c) Mechanical chamber for high-voltage electroporation.

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= −ikjjjjj

y{zzzzzR

VI

REPCESC

0i

(2)

The electroporation cell initial voltage (V) and residual voltage(V0) were calculated as in eqs 3 and 4, respectively,

= − ×V V I R0 ESC 0 i (3)

= ×V I REPC (4)

The energy of the single pulse of electroporation (Aimp) wascalculated using eq 5

=×

+ × −ikjjjjj

y{zzzzzA

V tR

C V V0.5 ( )imp

2i

EPCESC 0

2 2

(5)

After the treatment, the thalli were resuspended in 100 mL ofdistilled water into which they were suspended before applying PEF.The biomass was shaken at 32 °C and 150 rpm to allow theintracellular products to diffuse out into the aqueous medium(extraction). The thalli were removed from the water after 1 h, andthe water medium was filtered through a 50 μm nylon filter using aglass vacuum filtration system. The thalli at the end of filtration werereferred to as “PEF-treated biomass”, and the aqueous juice thusobtained was referred as “PEF-extract”. For control experiments, thewhole process was repeated but no pulses were applied in thechamber. The biomass thus obtained was called as “control-biomass”,and the liquid thus obtained was called as “control-extract”. In total,six replicates of 10 g of fresh U. ohnoi were treated each for PEF andcontrol treatment.Measurements of Conductivity, Starch, Protein, Total

Solids, and Ash Content in the PEF-Extract. The conductivityof the control-extract and PEF-extract was measured using an InLab731 ISM conductivity sensor mounted on the pH meter (Mettler-Toledo, Melbourne, Australia). All four components, control-extract,control-biomass, PEF-extract, and PEF-treated biomass, wereanalyzed for their ash, protein, and starch contents. The liquidextract was dried at 105 °C using a conventional oven to determinethe total solids (TS). Ash content was measured based on ISO5984.33 Biomass fractions were dried at 40 °C until constant masseswere reached. The presence of starch was analyzed by using theMegazyme’s total starch assay kit as described above (in theDetermination of Starch Content section).Total protein was analyzed using a modified version of the Lowry

method.35 The filtered liquid extract was centrifuged at 1800g for 10min. Solid biomass (∼15 mg) was accurately weighed in 2 mL tubesand filled to one-third with beads (zirconia, 2 mm, Sarstedt) and 1.5mL of 2 M sodium hydroxide (NaOH) solution. The tubes were runthrough three sessions of bead beating of 60 s each in a bead beater(BioSpec, OK, USA) with intermittent cooling for 10 min at 23 °C.The tubes were then centrifuged at 14,000 rpm for 20 min. Thesupernatants from all the tubes were appropriately diluted withultrapure water. Diluted samples were analyzed by adding 100 μL in awell of a 96-well plate. Biuret reagent was prepared by mixing 0.5 mLof 1% cupric sulfate with 0.5 mL of 2% sodium potassium tartrate,followed by the addition of 50 mL of 2% sodium carbonate in 0.1 NNaOH. Two hundred microliters of biuret reagent was added to eachwell and mixed thoroughly using a micropipet. The mixture was thenequilibrated at 23 °C for 10 min prior to the addition of 20 μL perwell of 1.0 N Folin−Ciocalteu reagent. Samples were mixedimmediately by repeated pipetting following each addition. Thecolor was allowed to develop for 30 min at 23 °C, following whichabsorbance was measured at 750 nm using a spectrophotometer(Infinite 200 Pro, TECAN, Switzerland). A standard curve wasproduced using bovine serum albumin (BSA) at different concen-trations (0−500 μg/mL). As a blank, water was used in place of thesample. Analyses were done in triplicate, and the results wereexpressed as BSA equivalent in mg/L.The fraction of ash and protein in TS of the extract was calculated

using eq 6

= ×ikjjj

y{zzz

Pash or protein(%of TS)

TS100f

(6)

where Pf is the amount of ash or protein in the extract (in g) and TS isthe amount of total solids in the extract (in g).

Total ash (TA) and total protein (TP) extracted in the liquid (inthe PEF-extract) from the initial biomass were calculated using eq 7

= ×ikjjjjj

y{zzzzz

PP

TA or TP(%) 100f

i (7)

where Pf is the amount of ash or protein (DM) present in PEF-extractand Pi is the amount of ash or protein present in initial Ulva biomass(DM).

Starch Extraction from U. ohnoi and Characterization.Starch was extracted from control-biomass and PEF-treated biomasssamples (10 g each, n = 5) of U. ohnoi using the homogenization andfiltration method as explained in our previous paper.10 Briefly, U.ohnoi was mixed with cold distilled water (1:20 (w/v)) andhomogenized into fine particulate suspension using a homogenizer(HG-300, Hasigtai Machinery Industry Co., Ltd., Taiwan). Therotation speed of the homogenizer blades (step 9) and duration (3min) were kept constant during the homogenization of each sample.The slurry thus obtained was filtered by sequentially passing throughnylon filter bags of 50 and 10 μm pore size with the help of thecentrifuge. Starch granules from the filtrate were collected using acentrifuge at 4500 rpm and 15 min (Rotanta 46 RSC, HettichInstruments, LP, Germany). The pellet obtained was purified bywashing with 30 mL of 80% ethanol and dried at 40 °C for 24 h. Thestarch-rich fractions extracted from PEF-treated biomass and fromcontrol-biomass were called as “US-PEF” and “US-C”, respectively.The quantity of extracted starch-rich fraction was measuredgravimetrically. The percentage of extracted starch-rich fraction(SE) was calculated using eq 8

= ×ikjjjjj

y{zzzzz

SS

SE (%) 100w

i (8)

where Sw is the mass (in g) of the extracted starch-rich fraction and Siis the initial mass (DM) of the U. ohnoi PEF-treated biomass.

The starch content in the extracted starch-rich fraction wasmeasured using the total starch assay kit (as mentioned above in thissection under Determination of Starch Content), and the starchextraction yield (SEY) was obtained using eq 9

= ×ikjjjjj

y{zzzzz

SS

SEY(%) 100f

i (9)

where Sf is the amount of starch (g) present in the extracted starch-rich fraction and Si is the amount of starch (g) present in the initialDM of harvested U. ohnoi biomass used in the experiment. Theattenuated total reflectance-Fourier transform infrared (ATR-FTIR)spectrum was recorded for extracted dry Ulva starch, US-PEF, andUS-C using a Bruker Platinum ATR-FTIR spectrometer from 400 to4000 cm−1. The scanning electron microscopy (SEM) image of Ulvastarch was obtained using a Quanta 200 FEG ESEM (Oregon, USA)after fixing the sample on a silicon tape and then coating with goldusing a sputter coater. AFM scans were obtained using NanoWizard IIBio-AFM (JPK Instruments, Germany) at 25 °C. Samples wereimaged in air in an intermittent contact mode using NSC21/NoAlsilicon tips (NT-MDT, spring constant between 8.5 and 33 N/m).

Protein Extraction from U. ohnoi. Following PEF treatment,protein was extracted from PEF-treated biomass and control-biomassof U. ohnoi (1 g fresh, corresponding to 0.261 g dry). Total proteinwas extracted using the thermochemical (TC) method (0.25 NNaOH solution at 60 °C for 1 h) as described by Mhatre et al.36 Theprotein-rich extract thus obtained was analyzed for protein contentusing the Lowry method,35 and the protein extraction yield (PEY)was obtained using eq 10

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= ×ikjjjjj

y{zzzzz

PP

SEY(%) 100f

i (10)

where Pf is the amount of protein (g) present in the extracted starch-rich fraction and Pi is the amount of starch (g) present in the initialDM of harvested U. ohnoi biomass used in the experiment.Statistical Analysis. For statistical analysis, a data analysis

package in Excel (ver. 13, Microsoft, WA) was used. All samplesand controls were prepared and measured, at least in triplicates, if notmentioned differently. The error bars are the standard error of themean (SEM). To compare the extracted starch yield from PEF-treated biomass to the controls, a two-tailed Student’s t-test wasperformed.

■ RESULTS AND DISCUSSIONDry Mass, Moisture, Ash, Starch, and Protein Content

in U. ohnoi. U. ohnoi, a species of light-green colored seaweedin the family Ulvaceae, was selected for this study as therepresentative species. Ulva sp. show a high growth rate andhave unique biochemical composition including attractivepolysaccharide composition, which in addition to the commoncellulose and starch, including large amounts of sulfatedpolysaccharide, Ulvan.37 In addition, Ulva sp. are distributedglobally from polar to tropical regions38 and have a relativeadvantage in warmer climate zones, thus being less sensitive toclimate changes.39,40 Several authors have studied Ulva sp. as apotential candidate for seaweed biorefinery.9,10,41 Dewatered,fresh (WM) U. ohnoi was characterized for dry mass andmoisture and ash contents. The DM and moisture contentwere 26.12 ± 0.94 and 73.88 ± 1.88%, respectively. The ashcontent was 27.8 ± 0.54% of the DM. Starch concentrationwas 7.35 ± 0.25%, and protein content was 8.41 ± 0.11% ofthe DM. The ash content in Ulva sp. can vary between 11 and57% of DM.42 The ash content in the present biomass wasslightly less than the values reported for laboratory grown Ulvasp., which showed 36.02%.43 The protein in Ulva is required tomaintain the nitrogen balance inside the cells. Its concentrationin natural environments can vary from 2.9 to 29% of DM.42,44

The protein value in this study was slightly less than other Ulvagrown in laboratory conditions.44 Starch is a storagepolysaccharide in Ulva, and its concentration in naturalenvironment can vary between 0.45 and 9.3% of DMdepending on nutrient conditions, season, and life cycle andcan be significantly increased upon nutrient starvation.10 Inthis study, the starch concentration in U. ohnoi, grown innutrient-rich conditions, was higher than observed in ourprevious study, which could be due to variation in abioticgrowth conditions such as temperature and light.10

PEF Treatment. U. ohnoi biomass, post-harvesting anddewatering (WM), was subjected to PEF treatment to extractash and protein, followed by starch extraction from the residualPEF-treated biomass. The whole flow process is shown inFigure 2. During the PEF treatment, resistivity of the biomassdecreased from 112.75 ± 0.35 ohm·cm during the first pulse ofthe treatment to 35.71 ± 0.87 ohm·cm at the end of the lastpulse. Similarly, the current increased while the voltagedecreased during the first pulse and the last pulse of thetreatment (current 7 ± 0.90 A and voltage 990 ± 5 V andcurrent 24.5 ± 2.3 A and voltage 965 ± 9.5 V during the firstand the last pulse of the treatment, respectively) (Figure 3),indicating that the biomass was electroporated. Suchphenomena were observed in earlier studies19,21,45,46 andoccur due to altered resistance and capacitance. The distancebetween the electrodes at the start of the first pulse was 10.5 ±

2.76 mm. The electric field strength at the first pulse was 98.5± 0.34 V mm−1, and the invested energy was 24.67 ± 3.43 Jpulse −1. The distance between the electrodes at the end of thelast pulse was 9.51 ± 2.37 mm. the electric field strength at thelast pulse was 98.5 ± 0.34 V mm−1, and the invested energywas 35.919 ± 2.33 J pulse −1. The distance between theelectrodes is adjusted only during the first pulse. It wasobserved that during the application of the pulse, ionic matterfrom the cytoplasm of the cell starts oozing out as indicated bythe increase in conductivity. Furthermore, due to thegravitational pressing during the application of the pulses,the distance between the electrodes spontaneously decreases.With the highest energy consumption of 35.919 J pulse −1, thetotal energy invested for pretreatment will be 1375 kJ/kg DWof Ulva biomass. Previous work on potato starch extractionshowed energy saving on cutting probably because of tissuesoftening and loss of turgor pressure. As the water content ofthe intracellular content of the biomass decreases due toextraction, this wilting could decrease the energy required formechanical breaking of thalli in the following steps. Ourprevious work22 has shown that PEF treatment is nonthermaland less energy-consuming compared to the conventionalthermal extraction and dehydration operations and enables tobetter valorize biomass.

Conductivity and Contents of Protein, Starch, TotalSolids, and Ash of the PEF-Extract. Conductivity. Theconductivity of the control-extract was 2988.33 ± 73.39 μS/cm, and that of the PEF-extract was 5075.60 ± 396.66 μS/cm,which was 69.84% higher than in the control-extract (Figure4a). The applied PEF affects the membrane permeability of theUlva cells, allowing free, nonselective passage of ions and saltsfrom the cellular cytosol. These salts are responsible for thehigher conductivity in the PEF-extract.19

Starch. Starch granules were expectedly absent in thecontrol-extract as well as in the PEF-extract, which is similar tothe results of the process when applied to potatoes.24 Byapplying PEF, it was possible to pierce the walls of the potatocells.24 It was observed in the literature that the tissue is alteredduring PEF treatment as the cell wall is affected by PEFtreatment.47 Whether cell wall components are changeddirectly due to the PEF treatment or due to cell membranedisintegration and the release of cytoplasm is still not clear.However, the content of the cell wall biopolymer lignindecreases during the PEF application, which may occur due tothe effective break of intermolecular and intramolecular bonds

Figure 2. Flow diagram of PEF-assisted process for the recovery ofstarch, salt, and protein from U. ohnoi. (DW = distilled water; PEF =pulsed electric fields).

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Figure 3. Electrical voltage and current shape for single pulse: (a) first pulse and (b) last pulse. Pulse strength, 1 kV cm−1; pulse number, 200; pulseduration, 50 μs; pulse frequency, 3 Hz; biomass load, 2 g.

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between or within the cellulose, hemicellulose, and lignin.47

Starch extraction from leafy biomass and macroalgae having asimilar leaf-like structure is challenging due to distribution ofstarch granules inside the chloroplast and their relative lowerconcentration and smaller size of granules as compared toother common starch from seeds and tuber plant sources.3,10

Above changes in the cell wall created by PEF could facilitatethe extraction of starch during a later starch extraction step.24

Protein. The protein content as % of TS was higher in PEF-extract (8.79%) compared to the control-extract (2.51%)(Figure 4b). The protein concentration in control-extract was84.58 ± 3.11 mg/L, whereas in PEF-extract, it was 329.62 ±23.10 mg/L (Figure 4c). This corresponds to the proteinconcentration in the PEF-extract, 289.7% higher than that inthe control-extract. The protein extracted out of the totalbiomass protein was 3.16 and 14.94% in the control-extractand in the PEF-extract, respectively (Figure 4d). The proteinyield in this study was higher than the protein yield reportedearlier by Robin et al.,19 who got only 2.93% extracted out oftotal biomass protein. This could be due the diffusion stepcarried out after the PEF treatment. However, the concen-tration of proteins in the PEF-extract was lesser than reportedby Robin et al.19 This could be due to the lower voltage andlesser number of pulses used for the PEF treatment in ourexperiments. Robin et al.19 showed that the protein extractedfrom Ulva can be significantly increased by increasing theapplied voltage from 20 to 50 kV, as well as the number ofpulses from 10 to 50. However, in our study, we could not gobeyond 1 kV due to current restrictions from the custom-madePEF device and also due to doubt of damaging the nativestructure of the starch granule at higher voltages.Notably, although PEF treatments seem to be effective for

ash removal (>68% of as extracted out of total biomass ash,

Figure 4e), the protein extracted out of total biomass protein(%) is still low (14.94%, Figure 4d). Therefore, this newmethod may not be sufficient to meet economical require-ments when it is compared with current methods of proteinextraction, due to the lower protein removal. However, itshould be also noted that protein content of raw Ulva biomassin this study was low (8.41 ± 0.11% of the DM), and thus, theefficiency may be higher when the protein content in thebiomass is high. In addition, although PEF extracted only14.94% protein out of initial total protein, it selectivelyextracted a protein fraction that has proteins within a certainmolecular weight range and specific functional properties (e.g.,high water solubility) as observed from our previous work onprotein extraction using PEF.19,20,30 Further, such proteinfractions are known to have the phenolic and antioxidantcompound and fractions of sulfated polysaccharides that can beuseful as an additional benefit during its use as a foodsupplement.19,30 Thus, the PEF method is still a useful methodfor protein extraction for various specific applications, whichmay require such a protein fraction. More importantly, thenotable feature of PEF extraction is that the tissue structure ofthe Ulva thallus is left intact after the PEF treatment, makingthe separation of protein and salts in liquid fraction from thesolid biomass much easier.19 To further increase the efficiencyof protein recovered in the PEF-extract, extraction conditionssuch as different polarity solvents, pH values, and diffusiontimes can be optimized in the future work. This may alsofurther facilitate starch extraction.

Total Solids and Ash Content. Total solids present in thecontrol-extract and the PEF-extract was 0.27 and 0.37%,respectively (Figure 4b). These values of TS were significantlylower compared to 2 and 3.8% in the control and PEF-treatedextract obtained in the study carried out by Robin et al.21 Ash

Figure 4. (a) Conductivity, (b) total solids (TS), (c, d) proteins concentration, and (e) ash concentration in control-extract and PEF-extract, n = 3,which is the number of repeated PEF treatments.

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contents were 67.97 ± 0.48 and 68.53 ± 0.44% out of the totalextracted solids in the control-extract and the PEF-extract,respectively (Figure 4b). This shows that the ash was a majorcomponent of the TS. The difference in the ash content inboth extracts was not significant, and the results arecomparable to those of Robin et al.21 However, there was ahigher amount of protein in the PEF-extract indicating thehigher fraction of organic content in the TS of the PEF-extract.The ash contents extracted out of the total ash in the initialbiomass in the control-extract and PEF-extract were 46.67 and68.52%, respectively (Figure 4e).The relative percentage content of ash and protein to the dry

raw material reflecting the yield is reported in Table 1. The

extraction yield of ash (68.5%) and protein (14.94%) observedin our study using PEF coupled with the mechanical pressfollowed by extraction into distilled water was much higherthan that by PEF followed by the mechanical press alone (46%ash and 2.9% protein) as observed by Robin et al.19 The higheryield could be due to a more effective extraction by distilledwater by washing the electroporated tissue during 1 h shakingcompared to mechanical pressing for a short time. Previously,it has been shown that PEF treatment on Ulva biomass releasesash (salts) and small proteins out of the cells.19−21 Our resultsof higher values for conductivity, ash, TS, and protein in thePEF-extract compared to the control-extract further supportthese findings. Furthermore, the salts and proteins from thePEF-extract can be separated using a suitable method, forinstance, ultrafiltration.19 The salts can be used for variouspurposes such as fertilizer or table salt, and the protein can beused in human and animal nutritional supplements.48−50

The mechanism behind the effect of PEF on the cell walland the cell membrane is complex and still only partiallyunderstood.51 Current theory explains that when PEF isapplied, small pores are created in the cell wall andmembranes. Through these pores, most of the salts, water-soluble proteins, and soluble carbohydrates exit the Ulvacells20,21,31 and bigger molecules are left inside the cells. Thereason behind the absence of starch in PEF-extract could bethat the size of the pores, created in the cell envelop by thePEF treatment in this work, was significantly smaller than thesize of the starch granules (5−7 μm, see ref 10). Moreover, thestarch granules are located inside the chloroplast,13 thusmaking it difficult for them to become extracted. Anotherreason could be lack of the chemical potential gradient fordiffusion and spontaneous release of the starch granules, aslarge molecules have very small molar concentration, leading tosmall motive force to diffuse out of the cells during extractionin distilled water.31 More radical PEF conditions for extractionor release of starch granules in the PEF-extract were not tested,as it might cause heating. Furthermore, higher PEF treatment(30−50 kV/cm) could cause dissociation, denaturation, andalso damage the starch granules, as observed in the case of purecorn and potato starch granules in earlier studies.52−54

The starch concentration in the control-biomass and PEF-treated biomass of U. ohnoi was measured and compared withthe starch concentration in freshly harvested biomass. As ashcontent in the biomass decreases with PEF treatment, proteinand starch contents in the biomass increase significantly(Figure 5).9,19 The starch concentrations in the harvested

biomass, control-biomass, and PEF-treated biomass were 7.35± 0.15, 9.21 ± 0.18, and 11.71 ± 0.23% of the DM,respectively. The increases in the starch concentration incontrol-biomass and PEF-treated biomass compared toharvested biomass were by 25.4 and 59.38%, respectively.This was further true for the protein concentration also, whichincreased from 8.41% in harvested biomass to 9.78 and 15.11%in control-biomass and PEF-treated biomass (an increase of16.28% and 79.68%), respectively (see Figure 5).

Protein and Starch Extraction and Characterization.The amount of protein extracted and protein extraction yield(PEY), similarly, the amount of starch extracted, that is, theactual starch content in extracted starch fraction and starchextraction yield (SEY) are shown in Table 2. Protein extractedfrom Ulva (1 g fresh, corresponding to 0.261 ± 0.008 g dry)control-biomass was 4.04 ± 0.05 mg and from PEF-treatedbiomass was 10.80 ± 0.24 mg. This corresponds to proteinextraction yields of 18.40 ± 0.21 and 49.15 ± 1.11% fromcontrol-biomass and PEF-treated biomass, respectively. Totalprotein extraction yields (combined from PEF and TC extractprotein) in this study were 21.56 ± 0.6 and 64.09 ± 1.6% fromcontrol-biomass and PEF-treated biomass, respectively. Aresult of protein extraction indicates that PEF treatment andmechanical pressing, followed by diffusion and TC extraction,significantly increased protein extraction by 197.26% overcontrol extraction with only mechanical pressing followed bydiffusion and TC extraction. The protein extraction yields fromUlva observed in this study using PEF, followed by aqueousdiffusion and alkaline TC extraction, were higher than onlychemical extraction those reported by ref 38 or ultrasonictreatment followed by chemical extraction reported by Kazir etal.55

Table 1. Relative Percentage Content of Ash and Protein toDry Raw Material

fraction extracted

(% dry biomass) raw biomass PEF-extract control-extract

ash 27.8 ± 0.54 19.05 ± 0.06 12.98 ± 0.15protein 8.41 ± 0.11 1.26 ± 0.29 0.27 ± 0.06

Figure 5. Ash, starch, and protein concentrations in the biomass (%DM) of U. ohnoi at various treatment steps. (Harvested biomass =fresh biomass (FM); control biomass = PEF treatment process onlywith mechanical press + diffusion + filtration; PEF-treated biomass =PEF treatment with electric pulses + mechanical press + diffusion +filtration). n = 3 (number of experimental repeats with applied PEFfor the analysis).

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The amounts of extracted starch-rich fractions were 6.71 ±0.11% (0.175 ± 0.03 g/2.61 ± 0.08 g Ulva DM) of DM fromthe control-biomass and 8.64 ± 0.21% of DM (or 0.22 ± 0.01g/2.61 ± 0.08 g Ulva DM) from the PEF-treated biomass. Theamount of extracted starch-rich fraction (as % DM) from thePEF-treated biomass was 28.85% higher (p-value of 0.0015)compared to the control-biomass (8.64% vs 6.71%).The purity of extracted starch-rich fraction from control-

biomass was 59.40 ± 2.47% and from PEF-treated biomass was53.05 ± 0.49% corresponding to 0.104 ± 0.008 and 0.12 ±0.006 g of actual starch, respectively. The purity of extractedstarch-rich fraction from PEF-treated biomass (53.05 ± 0.49%)compared to extracted starch-rich fraction from control-biomass (59.40 ± 2.47%) was less by 6.35 ± 1.48% of starchDM but less significantly (p-value of 0.077). However, the SEYin PEF-treated biomass increased to 59.54 ± 1.34%, comparedto 52.31 ± 3.21% from the control biomass on DM basis. Thedifference in the SEY between PEF-treated biomass andcontrol-biomass was 7.23 ± 2.28%. This corresponds to arelative increase of 13.85% in SEY from PEF-treated biomass.Although the purity of starch was lower, the SEY wassignificantly higher from PEF-treated biomass (p-value of0.029, Table 2). This could be due to the PEF-induceddamages to the cell wall cellulose and cytoskeleton,56 whichfurther favored the cell lysis in a more effective manner duringthe homogenization. Additionally, the increase in starchconcentration in the PEF-treated biomass, due to a higherrelease of salts, ions, and small protein molecules, may havefavored the higher starch extraction yield. ATR-FTIRabsorbance data showed that the extracted starch-rich fractionsfrom “PEF-treated biomass and “control-biomass” were similarand did not show a difference in the absorbance pattern (seeFigure S1 in the Supporting Information). SEM images alsoindicated similar spherical and ovoid morphology of granuleswith size ranging from 5 to 7 μm in both the starch samples(see Figure S2 in the Supporting Information).PEF is known to modify the tissue and the cell and to

disintegrate the cell wall by delignification by leakage of lignin-cellulose bonds.34,47,54 The increase in starch and proteinextraction yields may be due to one of the reasons such asweakening and irreversible damage to the cell wall andmembrane,34,47,54 osmotic dehydration, and loss of turgorpressure, followed by an increased osmotic flow of salts, ions,and small sized proteins and by moisture redistribution insidethe U. ohnoi cell. Additionally, increased protein and starchconcentration per DM of PEF-treated biomass (see Figure 5)could have influenced protein and starch extraction yields dueto increased availability, as observed in the literature forproteins, where pretreatment with cell-disruption techniques

showed higher protein yields during the later extractionprocess by increasing the availability of proteins.57 However,this hypothesis requires further investigation. Our resultssuggest that the PEF process can be effectively used as apretreatment to increase the protein and starch concentrationin the U. Ohnoi biomass before further processing for theextraction. Several native starches and modified starches areused for commercial applications in various industriesincluding food, fermentation, textile, cosmetics, pharmaceut-icals, packaging, and synthetic polymer industries.3 Certainmodifications are done on starch to enhance its specificproperties for unique applications.2 PEF-assisted modificationof starch has been achieved for several starches;58,59 however, itwas not studied in this work.

■ CONCLUSIONS

Extracting multiple products from the same biomass is a crucialstep in a marine biorefinery. Harvesting starch from seaweedswill reduce the pressure on terrestrial agriculture and has thepotential to feed the growing global population. However,macroalgae biomass fractionation is challenging, and newsustainable technologies and processes are needed. In thepresent study, the use of PEF for nonthermal fractionation ofmarine biomass and increasing the starch extraction yield fromU. Ohnoi was studied for the first time. PEF concentrated theprotein by 79.68% and starch by 59.38% in the pretreatedbiomass, compared to their initial concentrations. PEFpretreatment allowed higher yields of starch extraction fromU. ohnoi. Starch and protein extraction yields from PEF-treatedbiomass were 13.85 and 197.26% higher relative to the yieldfrom control biomass (percentage increase), respectively.Furthermore, using PEF coupled to mechanical pressing,15% protein and 69% salt (from initial biomass protein andash) were extracted from U. ohnoi compared to mechanicalpressing alone. This novel study provides missing informationand suggests that the PEF can be used as a chemical-free,nonthermal method for the macroalgae fractionation into salt,protein, and starch-rich residue. At the same time, it can beused as a pretreatment method for a more efficient extractionof protein and starch from a strenuous macroalgae biomass.Future wok will focus on increasing the yields of products byoptimizing various conditions involved in the process. Thedeveloped devices and pretreatment approach provide a newprocess for marine biomass fractionation important for marinebiorefinery.

Table 2. Comparison of Starch Extraction Parameters and Protein from Control and PEF-Treated U. ohnoi Biomassa

parameters/sample control-biomass PEF-treated biomass p-value(n = 5, x = 0.05)

protein extracted (in mg) from 1 g of fresh biomass 4.04 ± 0.05 10.80 ± 0.24 <0.001protein yield (%) from initial biomass total protein 18.40 ± 0.21 49.15 ± 1.11 0.0007total protein yield (PEF extract + TC extract) 21.56 ± 0.6 64.09 ± 1.6 <0.001

starch content in initial biomass (% DM) 7.35 ± 0.25 7.35 ± 0.25 NAstarch content in initial DM (g) 0.20 ± 0.01 0.20 ± 0.01 NAextracted starch-rich fraction (g/2.61 ± 0.08 g Ulva DM) 0.175 ± 0.003 0.224 ± 0.01 2.43 × 10−6

extracted starch-rich fraction (% DM) 6.71 ± 0.12 8.64 ± 0.21 0.0015purity of the extracted starch-rich fraction (%) 59.40 ± 2.47 53.05 ± 0.49 0.077starch extraction yield (SEY) (%) 52.31 ± 3.21 59.54 ± 1.34 0.029

an = number of experimental repeats with PEF; NA = not applicable

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■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acssusche-meng.9b04669.

ATR-FTIR absorbance pattern and SEM image ofextracted starch-rich fractions (PDF)

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: mehanath@tauex.tau.ac.il (M.S.P).*E-mail: agolberg@tauex.tau.ac.il (A.G.).ORCIDMeghanath S. Prabhu: 0000-0003-2650-0217Yoav D. Livney: 0000-0001-8273-9631Present Address⊥Department of Biotechnology, Goa University, TaleigaoPlateau 403206, Goa.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSAll authors are thankful to Israel Ministry of Science for thefunding (grant no. 57514).

■ REFERENCES(1) Godfray, H. C. J.; Beddington, J. R.; Crute, I. R.; Haddad, L.;Lawrence, D.; Muir, J. F.; Pretty, J.; Robinson, S.; Thomas, S. M.;Toulmin, C. Food Security: The Challenge of Feeding 9 BillionPeople. Science 2010, 327, 812−818.(2) Alcazar-Alay, S. C.; Meireles, M. A. A. PhysicochemicalProperties, Modifications and Applications of Starches from DifferentBotanical Sources. Food Sci. Technol 2015, 35, 215−236.(3) Santana, A. L.; Angela, A.; Meireles, M. New Starches Are theTrend for Industry Applications: A Review. Food Public Heal. 2014, 4,229−241.(4) McWilliams, A. Biodegradable Polymers; BCC Research:Wellesley, MA, 2017; Vol. 7215.(5) Sullivan-Trainor, M. Starches/Glucose: Golbal Markets; BCCRresearch; Wellesley, MA, 2013.(6) Spiertz, J. H. J.; Ewert, F. Crop Production and Resource Use toMeet the Growing Demand for Food, Feed and Fuel: Opportunitiesand Constraints. NJAS - Wageningen J. Life Sci. 2009, 56, 281−300.(7) Gajaria, T. K.; Suthar, P.; Baghel, R. S.; Balar, N. B.; Sharnagat,P.; Mantri, V. A.; Reddy, C. R. K. Integration of Protein Extractionwith a Stream of Byproducts from Marine Macroalgae: A ModelForms the Basis for Marine Bioeconomy. Bioresour. Technol. 2017,243, 867−873.(8) Lehahn, Y.; Ingle, K. N.; Golberg, A. Global Potential ofOffshore and Shallow Waters Macroalgal Biorefineries to Provide forFood, Chemicals and Energy: Feasibility and Sustainability. Algal Res.2016, 17, 150−160.(9) Magnusson, M.; Carl, C.; Mata, L.; de Nys, R.; Paul, N. A.Seaweed Salt from Ulva: A Novel First Step in a CascadingBiorefinery Model. Algal Res. 2016, 16, 308−316.(10) Prabhu, M.; Chemodanov, A.; Gottlieb, R.; Kazir, M.; Nahor,O.; Gozin, M.; Israel, A.; Livney, Y. D.; Golberg, A. Starch from theSea: The Green Macroalga Ulva Ohnoi as a Potential Source forSustainable Starch Production in the Marine Biorefinery. Algal Res.2019, 37, 215−227.(11) Baweja, P. Cladophora - an overview. ScienceDirect. http://www.sciencedirect.com/topics/agricultural-and-biological-sciences/cladophora (accessed Nov 20, 2017).

(12) Cian, R.; Drago, S.; De Medina, F.; Martínez-Augustin, O.Proteins and Carbohydrates from Red Seaweeds: Evidence forBeneficial Effects on Gut Function and Microbiota. Mar. Drugs2015, 13, 5358−5383.(13) Farias, D. R.; Simioni, C.; Poltronieri, E.; Bouzon, Z. L.;Macleod, C. K. Fine-Tuning Transmission Electron MicroscopyMethods to Evaluate the Cellular Architecture of Ulvacean Seaweeds(Chlorophyta). Micron 2017, 96, 48−56.(14) Korzen, L.; Abelson, A.; Israel, A. Growth, Protein andCarbohydrate Contents in Ulva Rigida and Gracilaria Bursa-PastorisIntegrated with an Offshore Fish Farm. J. Appl. Phycol. 2016, 28,1835−1845.(15) Løvlie, A.; Braten, T. On the Division of Cytoplasm andChloroplast in the Multicellular Green Alga Ulva Mutabilis Føyn. Exp.Cell Res. 1968, 51, 211−220.(16) Yu, S.; Blennow, A.; Bojko, M.; Madsen, F.; Olsen, C. E.;Engelsen, S. B. Physico-Chemical Characterization of FlorideanStarch of Red Algae. Starch/Staerke 2002, 54, 66−74.(17) Korzen, L.; Pulidindi, I. N.; Israel, A.; Abelson, A.; Gedanken,A. Marine Integrated Culture of Carbohydrate Rich Ulva Rigida forEnhanced Production of Bioethanol. RSC Adv. 2015, 5, 59251−59256.(18) Yang, H.; Gozaydın, G.; Nasaruddin, R. R.; Ren, J.; Har, J. R.G.; Chen, X.; Wang, X.; Yan, N. Toward the Shell Biorefinery:Processing Crustacean Shell Waste Using Hot Water and CarbonicAcid. ACS Sustainable Chem. Eng. 2019, 7, 5532−5542.(19) Robin, A.; Kazir, M.; Sack, M.; Israel, A.; Frey, W.; Mueller, G.;Livney, Y. D.; Golberg, A. Functional Protein Concentrates Extractedfrom the Green Marine MacroalgaUlvasp., by High Voltage PulsedElectric Fields and Mechanical Press. ACS Sustainable Chem. Eng.2018, 6, 13696−13705.(20) Polikovsky, M.; Fernand, F.; Sack, M.; Frey, W.; Muller, G.;Golberg, A. Towards Marine Biorefineries: Selective ProteinsExtractions from Marine Macroalgae Ulva with Pulsed Electric Fields.Innovative Food Sci. Emerging Technol. 2016, 37, 194−200.(21) Robin, A.; Sack, M.; Israel, A.; Frey, W.; Muller, G.; Golberg, A.Deashing Macroalgae Biomass by Pulsed Electric Field Treatment.Bioresour. Technol. 2018, 255, 131−139.(22) Golberg, A.; Sack, M.; Teissie, J.; Pataro, G.; Pliquett, U.;Saulis, G.; Stefan, T.; Miklavcic, D.; Vorobiev, E.; Frey, W. Energy-Efficient Biomass Processing with Pulsed Electric Fields forBioeconomy and Sustainable Development. Biotechnol. Biofuels2016, 9, 1−22.(23) Topfl, S. Pulsed Electric Fields (PEF) for Permeabilization of CellMembranes in Food- and Bioprocessing. Applications, Process andEquipment Design and Cost Analysis; Technical University of Berlin:Berlin, Germany, 2006.(24) Loeffler, M. J. In Commercial Pulsed Power Applications inGermany, Papers of Technical Meeting on Plasma Science andTechnology; IEE: Tokyo, 2002; pp 95−100.(25) Aoshima, H. In Structure and Function of Food Engineering;Eissa, A. A., Ed.; Intech, London, 2012; pp 275−306.(26) Schirmer, M.; Jekle, M.; Becker, T. Starch Gelatinization and ItsComplexity for Analysis. Starch/Staerke 2015, 67, 30−41.(27) Wang, S.; Li, C.; Copeland, L.; Niu, Q.; Wang, S. StarchRetrogradation: A Comprehensive Review. Compr. Rev. Food Sci. FoodSaf. 2015, 14, 568−585.(28) Collado, L. S.; Mabesa, R. C.; Corke, H. Genetic Variation inthe Physical Properties of Sweet Potato Starch. J. Agric. Food Chem.1999, 47, 4195−4201.(29) GEA. Wheat Starch Processing Engineering Excellence for Custom-Fit Solutions Engineering for a Better World GEA Mechanical Equipment;GEA: Oelde, Germany, 2013.(30) Polikovsky, M.; Fernand, F.; Sack, M.; Frey, W.; Muller, G.;Golberg, A. In Silico Food Allergenic Risk Evaluation of ProteinsExtracted from Macroalgae Ulva Sp. with Pulsed Electric Fields. FoodChem. 2019, 276, 735−744.(31) Postma, P. R.; Cerezo-Chinarro, O.; Akkerman, R. J.; Olivieri,G.; Wijffels, R. H.; Brandenburg, W. A.; Eppink, M. H. Biorefinery of

ACS Sustainable Chemistry & Engineering Research Article

DOI: 10.1021/acssuschemeng.9b04669ACS Sustainable Chem. Eng. 2019, 7, 17453−17463

17462

the Macroalgae Ulva Lactuca: Extraction of Proteins and Carbohy-drates by Mild Disintegration. J. Appl. Phycol. 2018, 1281−1293.(32) Nimrod Krupnik, B. R.; Paz, G.; Douek, J.; Lewinsohn, E.;Israel, A.; Nava Carmel, F. M.; Maggs, C. A. Native, Invasive andCryptogenic Ulva Species from the Israeli Mediterranean Sea: Riskand Potential. Mediterr. Mar. Sci. 2018, 19, 132−146.(33) ISO-5984. Animal Feeding Stuffs Determination of Crude AshReference. Kampala Uganda. ISO: Geneva, Switzerland, 2009.(34) Janositz, A.; Semrau, J.; Knorr, D. Impact of PEF treatment onquality parameters of white asparagus (Asparagus officinalis L.). Innov.Food Sci. Emerg. Technol. 2011, 12, 269−274.(35) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J.Lowry’s handbook of right-to-know emergency planning. J. Biol.Chem. 1992, 193, 265−275.(36) Mhatre, A.; Gore, S.; Mhatre, A.; Trivedi, N.; Sharma, M.;Pandit, R.; Anil, A.; Lali, A. Effect of multiple product extractions onbio-methane potential of marine macrophytic green alga Ulva lactuca.Renewable Energy 2019, DOI: 10.1016/j.renene.2018.08.012.(37) Chemodanov, A.; Jinjikhashvily, G.; Habiby, O.; Liberzon, A.;Israel, A.; Yakhini, Z.; Golberg, A. Net Primary Productivity, BiofuelProduction and CO2 Emissions Reduction Potential of Ulva Sp.(Chlorophyta) Biomass in a Coastal Area of the EasternMediterranean. Energy Convers. Manage. 2017, 148, 1497−1507.(38) Wiencke, C.; Bischof, K. Seaweed Biology: Novel Insights IntoEcophysiology. Ecology And Utilization; Springer: Berlin, Germany,2012.(39) van den Burg, S.; Stuiver, M.; Veenstra, F.; Bikker, P.; LopezContreras, A.; Palstra, A.; Broeze, J.; Jansen, H.; Jak, R.; Gerritsen, A.;et al. A Triple P Review of the Feasibility of Sustainable Offshore SeaweedProduction in the North Sea. 2013, Research report number 442638,ISBN 9789086156528−108.(40) Gao, G.; Clare, A. S.; Rose, C.; Caldwell, G. S. Eutrophicationand Warming-Driven Green Tides (Ulva Rigida) Are Predicted toIncrease under Future Climate Change Scenarios. Mar. Pollut. Bull.2017, 114, 439−447.(41) Glasson, C. R. K.; Sims, I. M.; Carnachan, S. M.; de Nys, R.;Magnusson, M. A Cascading Biorefinery Process Targeting SulfatedPolysaccharides (Ulvan) from Ulva Ohnoi. Algal Res. 2017, 27, 383−391.(42) Chemodanov, A.; Robin, A.; Jinjikhashvily, G.; Yitzhak, D.;Liberzon, A.; Israel, A.; Golberg, A. Feasibility Study ofUlvasp.(Chlorophyta) Intensive Cultivation in a Coastal Area of the EasternMediterranean Sea. Biofuels, Bioprod. Biorefin. 2019, 1−14.(43) Chemodanov, A.; Jinjikhashvily, G.; Habiby, O.; Liberzon, A.;Israel, A.; Yakhini, Z.; Golberg, A. Net Primary Productivity, BiofuelProduction and CO 2 emissions Reduction Potential of Ulva Sp.(Chlorophyta) Biomass in a Coastal Area of the EasternMediterranean. Energy Convers. Manage. 2017, 148, 1497−1507.(44) Chemodanov, A.; Robin, A.; Golberg, A. Design of MarineMacroalgae Photobioreactor Integrated into Building to SupportSeagriculture for Biorefinery and Bioeconomy. Bioresour. Technol.2017, 241, 1084−1093.(45) Ghosh, S.; Gillis, A.; Sheviryov, J.; Levkov, K.; Golberg, A.Towards Waste Meat Biorefinery: Extraction of Proteins from WasteChicken Meat with Non-Thermal Pulsed Electric Fields andMechanical Pressing. J. Cleaner Prod. 2019, 208, 220−231.(46) Golberg, A.; Laufer, S.; Rabinowitch, H. D.; Rubinsky, B. InVivo Non-Thermal Irreversible Electroporation Impact on Rat LiverGalvanic Apparent Internal Resistance. Phys. Med. Biol. 2011, 56,951−963.(47) Janositz, A.; Noack, A.; Knorr, D. Pulsed Electric fields andTheir Impact on the Diffusion Characteristics of Potato Slices. FoodSci. Technol. 2011, 44, 1939−1945.(48) Wells, M. L.; Potin, P.; Craigie, J. S.; Raven, J. A.; Merchant, S.S.; Helliwell, K. E.; Smith, A. G.; Camire, M. E.; Brawley, S. H. Algaeas Nutritional and Functional Food Sources: Revisiting OurUnderstanding. J. Appl. Phycol. 2017, 29, 949−982.

(49) Fitzgerald, C.; Gallagher, E.; Tasdemir, D.; Hayes, M. HeartHealth Peptides from Macroalgae and Their Potential Use inFunctional Foods. J. Agric. Food Chem. 2011, 59, 6829−6836.(50) Vanthoor-Koopmans, M.; Wijffels, R. H.; Barbosa, M. J.;Eppink, M. H. M. Biorefinery of Microalgae for Food and Fuel.Bioresour. Technol. 2013, 135, 142−149.(51) Vega-mercado, H.; Gongora-Nieto, M. M.; Barbosa-Canovas,G. V.; Swanson, B. G. Pulse Electric Fields in Food Preservation.Handb. food Preserv. 2007, 783−814.(52) Han, Z.; Zeng, X. A.; Zhang, B. S.; Yu, S. J. Effects of PulsedElectric Fields (PEF) Treatment on the Properties of Corn Starch. J.Food Eng. 2009, 93, 318−323.(53) Han, Z.; Zeng, X. A.; Yu, S. J.; Zhang, B. S.; Chen, X. D. Effectsof Pulsed Electric Fields (PEF) Treatment on PhysicochemicalProperties of Potato Starch. Innovative Food Sci. Emerging Technol.2009, 10, 481−485.(54) Vorobiev, E.; Lebovka, N. Pulse Electric Field-AssistedExtraction. In Enhancing Extraction Processes in the Food Industry;Vorobiev, E., Lebovka, N., Chemat, F., Eds.; Taylor & Francis:London, 2011; pp 25−84.(55) Kazir, M.; Abuhassira, Y.; Robin, A.; Nahor, O.; Luo, J.; Israel,A.; Golberg, A.; Livney, Y. D. Extraction of Proteins from Two MarineMacroalgae, Ulva Sp. and Gracilaria Sp., for Food Application, andEvaluating Digestibility, Amino Acid Composition and AntioxidantProperties of the Protein Concentrates. Food Hydrocolloids 2019, 87,194.(56) Bodenes, P. Study of the Application of Pulsed Electric Fields (PEF ) on Microalgae for the Extraction of Neutral Lipids; Universite Paris-Saclay: Saint-Aubin, France, 2017.(57) Bleakley, S.; Hayes, M. Algal Proteins: Extraction, Application,and Challenges Concerning Production. Foods 2017, 6, 1−34.(58) Hong, J.; Zeng, X. A.; Buckow, R.; Han, Z.; Wang, M. S.Nanostructure, Morphology and Functionality of Cassava Starch afterPulsed Electric Fields Assisted Acetylation. Food Hydrocolloids 2016,54, 139−150.(59) Hong, J.; Zeng, X. A.; Han, Z.; Brennan, C. S. Effect of PulsedElectric Fields Treatment on the Nanostructure of Esterified PotatoStarch and Their Potential Glycemic Digestibility. Innov. Food Sci.Emerg. Technol. 2018, 45, 438−446.

ACS Sustainable Chemistry & Engineering Research Article

DOI: 10.1021/acssuschemeng.9b04669ACS Sustainable Chem. Eng. 2019, 7, 17453−17463

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