microorganisms

Review
Separation, Characterization, and Handling of
Microalgae by Dielectrophoresis
V inzenz Abt 1 , Fabian Gringel 2 , Arum Han 3 , Peter Neubauer 1 and Mario Birkholz 2 , *
1
Chair of Biopr ocess Engineering, Department of Biotechnology , T echnische Universität Berlin, Ackerstr . 76,
ACK24, 13355 Berlin, Germany; v [email protected] (V .A.); peter [email protected] (P .N.)
2 IHP–Leibniz-Institut für innovative Mikroelektr onik, Im T echnologiepark 25,
15236 Frankfurt (Oder), Germany; [email protected]
3 Department of Electrical and Computer Engineering, T exas A&M University ,
College Station, TX 77843, USA; [email protected]
* Correspondence: birkholz@ihp-micr oelectr onics.com
Received: 9 March 2020; Accepted: 5 April 2020; Published: 9 April 2020
       
  

Abstract:
Micr oalgae biotechnology has a high potential for sustainable bioproduction of diverse
high-value biomolecules. Some of the main bottlenecks in cell-based biopr oduction, and mor e
specifically in micr oalgae-based biopr oduction, ar e due to insu ffi cient methods for rapid and e ffi cient
cell characterization, which contributes to having only a few industrially established microalgal
species in commer cial use. Dielectr ophoresis-based micr ofluidic devices have been long established as
pr omising tools for label-free handling, characterization, and separation of br oad ranges of cells. The
technique is based on di ff er ences in dielectric properties and sizes, which r esults in di ff erent degr ees
of cell movement under an applied inhomogeneous electrical field. The method has also earned
inter est for separating microalgae based on their intrinsic pr operties, since their dielectric properties
may significantly change during biopr oduction, in particular for lipid-pr oducing species. Here, we
pr ovide a comprehensive r eview of dielectrophor esis-based microfluidic devices that ar e used for
handling, characterization, and separation of microalgae. Additionally , we pr ovide a perspective on
r elated areas of r esearch in cell-based biopr oduction that can benefit from dielectr ophoresis-based
micr odevices. This work provides key information that will be useful for micr oalgae resear chers to
decide whether dielectr ophoresis and which method is most suitable for their particular application.
Keywords: micr oalgae; dielectrophor esis; cell sorting; microfluidics
1. Recent Advances in Microalgae Research and Microalgae-Based Bioprocess Development
Micr oalgae are photosynthetic micr oorganisms that can utilize sunlight and CO
2
to pr oduce
diverse ranges of biopr oducts, including various high-value lipids and pigments to name a few [
1
–
3
].
Several applications, especially sustainable biofuel production fr om microalgae, ar e consider ed
essential in the United Nation’s (UN) Sustainable Development Goals and for the European Union’s
(EU) bioeconomy strategy [
4
,
5
]. While the curr ent generation of microalgae-based biofuels is not
economically competitive yet, next-generation fuels coming from genetically modified micr oalgae
with much higher yields and much higher quality lipids, are emer ging thanks to the advances made in
biotechnology and synthetic biology . An example is Nanochloropsis gaditana , which was r ecently shown
to double its pr oduction rate by simply decreasing the expr ession of a single regulator [
6
]. Besides
biofuel, micr oalgae are inter esting cell factories in many other fields [
7
], such as the cosmetic industry ,
as well as for pharmaceutical applications wher e micr oalgae are sour ces of large-scale pr oduction of
anti-inflammatory , anti-micr obial, anti-viral, and anti-tumor molecules [
8
]. Additionally , the use of
micr oalgae as either animal feedstock or as a nutritional source for the gr owing global population is
Microor ganisms 2020 , 8 , 540; doi:10.3390 / microor ganisms8040540 www .mdpi.com / journal / micr oorganisms

Microor ganisms 2020 , 8 , 540 2 of 19
pr omising [
9
] (Figur e 1 ). For example, microalgae ar e an important source of polyunsaturated fatty acid
(PUF A). Currently , docosahexaenoic acid (DHA) fr om microalgae is contained in baby food, but also
DHA-pr oducing microalgae can be dir ectly applied as fish feed in aquaculture if e ff ective pr oduction
pr ocesses at a larger scale can be established [
7
,
10
,
11
]. Many commer cialization e ff orts in this field are
also ongoing. For example, r ecently a joint venture of Evonik and DSM was established (V eramaris
™
),
aiming at the development of lar ge-scale processes of DHA with the micr oalgae Schizochytrium
sp. to pr ovide 15% of the global need of DHA. In parallel, discovering previously unknown or
unidentified micr oalgae fr om nature continues [
12
]. Ongoing recent biopr ospecting e ff orts [
13
,
14
]
show that ther e us still a large number of unexplor ed micr oalgae that we are not significantly awar e of,
leaving the possibility of further biopr ospecting e ff orts to uncover microalgal strains with inter esting
biopr oduction capabilities.
Microorganisms 2 020 , 8 , x FOR PEER REVIEW 2 of 19
(PU F A) . Curr ent l y, docos a hexaeno i c ac i d (DHA ) fro m microa lga e is cont ain ed in baby foo d , but also
DHA-prod uc ing m i cro a lg a e can be d i re ct ly applie d a s f i sh fe ed in aq ua cu lt ure i f e f fect ive pr oduct i on
processes at a lar g er s c a l e c a n be est a bli s hed [ 7 , 1 0 , 1 1 ] . Many comme rcia li z a t i on e f fort s in t h i s f i e ld are
also on going . For ex ample, recently a joint ventur e of Evonik and D S M was e s tab lish ed (Ver amaris™),
aim i ng at the developmen t of lar g e-scale processe s o f DHA w i th the microalg ae Schizochytri u m sp. to
provide 15% of the glob al need of DH A . In par a llel, discover ing p r eviously un known or un identified
microa lg ae fr om nat u re c o nt inues [ 1 2] . Ongoing rece nt bioprospec ting e ffo rts [13, 14] show that there u s
still a larg e number of unexplored m i croalgae that we a r e not sign if icant l y awa r e o f , lea v ing t h e
possibi lit y o f furt her biopr o spect i ng ef fo rt s t o uncover micro a l g a l st rain s w i t h int e rest ing biopr o duct ion
capabi lit ies.

Figure 1. Re se arch areas of publications a ssociated w i th the term “microalgae“. Bub b les show the
nu m b er of results of com b ined searches “m i c roalg a e + indi ca ted term” base d on pu blicatio ns of the past
five years [Sou rce: web o fknowledge.com].
Despit e t h ese advance s , m a ny ch al leng es st il l rem a i n [1 5– 1 7 ] . Ide n t ifyin g an d developin g st rain s
with higher productivity , improved understand i ng of their be havior s und er diver s e c u ltiv ation
condi t i o ns, and better i n sights i n to the heterogeneous population are all some o f the ke y adv a nces that
h a v e t o b e m a d e i n t h e u p s t r e a m p r o c e s s o f m i c r oa lga e b i otechnol o gy a n d b i oprocessi ng [1 6,18– 22].
Yet, these dev elopment processes ar e oft e n time-consu ming and l a bor-int e ns ive, posing as a si gnif ic ant
bottleneck. P a rt of these c h allenge s is d u e to the lac k of r a pi d and ef fic ient in st rument s and met h ods
t h at have bee n lim it ing rap i d advanc ement s in t h i s fi eld [1 2, 2 3 ] .
Development of var i ou s microf lu idic devices ha s revolut i on i z e d t h e are a of ce ll an a l y s is ,
separ a t i on, a n d c u lt iv at io n in m a ny f i e l ds of biot ec h n ology in the past two dec ades d u e to t h e sing le-
cell reso lut i o n and h i gh-t hroughput c a pabi lit ies o f such syst em s [ 2 4 – 2 6 ] . Th e appl icat ion of t h es e
powerfu l t e c hnologi es h a s recent ly be en misse d in vario u s m i c r oal g a e biot e c hnology fie l d s [ 2 7 , 2 8 ] .
Genera l a pplica t i o ns a n d impa cts of microf lu i d i c de vices for mic r oalgae have been present ed [28]. In
t h is rev i ew paper, we wil l spec if ic a lly foc u s on die l ect r oph o resi s (DEP )- based cha r ac t e riz a t i on ,
ma ni pula ti on, a n d sep a ra tion techni ques i n mi croa lga e bi otechnology a ppl i c a t ions.
2. DEP T e chn o lo gy f o r Cell Po pulatio n Analys is
Flow cyt o me t r y, in gene r a l , is t h e cu r r ent gold st a n dard for m a ny hi gh-t hr oughput s i ng le-ce ll
ana l y s is appl icat ion s [ 2 9] . Fl uorescen c e -ba s ed me t h ods s u ch a s f l uore scenc e -act ivat ed c e ll sort ing
(FAC S) are widely utilized to determ i ne the ch aracteri stics o f c ells b a sed on fluor escent s t ainin g o f
part icu l a r t a r g et molec u l e s , s u ch a s int r a c ell u l a r l i pid s in mic r oa lg a e [ 3 0, 31 ]. Be si des fl uore sce n ce, d i rect
imag ing o f c ells to deter mine their p h enotypes an d/ or char acteristic s is also becoming in creas i ng ly
popul a r tha n ks to the a d v a nces i n i m a g i n g fl ow cytometry [ 3 2 , 33 ]. Al though extremel y powerf ul a n d
versat i l e, s e vera l limit at io ns ex ist [1 8 ] . Most of t h e s e ana l y s es r e ly on labe li ng ce lls w i t h var i ou s
marker s, wh i c h req u ir e s a mple prep a r at ion t h at is t i me-cons u ming and m a y ch ange t h e nat u r a l
char acterist ic s of ce lls. Ad dition ally, flo w cytome try-ba sed methods come wi t h rela ti vel y expensi v e
inst rument s , which f u rt her lim it s t h ei r a pplicat i o ns.
In DEP-ba sed cell m a nip u l a t i on and sor t ing, t h e movement of pol a ri z a ble p a rt i c les, such as cell s ,
in an inhomo geneous e l ect r ic field is de pendent on t h eir intrin sic dielectric pro p erties in co mparison to

Figure 1.
Research ar eas of publications associated with the term “microalgae“. Bubbles show the
number of results of combined sear ches “microalgae + indicated term” based on publications of the
past five years [Source: webofknowledge.com ].
Despite these advances, many challenges still remain [
15
–
17
]. Identifying and developing
strains with higher pr oductivity , improved understanding of their behaviors under diverse cultivation
conditions, and better insights into the heter ogeneous population are all some of the key advances that
have to be made in the upstr eam process of micr oalgae biotechnology and bioprocessing [
16
,
18
–
22
].
Y et, these development processes ar e often time-consuming and labor-intensive, posing as a significant
bottleneck. Part of these challenges is due to the lack of rapid and e ffi cient instruments and methods
that have been limiting rapid advancements in this field [ 12 , 23 ].
Development of various micr ofluidic devices has revolutionized the ar ea of cell analysis, separation,
and cultivation in many fields of biotechnology in the past two decades due to the single-cell
r esolution and high-thr oughput capabilities of such systems [
24
–
26
]. The application of these powerful
technologies has r ecently been missed in various microalgae biotechnology fields [
27
,
28
]. General
applications and impacts of micr ofluidic devices for micr oalgae have been pr esented [
28
]. In this review
paper , we will specifically focus on dielectrophor esis (DEP)-based characterization, manipulation, and
separation techniques in micr oalgae biotechnology applications.
2. DEP T echnology for Cell Population Analysis
Flow cytometry , in general, is the curr ent gold standard for many high-thr oughput single-cell
analysis applications [
29
]. Fluorescence-based methods such as fluor escence-activated cell sorting
(F ACS) are widely utilized to determine the characteristics of cells based on fluor escent staining of
particular tar get molecules, such as intracellular lipids in microalgae [
30
,
31
]. Besides fluor escence, direct
imaging of cells to determine their phenotypes and / or characteristics is also becoming incr easingly
popular thanks to the advances in imaging flow cytometry [
32
,
33
]. Although extremely powerful
and versatile, several limitations exist [
18
]. Most of these analyses rely on labeling cells with various
markers, which requir e sample preparation that is time-consuming and may change the natural

Microor ganisms 2020 , 8 , 540 3 of 19
characteristics of cells. Additionally , flow cytometry-based methods come with relatively expensive
instruments, which further limits their applications.
In DEP-based cell manipulation and sorting, the movement of polarizable particles, such as cells,
in an inhomogeneous electric field is dependent on their intrinsic dielectric pr operties in comparison
to the dielectric pr operties of the surrounding liquid. Thus, DEP is a noninvasive and label-free cell
manipulation method. In contrast to electr ophoresis, wher e only charged particles can be manipulated,
DEP enables the interaction with unchar ged particles through the induction of a dipole moment p
under an inhomogeneous electric field. The included dipole interacts with the electrical field gradient
∇
E [
34
,
35
], resulting in the dielectr ophor etic for ce
F DEP
that depends on the polarizability of the
particle (index p ) within the medium ( m ), where the latter can be expr essed by a function of their
complex dielectric constants
ε ∗
p ( ω )
and
ε ∗
m ( ω )
. These functions depend on the r eal part of permittivity
ε 0
and electrical conductivity
σ
, and ar e, therefor e, dependent on the frequency f or angular fr equency
ω =
2
π f
, r espectively , by which the applied voltage is oscillating, since
ε ∗ = ε 0 + i σ / ω
. Accor dingly ,
F DEP exerted upon a spherical particle in an AC field is given by:
Microorganisms 2 020 , 8 , x FOR PEER REVIEW 3 of 19
the d i elec tr ic properties o f the sur r ou nding li qu id. Thus, DE P i s a non i nva s ive and l a bel -fre e cel l
ma nipula tion met h od. In cont rast to e l ec trophoresis, where only c h arge d partic les c a n be manipulated,
DEP en abl e s t h e in ter a c t ion wi th unch arg ed p a rt icl e s t h rough th e in duct ion of a d i pole moment p un der
an inhomoge neous e l ectric field. The include d dipo le in ter a ct s with the elec tric a l fie ld gr adi e nt ∇ E
[3 4, 3 5 ], res u l t ing in th e d i e l ect r ophoret i c force F
DEP
that depends on the po la r i zab il it y of th e par t ic le
(index p ) w i t h in th e med i um ( m ), whe r e the latter can be expr e ssed by a function o f the i r comple x
dielectr ic con s tants 𝜀 
∗ (𝜔 ) and 𝜀 
∗ (𝜔 ) . These functions depend on the re al part of perm ittivity 𝜀 󰆒 an d
elec tric al con d uctivity 𝜎 , an d are , there f o r e, depend e n t on the frequency f or an gular fre q uen c y 𝜔=
2𝜋𝑓 , re spect i vel y , by which th e app lied vol t a g e is osc i l l a t ing , since 𝜀 ∗ =𝜀 󰆒 +𝑖 𝜎 / 𝜔 . Accordin gly, F
DEP

exerted upon a sphe ric a l pa rtic le in an AC fie ld is give n by:

𝐹  = 
 𝑑 
 ∙𝜀 
󰆒 𝑅𝑒  

∗


∗


∗


∗
∙∇  𝐸
󰇍



 
, (1)

where d
p
stan ds for the p a r t ic le diame t er an d Re in dic a te s the re a l p a rt o f the co m p lex term i n side the
parenth e sis. From each of the thre e fa ct ors, a specif i c fea t u r e of F
DEP
can be der ive d :
1. The par t ic le d i ame t er d is t h e sin g le mos t impor t ant c o ntrib u tor to F
DEP
.
2. F
DEP
depen ds on 𝜀 and 𝜎 of bot h t h e pa rt ic l e a n d me dia ( i n de x p an d m ). C h ang e s of ang u l a r
freq uenc y 𝜔 may either c a use a positive or neg a tive DEP fo rc e, d e pendin g on wh ether
𝑅𝑒 𝜀 
∗ −𝜀 
∗ 0 o r 𝑅 𝑒  𝜀 
∗ −𝜀 
∗ <0 holds.
3. The loc a l e l ec tric a l fie ld de pends on th e applie d vo lt a g e V as well as the e l ec tro de de sign an d
geometr y . Th e layout o f the flow ch an nel and the elec trodes th us o ffe r various d e gree s of
freedom for o p timizing the DEP force.
Equation (1) represen ts th e simp lest ap proach for m o delin g the DEP e ffec t o n a homo gen e ous
part icle . A m o re adv a nced approach is ut il iz ing a co re-she ll mod e l (in st ead o f c o nsider ing th e part icl e
as a homogen e ous spher i ca l objec t ) , wh ic h is va lu abl e i f ana l y z in g in divid u a l c e l l u l ar componen ts such
as c e l l m e m b ranes an d va riou s in tr acel lul a r com p on ents . Oth e r e x tens ions o f t h e b a s i c form ul a g i v e n
above inc l ud e the conside ration o f the aspher icity o f cell s and the i r mode ling c e ll s a s e l l i pso i ds [3 6 ] .
More det a ils reg a rd ing complex c a l c ul at ions on the inte rac t ion of e l l i p tic par t ic le s w i th in
inhomogeneo u s e l ec tric fields an d simulation thereo f are given in p u blic a t ion s fo cused on DE P theo ry
[3 7, 3 8 ] (F igu r e 2 ) .

Figure 2. Simulation of diele c t r ophoresis (DEP) cel l separati on in a mi crofl u idic flow . ( a ) b e f o re a n d ( b )
after microalg ae Chlamydo mona s reinha r dtii have passed a defl ector ele c t r ode conf igura t ion (top and
bottom ele c tro d e).
Because o f th e possibility o f manip u latin g cells bas ed on their intrin sic dielec tr ic propertie s an d the
ease of int e g r at ing in to microf lu idic s forma t , DE P ha s been widel y appli ed a s a l a be l- free ce ll
manipulation and sep a ration method. T h orough rev i ews on the c u rrent state an d poten t ial o f DEP ar e
ava i l a ble [ 3 9– 41 ] and prog ress in in tegr ate d m i crof lu idic DEP dev ices for l ife s c ience appl ic at ions , i n
genera l, h a ve al so been pre v ious ly revie w ed [ 4 2– 4 4 ] . Other review artic l es consider the po ten t ial o f D E P
for nex t -g ene r at ion ce ll sor t ing [ 4 5 ] . A g e nera l overvi ew of d i f f ere n t micro f l u id i c separ a tion t e chniq u es
1 2 3

wher e d
p
stands for the particle diameter and Re indicates the r eal part of the complex term inside the
par enthesis. From each of the thr ee factors, a specific feature of F DEP can be derived:
1. The particle diameter d is the single most important contributor to F DEP .
2. F DEP
depends on
ε
and
σ
of both the particle and media (index p and m ). Changes of
angular fr equency
ω
may either cause a positive or negative DEP for ce, depending on whether
Re  ε ∗
p − ε ∗
m  > 0 or Re  ε ∗
p − ε ∗
m  < 0 holds.
3.
The local electrical field depends on the applied voltage V as well as the electrode design and
geometry . The layout of the flow channel and the electr odes thus o ff er various degrees of fr eedom
for optimizing the DEP for ce.
Equation (1) r epresents the simplest appr oach for modeling the DEP e ff ect on a homogeneous
particle. A more advanced approach is utilizing a cor e-shell model (instead of considering the particle as
a homogeneous spherical object), which is valuable if analyzing individual cellular components such as
cell membranes and various intracellular components. Other extensions of the basic formula given above
include the consideration of the asphericity of cells and their modeling cells as ellipsoids [
36
]. Mor e
details r egarding complex calculations on the interaction of elliptic particles within inhomogeneous
electric fields and simulation ther eof are given in publications focused on DEP theory [
37
,
38
] (Figur e 2 ).
Because of the possibility of manipulating cells based on their intrinsic dielectric properties and
the ease of integrating into microfluidics format, DEP has been widely applied as a label-free cell
manipulation and separation method. Thorough r eviews on the current state and potential of DEP
ar e available [
39
–
41
] and pr ogress in integrated micr ofluidic DEP devices for life science applications,
in general, have also been pr eviously reviewed [
42
–
44
]. Other review articles consider the potential
of DEP for next-generation cell sorting [
45
]. A general overview of di ff erent micr ofluidic separation
techniques applicable to micr oorganisms is pr ovided in [
46
], especially focusing on bacteria and yeast
cells. Here, we r eview DEP applications for micr oalgae r esearch and development, including a critical
analysis of the advantages and disadvantages of the various DEP micr ofluidic configurations.

Microor ganisms 2020 , 8 , 540 4 of 19
Microorganisms 2 020 , 8 , x FOR PEER REVIEW 3 of 19
the dielectr ic properties o f the sur r ounding liqu i d . Thu s , DEP is a noni nvasi v e a n d la bel- f r ee cell
ma ni pula ti on method. In contra st to el ectrophoresi s, where only c h arge d partic les c a n be manipulated,
DEP en abl e s t h e int e r a ct ion wit h unch arg ed p a rt icl e s t h rough t h e in duct ion of a d i pole moment p un de r
an inhomoge neous e l ectric field. The include d dipo le int e r a ct s wit h t h e elec t r ica l fie ld gr adi e nt ∇ E
[3 4, 3 5 ], res u lt ing in t h e d i e l ect r ophoret i c force F
DEP
t h at depends on the pola r i zab il it y of t h e part ic le
(index p ) w i t h in t h e med i um ( m ), whe r e the latter can be expr e ssed by a function of the i r complex
dielectric con s tants  
∗ ( ) and  
∗ ( ) . These functions depend on the real part of perm ittivity  󰆒 an d
el ectri c a l conducti vi ty  , an d are , there f o r e, depend en t on the frequency f or an gular fre q uen c y =
2 , re spect i vel y , by which t h e app lied vol t age is osc i l l a t ing, since  ∗ = 󰆒 +  /  . Accordin gly, F
DEP

exerted upon a sphe ric a l particle in an AC fie ld is give n by:
  = 
  
 ∙ 
󰆒   

∗


∗


∗


∗
∙   
󰇍



 
, (1 )

where d
p
stan ds for the p a r t icle diameter an d Re i n di ca tes the real pa rt of the compl e x term insi de the
parenthesis. From each of t h e thr ee facto r s, a specific feature of F
DEP
can be der ive d :
1. The partic le d i ameter d i s the si ngl e most i m porta n t contri butor to F
DEP
.
2. F
DEP
depen ds on  and  of both the pa r t ic l e a n d me dia ( i n de x p an d m ). C h ang e s of ang u l a r
freq uenc y  may either c a use a posit i ve or neg a tive DEP fo rc e, d e pendin g on wh ether
  
∗ − 
∗ 0  o r      
∗ − 
∗ <0 holds.
3. The loc a l e l ec t r ica l fie ld de pends on t h e applie d vo lt a g e V as well as the e l ectro de de sign an d
geometry. Th e layout o f t h e flow ch an nel and the electrodes thus o ffe r various d e gree s of
freedom for o p timizing the DEP force.
Equation (1) represents th e si mp lest ap proach for m o delin g t h e DEP e ffect o n a homo gen e ous
part icle . A m o re adv a nced approach is ut il iz ing a co re-she ll mod e l (in st ead o f c o nsider ing t h e part icl e
as a homogen e ous spher i ca l object ) , wh ic h is va lu abl e i f ana l y z in g in divid u a l c e l l u l ar componen t s such
as c e l l m e m b ranes an d va riou s int r acel lul a r com p on ents. Other extensions o f t h e basic form ula given
above inc l ud e the conside r ation o f the aspher icity o f cell s and t h e i r mode ling c e ll s a s e l l i pso i ds [3 6 ] .
More det a ils reg a rd ing complex c a l c ul at ions on t h e int e rac t ion of e l l i p t i c part ic le s w i t h in
inhomogeneo u s e l ectric fields an d simulation thereo f are given in p u blic at ion s fo cused on DE P t h eory
[3 7, 3 8 ] (F igu r e 2 ) .

Figure 2. Simulation of diele c t r ophoresis (DEP) cel l separati on in a mi crofl u idic flow . ( a ) b e f o re a n d ( b )
after microalg ae Chlamydo mona s reinha r dtii have passed a defl ector ele c t r ode conf igura t ion (top and
bottom ele c tro d e).
Because o f the possibility o f manip u latin g cells bas ed on their intrin sic dielectr ic properties an d the
ease of int e g r at ing int o microf lu idic s format , DE P ha s been widel y appli ed a s a l a be l- free ce ll
ma ni pula ti on a n d sep a ra tion method . Thorough rev i ews on the current st at e an d p o t e nt ial o f DEP ar e
ava i l a ble [ 3 9– 41 ] and prog ress in int e gr at ed m i crof lu idic DEP dev ices for l ife s c ience appl ic at ions , i n
genera l, h a ve al so been pre v ious ly revie w ed [ 4 2– 4 4 ] . Other review artic l es consider the potent ial o f D E P
for next -g ene r at ion ce ll sor t ing [ 4 5 ] . A g e nera l overvi ew of d i f f ere n t microfl u id i c separ a t i on t e chniq u es
applic able t o microorg a ni s m s i s provi d e d in [ 4 6], esp e cia l l y focu si ng on b a ct eri a and ye ast c e ll s. Here ,
1 2 3

Figure 2.
Simulation of dielectrophor esis (DEP) cell separation in a microfluidic flow . (
a
) before and (
b
)
after microalgae Chlamydomonas r einhardtii have passed a deflector electrode configuration (top and
bottom electrode).
3. Overview of DEP Microfluidic Systems for Microalgae Research
Despite a significant amount of work in applying DEP for various micr ofluidic cell manipulation
and separation applications, especially for mammalian cells and bacterial cells, relatively little work
has been published in applying the technology for micr oalgae resear ch [
47
]. Early works started by
Pohl et al.
[
48
,
49
] involved not only describing the basic DEP formula that governs cell movement
under the DEP force but also towar ds attempting continuous separation of Chlorella vulgaris and
understanding the dependency of cell movement on media salinity . Some of the earlier works focused
on dielectric spectra analysis of cells [
50
–
52
], as well as basic system development and accompanying
electr ode designs [ 53 , 54 ].
One of the most interesting and unique characteristics of micr oalgae compared to other cells that
makes them inter esting for DEP-based cell manipulation, is that they can accumulate large amounts of
intracellular lipid (Figur e 3 ) [
55
]. As lipid has rather di ff er ent dielectric pr operties than typical cytosol,
the DEP for ce generated can be vastly di ff er ent, making it ideal for applying DEP-based separation
and characterization techniques. Additionally , the size of microalgae can also be an indicator of their
characteristics or changes in physiology , which o ff ers another excellent opportunity for DEP-based
separation. For this review , DEP microfluidic devices for micr oalgae applications will be critically
analyzed fr om two di ff er ent aspects; first, based on the micr ofluidic structures being utilized, and
second, based on the applications of such DEP-based micr ofluidic devices.
Microorganism s 2 020 , 8 , x FOR PEER REVIEW 4 of 19
we review D E P applic at io ns for mic r oa lga e re se arch and develop m ent , incl ud i n g a cr it ica l a n aly s i s o f
t h e adv a nt ag es and d i s a dv ant a ge s o f t h e v a r i ous DEP m i crof lu idic c o nfig ur at ions .
3. Ov erv i ew of DE P M i cr oflu idic Sys t em s for M i cr oal g ae Res e a r ch
Despi t e a si gni f ica n t a m ou nt of work in a ppl yi ng DEP f o r vari ous mi crof lu i d i c cell ma ni pula ti on
and sep a rat i o n app lic at ion s , especi a lly f o r mamm al i a n cel l s and ba ct eria l c e l l s, r e lat i ve ly lit t l e work h a s
been pub lish e d in appl yin g t h e t e chnol o gy for mic r o a lg ae re se arc h [ 4 7] . Ear l y works st a r t e d b y Poh l et
al . [ 4 8 , 4 9 ] inv o lved not onl y descr i bing t h e basic D E P formu l a t h at governs ce ll movement under t h e
DEP f o rce b u t a l so towards a ttempti ng conti n uous sepa ra t i on of Ch lorella v u l g aris and un de rst a nd ing
the dependency of c e ll m o vement on media sa linit y . Some of the ea rl ie r work s f o cu se d on d i el e c tr i c
sp ect r a an aly s is of cel l s [ 5 0– 5 2 ] , as w e l l as b a s i c s y s t em dev e lop m ent and ac com p anyin g elect r ode
designs [53, 54].
One of t h e m o st int e rest in g and un i q ue charact e r i st ic s of m i cro a l g ae com p are d t o ot her cells t h at
makes t h em i n t e rest ing for DEP - ba sed c e ll man i pu lat i on, i s tha t they ca n a c cu mu la t e l a r g e a m o u n t s of
i n tra c el lu la r li pi d ( F i g u r e 3) [ 5 5 ] . As li pid has ra ther different die l ectric propert i es than typic a l cytoso l,
the DEP force generated c a n be vastly different, m a ki ng it ide a l fo r apply i ng DE P-based sep a r a tion an d
char act e ri zat i o n t e chni que s . Addit i ona l ly, t h e s i ze o f m i c r oa lg ae can a l so b e an ind i c a t o r of t h eir
char act e rist ic s or chang e s in physio log y , which of fe rs another ex cellent oppor t unity for D E P-b a sed
separ a t i on. F o r t h is rev i e w , DEP mic r ofl u id ic devi ces for m i cro a lg ae applic a t ions w i l l be crit ic a lly
analy z ed fro m two differ ent aspects; first, b a sed o n t h e microf l u id ic st ru ct u r es bein g ut i l i ze d, and
second, b a se d on the application s o f such DEP-b a se d micro f luid ic device s.

Figure 3. Lig h t m i croscopi c im ag es of the m i c r oalg a Crypthecodinium cohnii.
A sum m ary o f t h e an al yz e d p u b lic at ion s is li st ed in Tab l e 1. The t a b l e cont ain s i n form at ion a b out
t h e microa lg ae spec ies ut ili ze d, dev i ce desi gn, an d applic at ion are a s. Two s e t s of in form at ion are
provi d ed i n deta il : the fi rst one f o cuses on the device and DEP electrode con f ig ur ation, important
a s pects when developing DEP-b a sed cel l ma ni pul a tion, sepa ra ti on, a n d cha r a c teri za ti on devi ces. T h e
s e c o n d o n e p r o v i d e s d e t a i l s o f e x p e r i m e n t a l p a r a m e t e r s s u c h a s f l o w v e l o c i t y , v o l t a g e s a n d f r e q u e n c i e s
ap p lie d, cel l p a ram e t e r s s u ch as ce ll s i ze and d i el ectri c properties. T a ken to gether, these t w o sets of
inform at ion c a n serve a s a qu ick loo k up t a ble t h at w i l l be u s ef ul in desi gning sp ecif ic D E P de vices for
desir e d applications of int e rest. Notatio n and un its are given in Table 2.

Figure 3. Light microscopic images of the micr oalga Crypthecodinium cohnii .

Microor ganisms 2020 , 8 , 540 5 of 19
A summary of the analyzed publications is listed in T able 1 . The table contains information about
the micr oalgae species utilized, device design, and application ar eas. T wo sets of information are
pr ovided in detail: the first one focuses on the device and DEP electr ode configuration, important
aspects when developing DEP-based cell manipulation, separation, and characterization devices. The
second one pr ovides details of experimental parameters such as flow velocity , voltages and frequencies
applied, cell parameters such as cell size and dielectric pr operties. T aken together , these two sets of
information can serve as a quick lookup table that will be useful in designing specific DEP devices for
desir ed applications of interest. Notation and units are given in T able 2 .
Fr om the information summarized in T able 1 , several assessments of the current status of the
field can be made. First, most of the investigated micr oalgae belong to the group of gr een microalgae,
although ther e are some belonging to diatoms (Figur e 4 ). Additionally , most published work has
focused on static experiments (
.
V =
0), despite the advantages of continuous-flow operations, such
as higher thr oughput. This shows the challenge of successfully combining dielectrophor etic cell
manipulation and separation systems with continuous-flow micr ofluidic setups. Lastly , in most cases,
prior to dielectr ophoretic cell analysis or cell manipulation, regular cultur e media ar e diluted or
exchanged. This can be done by pressur e-driven membrane operations, which also show potential
in r ecovering functional molecules during downstr eam processing [
74
–
76
]. The decr ease in media
conductivity r esults in higher contrast in polarizability
 

 ε ∗
p − ε ∗
m 

 
and thus a lar ger DEP for ce. This
shows the challenges and limitations of DEP-based cell handling, as in situ applications in normal
cultur e media may become challenging.
Microorganisms 2 020 , 8 , x FOR P E E R REVIE W 19 of 19

From t h e inf o rmat ion su mmari zed in Table 1 , seve ra l a s sessments of the current sta t us of the
fie ld c a n be made. Fir s t , mo st of t h e inves t igat ed mic r o a lg ae be long t o t h e group o f green m i cro a lg ae ,
although ther e are some belongin g to d i atoms (F ig ur e 4). A dditio n ally, most published wor k h a s
focused on st atic exper i me nts (  󰇗 =0 ), despit e t h e advant a g es of cont in u o us- flow ope r at ions , s u ch as
higher throughput. This shows the c h allenge o f successfully combining d i electrophore tic cell
manipu l a t i on an d sepa rat i on sy st ems wit h cont in u o us- flow m i c r ofl u id ic set u ps. L a st ly, in most
case s, prior t o dielectroph o retic cell an alysis or cel l ma ni pula ti on, regula r culture media ar e diluted
o r e x c h a n g e d . T h i s c a n b e d o n e b y p r e s s u r e - d r i v e n m e mbrane operations, wh ich also show pot e ntial
in r e coverin g f u nct i on al m o lecu les d u ri ng down st re am proce s sin g [74–76]. Th e decr e ase in media
conduct i vit y resu lt s in h i g h er cont rast i n polar i z a bi li t y  
∗ − 
∗  and t h u s a l a rge r DEP f o rce. This
shows t h e ch al lenge s an d limit at ions o f DEP-b a sed c e ll h a nd ling , as in s i t u applic at ions in n o rmal
cult ur e med i a m a y becom e cha llen g ing .

Figure 4. Ov erview of mi croa lgae DEP re sea r ch based on 1 6 pu blications (Table 1). ( a ) U s ed speci e s
of algae, ( b ) A pplication types of DEP in mi c r oalgae researc h .
4. DEP Micr o f lui d ic D e vic e s Ca t e gor i ze d Bas e d on Working Principles of Devic e s
There are b r oad l y two different de vice c a tegor i es in how DEP-b a sed microalg al c e l l
manipulation s ar e conduc ted. The first device c a te gory tra p s desi red ta rget cel l s onto the DEP
elect r odes fro m cells flow i ng t h rough a channel us in g a pos i t i ve DEP force , es sent ia ll y f u nc t i oning
as a fi lt rat i on device t h at t a rget s speci fic cell s b a se d on their d i electric propertie s . The secon d c a tegory
defl e ct s t h e c e ll s flow i ng i n a mic r of lu i d ic ch anne l t h rough eit h er a pos i t i ve D E P force or a negat i ve
DEP forc e, w h ere the applied force c a uses the ce ll tr a j ectori es to cha n ge, resul t ing i n sepa ra t i on of
microalg ae ce lls based on their d i e l ectric properties.
4. 1. T r ap pi n g Desi gns Usi n g pD EP F o rce
In these desig ns, electrodes positioned in side a mic r of l u id ic ch annel applie s a pDE P force t o ce ll s
pa ssi n g through a n d tra p s ta rget cel l s ont o the el ec trod es. Three different electrod es design s are most
commonly ut iliz e d.
4.2. Planar Pa rallel Surface Electro d es for Cell Trappin g
The first dev ice catego ry is those that trap desired t a rg et cells onto t h e DEP electr odes from ce lls
flow i ng thro ugh a chann e l. Two DE P electro de st ructures most commonly used are the planar
paralle l e l ectr ode structure (Figur e 5a) and the plan ar int e rdi git at e d elect r ode st ruct ure (F igu r e 5b) .
The plan ar electrode struct ure is con f igured to have a f l ow cell wi th two pa ra ll el electrodes at both
side s of t h e microf lu idic channel . Her e , t h e DE P force is applied between the electrode s , thus
perpendicu l a r t o t h e flow direct ion . S u s c il lo n et. al. utilized such a structure to t r ap Ch lamyd o monas
reinhar dtii ce lls to the DE P electrode s at an e l ectric field o f 20 V m m
− 1
and fre q uency o f 1 k H z [6 0 ] .

Figure 4.
Overview of micr oalgae DEP r esear ch based on 16 publications (T able 1 ). (
a
) Used species of
algae, ( b ) Application types of DEP in microalgae r esearch.

Microor ganisms 2020 , 8 , 540 6 of 19
T able 1. Key publications reviewed in this article, with a summary of key featur es and applications.
Algal Species T axonomy and
No. Flagella Application T ype and Description Device Structure Electric Field, Flow rate,
and Cell Concentration
Dielectric
Properties of
the Medium
Dielectric Properties
of Microalgae Ref.
(a) Chlamydomonas
reinhardtii
(b) Synechocystis sp.
(c) Cyclotella
meneghiniana
(a) green alga, 2
(b) cyanobac., 0
(c) diatom, 0
ass.
rot.
measuring e ff ects of AC
field intensity , frequency
and duration on chaining
e ffi ciency and chain lengths
chamber ( d ch = 350 ) , coplanar
electrodes  Au, d eg = 2000 
E = 15 ... 25
f = 10 − 4 ... 0.5
.
V = 0
c = 5 · 10 6 ... 5 · 10 7
Geneva lake water
σ m = 320
ε m = 80
( a ) d = 13, t w = 500,
σ i = 0.008, σ w = 50,
ε i = 150, ε w = 70
( b ) d = 3.98, t w = 130,
σ i = 0.19, σ w = 680,
ε w = 60, ε i = 61
( c ) d = 17.72, t w = 500,
σ i = 0.008, σ w = 10 − 17
ε i = 150, ε w = 3.9
[
56
]
(a) Chlorella
vulgaris
(b) Raphidocelis
subcapitata
(c) Dunaliella salina
(a) green alga, 0
(b) green alga, 0
(c) green alga, 2
sep. separation by size and
species
PDMS channels ( d cw = 90 . . . 300,
d ch = 25 ) with overall field gradient
U < 295
f = 0
sodium borate
bu ff er solution
pH 7.5
( a ) d = 2 ... 4
( b ) d = 3.7 ... 6.25
( c ) d = 3.8 ... 6.0
[
57
]
Chlamydomonas
reinhardtii
(a) high lipid
(b) low lipid
green alga, 2 sep.
high-frequency DEP in
continuous-flow cell
screening device for
separation based on lipid
content
PDMS channel ( d ch = 20, d cw = 1000 ) ,
4 interdigitated electr ode arrays 10
electrodes each ( Au, d ew = 50,
d eg = 50 ) by etching
U = 30
f = 50
.
V = 9
c = 6.7 · 10 6
KCl solution
85 g L − 1 Glc
0.1
% serum albumin
σ m = 10.6
ε m = 80
d = 10 . . . 15
( a ) σ i = 0.095
( b ) σ i = 0.2267
ε i = 50, ε mem = 8
σ mem = 2 · 10 − 6
[
58
]
Chlamydomonas
reinhardtii gr een alga, 2 ass.
high-frequency DEP to
determine upper crossover
frequency of cells with
varying lipid content
glass slide with needle patterned
electrodes (Au)
U = 30
f = 10 ... 110
.
V = 0
KCl solution;
85 g L − 1 Glc;
σ m = 64
ε m = 80
d = 12, t mem = 7
ε i = 50, ε mem = 8,
σ i = 0.5, σ mem = 0.02
[
59
]
Chlamydomonas
reinhardtii gr een alga, 2 ass.
characterization of e ff ects
of freshwater composition
on the DEP response
chamber ( d ch = 250 ) , coplanar
electrodes  Au, d eg = 2000  by
vapor deposition
E = 20
f = 0.001
.
V = 0
n = 10 6
fresh water;
σ m = 32 . . . 56
[
60
]
Chlamydomonas
reinhardtii gr een alga, 2 ass.
rapid tool for capture and
screen with fluor escence for
the e ff ect of contaminants
chamber ( silicone, d ch = 2000 ) ,
four orthogonally needle
electrodes ( stainless steel, d eg = 5000 )
E = 10
f = 0.0001
.
V = 0
n = 5 · 10 6
water;
0.0001 M MOPS
+ various
contaminants
[ 61 ,
62 ]
Chlorella vulgaris green alga, 0 sep.
studies on solution
conductivity and lipid
content, microfluidic chip
to sort the microalgae with
di ff erent lipid contents
channel ( JSR THB151N, d ch = 15 ) by
spin coating, symmetrical deflector
electrodes ( T i 0.2 Au, d eh = 0.03 )
by vapor deposition and
photolithography
U = 10
f = 7, 10, 20
.
V = 250
KH 2 PO 4
bu ff er solution;
σ m = 290
ε m = 80
d = 5.2
d lip = 1.23 ... 2.05
σ w = 10 − 8 , σ i = 0.5
σ lip = 0.0001
ε w = 5, ε i = 60
ε lip = 3
t w = 100
[ 63 ,
64 ]

Microor ganisms 2020 , 8 , 540 7 of 19
T able 1. Cont.
Algal Species T axonomy and
No. Flagella Application T ype and Description Device Structure Electric Field, Flow rate,
and Cell Concentration
Dielectric
Properties of
the Medium
Dielectric Properties
of Microalgae Ref.
Chlorella vulgaris green alga, 0 att.
rep.
screening for highest
radionuclide
bio-decontamination by
n- and p-DEP
PDMS Chamber, electrodes
( Au, d eh = 0.3, d ew = 30 )
on glass by lithography
U = 10
f = 0.1 ... 5
c = 4.8 · 10 6
3 mM NaHCO 3
σ m = 335
[
65
]
Coscinodiscus
wailesii diatom, 0 att.
rep.
2D dielectrophor etic
signature
PDMS microfluidic well, inter digitated
electrode pattern
U = 1 ... 10
f = 0.001 ... 100
.
V = 0
f /
2
culture medium
σ m = 47
ε m = 79
d = 20, 75 . . . 80
σ i = 0.06, σ mem = 0.03
ε i = 48, ε mem = 20
t mem = 9
[
66
]
Eremosphaera viridis
green alga, 0 att.
rep.
tool for spatial
manipulation
commercially available single electr ode,
etched elgiloy tip with porous
metal-oxide coating
U = 1 ... 5
f = 0.05 ... 10
.
V = 0
low calcium
Dickinson medium
[
67
]
Karenia br evis dinoflagellate, 2 ass. dielectrophor etic
concentration
glass slide, 3x4mm array of castellated
interdigitated electr odes
( Pt, d eh = 0.2, d ew = 20, d eg = 20 )
U = 1
f = 0.2
.
V = 0
n = 3 · 10 5
280mM mannose;
0.5% T ween
σ m = 10.5
[
68
]
Raphidocelis
subcapitata green alga, 0 sep. concentrate and separate
live and dead cells
glass chambers ( d cw = 2000, d ch = 20 )
with cylinders ( 470 . . . 520 µ m ) by wet
etching, overall field gradient
E = 10 ... 25
f = 0
n = 1.7 · 10 7
( i ) bidistilled water
( ii ) 1mM KH2PO4
σ m = 0.225, 18.7
[
69
]
T etraselmis sp. green alga, 4
trans.
twDEP used to estimate the
dielectric properties
glass slide, octa-pairs interdigitated
electrode ( Au, d ew = 50, d eg = 50,
d eh = 0.5 ) by photolithography
and wet-etching
U = 1.5 ... 14
f = 0.005 ... 4
.
V = 2.4
c = 10 6
0.5 M sorbitol
+ 0.1 M KCl
σ m = 3 ... 370
[
70
]
T etraselmis sp.
(a) control
(b) As treated
(c) boiled
green alga, 4
trans.
determination of dielectric
properties and e ff ects
of arsenic
glass slide, octa-pair interdigitated
electrodes (Au, d el = 200;
d ew = 100, d eh = 0.2; d eg1 = 100, 300)
U = 2 ... 10
f = 0.015 ... 0.5
.
V = 0
c = 4 · 10 5 ... 9.2 · 10 6
σ m = 10 ... 250
ε m = 78
shperic : d a = 20
d b = 16
( a ) σ i = 0.37
σ mem = 0.00017
ε i = 48, ε mem = 8
( b ) σ i = 0.013 ... 0.03
σ mem = 0.003 ... 0.0038
ε i = 48, ε mem = 10 ... 32
( c ) σ i = 0.06
σ mem = 0.03
ε i = 91, ε mem = 20
t mem = 13
[
71
]

Microor ganisms 2020 , 8 , 540 8 of 19
T able 1. Cont.
Algal Species T axonomy and
No. Flagella Application T ype and Description Device Structure Electric Field, Flow rate,
and Cell Concentration
Dielectric
Properties of
the Medium
Dielectric Properties
of Microalgae Ref.
heterogeneous
population various ass.
technique to monitor the
concentration of algae in
fresh water to avoid mass
contaminations
chamber , four electrodes
U < 1.65
f < 1.2
.
V = 0
d = 15
[
72
]
(a) Platymonas sp.
(b) Closterium sp.
(a) green alga, 2
(b) green alga, 0 sep.
continuous separation of
di ff erent micr oalgae from
microplastics by
multi-electrode n- and
p-DEP
PDMS chamber ( d cw = 100 ) ,
electrodes on IT O ( Ag −
PDMS mixture, d ew1 = 1900,
d ew2 = 100, d eg = 100 )
f = 30
.
V = 0.083
PBS bu ff er solution
σ m = 300
[
73
]
T able 2. Notation and Units.
T erm Meaning Unit T erm Meaning Unit / V alue T erm Meaning Unit
d Cell diameter µ m σ m Suspending medium conductivity mS · m − 1 t w Cell wall thickness nm
d ew Electrode width µ m σ mem Cell membrane conductivity mS · m − 1 t mem Cell membrane thickness nm
d eh Electrode height µ m σ i Cell interior conductivity S · m − 1 c Cell concentration ml − 1
d el Electrode length µ m σ lip Cell lipid body conductivity S · m − 1 n Cell number -
d ed Electrode diameter µ m σ w Cell wall conductivity S · m − 1 .
V Flow rate µ l · s − 1
d eg Gap to next electrode µ m ε m Suspending medium relative permittivity - f frequency MHz
d cw Channel / chamber width µ m ε i Cell interior relative permittivity - E Field str ength V · mm − 1
d ch Channel / chamber height µ m ε lip Cell lipid body relative permittivity - U V oltage V
d cl Channel / chamber length µ m ε w Cell wall relative permittivity
d cl Channel / chamber length µ m ε 0 Permittivity of free space 8.854 × 10 − 12 F · m − 1
d cd
Channel / chamber diameter
µ m

Microor ganisms 2020 , 8 , 540 9 of 19
4. DEP Microfluidic Devices Categorized Based on W orking Principles of Devices
Ther e are br oadly two di ff erent device categories in how DEP-based micr oalgal cell manipulations
ar e conducted. The first device category traps desired tar get cells onto the DEP electrodes fr om cells
flowing thr ough a channel using a positive DEP force, essentially functioning as a filtration device
that tar gets specific cells based on their dielectric proper ties. The second category deflects the cells
flowing in a micr ofluidic channel through either a positive DEP for ce or a negative DEP force, wher e
the applied for ce causes the cell trajectories to change, resulting in separation of micr oalgae cells based
on their dielectric pr operties.
4.1. T rapping Designs Using pDEP For ce
In these designs, electr odes positioned inside a micr ofluidic channel applies a pDEP for ce to cells
passing thr ough and traps target cells onto the electr odes. Three di ff er ent electrodes designs ar e most
commonly utilized.
4.2. Planar Parallel Surface Electr odes for Cell T rapping
The first device category is those that trap desir ed tar get cells onto the DEP electrodes fr om cells
flowing thr ough a channel. T wo DEP electrode str uctures most commonly used ar e the planar parallel
electr ode structure (Figur e 5 a) and the planar interdigitated electr ode structur e (Figure 5 b). The planar
electr ode structure is configur ed to have a flow cell with two parallel electrodes at both sides of the
micr ofluidic channel. Her e, the DEP force is applied between the electr odes, thus perpendicular to
the flow dir ection. Suscillon et al. utilized such a structur e to trap Chlamydomonas reinhardtii cells to
the DEP electr odes at an electric field of 20 V mm
− 1
and fr equency of 1 kHz [
60
]. Here the electr ode
material was gold, which is most commonly used due to its chemical inertness and stability in the
solution phase.
Microorganisms 2 020 , 8 , x FOR P E E R REVIE W 20 of 19

Here the elec trode material was gold, w h ich is mo st commonly used due to its chemical ine r tness
and st ab i lit y i n t h e sol u t i on p h ase .

Figure 5. Sche matics o f chan nel-electro d e configuratio ns u s ed in DEP st u d ies on m i cro a lg ae.
Electro d e confi g urations are (a ) planar parallel, (b) p l anar in terdigitated , (c ) mod i fied p l anar
interdigitated , (d) m i cro-po st and (e) angled
Siebm a n et. al. ut ilize d two pair s of w i r e electr ode s p o sit i oned at a 1 8 0 ° ang l e t o creat e a hi g h
electric field zone in the middle, into wh ich ce lls co uld be tr apped [56,61,62] . He re, the electro de w a s
a p a ir of st ain l ess-stee l needle s . In these studies, green micr oalga C h l a m ydo monas rei nhar dti i ,
cyanobacterium Syn e c h ocystis s p . , a n d di a t om C y clotella menegh iniana wer e ut il i z ed, and fre q u e ncie s
from 0. 1 t o 5 0 0 kH z were ap p lie d t o ch ar act e ri ze the c e ll s a n d thei r move ment. Al though easy to
ass e mble a s no micro f abr i cat i on is inv o lved (st a nd ard need les were used ), i t wil l be d i f f i cu lt t o
fabr icate suc h device s rep e atedly d u e t o difficulties in a ssemb lin g t h ese four wire e l ect r od es in a
reproduc ible manner, espe cia l l y when p r ecise cel l m a nipul a t i on is requ ire d .
Bahi et al . ut ili ze d an int e rdig it at ed (I DT) e l ectrod e design , wh ere arr a ys of fin ger - sh ape d
electrode structures facin g each other w e re utilized [68]. He re th e electrode mat e rial was p l at inum .
This IDT elec trode design is on e o f the most clas sic a l DEP electro de de sign s used fo r tr apping [77],
as the man y interdigit ated finge r structure can gen e rate arrays o f high electric field region s, to which
cel l s ca n be attra c ted through a posi ti v e DEP f o rce (Fig ure 5b). A s a large are a can be cover ed with
such an electr ode structur e , this is a ve r y effic i ent wa y of t r appin g cell s. In t h is applic at ion , marine
microalga Ka renia brevis wa s u t il iz ed.
Wang et . a l a l so operat ed wit h s u ch an IDT st r u ct ur e [ 7 3] in a m i crof lu idic flo w cel l t o t r a p
Ch lorella v u lgaris cells at a volt age o f 1 0 V
pp
in a fre q uency ran g e of 0.1 to 5 MH z. Here the ele c trode
ma teria l was gold.
Another plan ar e l ectrode design show n by Kum a r et. al had an arr a y o f p a r a llel electrod es
(modi fied ID T des ign) pl a c ed on bot h s i des of a f l ow channe l [6 6 ] , a s shown in Fig u re 5c. He re, ce ll s
f l o wi ng through the m i ddl e p a rt coul d be a ttra c ted to both si des of the m i croflui d i c cha nnel under
the influence of d i electric force, tr appin g tar g et ce lls to the side o f the main flo w stre am. Sin c e the
microf lu idic c h annel wid e n ed t o bot h s i d e s o f t h e flow channe l, t h e flow is we ak on bot h sid e s, t h us
tra ppi ng the cel l s wa s easier tha n ha v i ng a stra ig ht m i crochann el d e sign . The ele c trode mater i al was
gold. This de sign w a s u s e d t o invest igate the trappin g of green m i croalga Cosci n odiscu s waile sii cells
at a volt a g e r a nge of 1 t o 1 0 V and a fre q uenc y r a nge of 1 kH z t o 1 0 0 MH z.

Figure 5.
Schematics of channel-electrode configurations used in DEP studies on micr oalgae. Electrode
configurations are (
a
) planar parallel, (
b
) planar interdigitated, (
c
) modified planar interdigitated,
( d ) micro-post and ( e ) angled.
Siebman et al. utilized two pairs of wire electr odes positioned at a 180
◦
angle to cr eate a high
electric field zone in the middle, into which cells could be trapped [
56
,
61
,
62
]. Her e, the electr ode
was a pair of stainless-steel needles. In these studies, gr een micr oalga Chlamydomonas r einhardtii ,

Microor ganisms 2020 , 8 , 540 10 of 19
cyanobacterium Synechocystis sp. , and diatom Cyclotella meneghiniana were utilized, and fr equencies
fr om 0.1 to 500 kHz were applied to characterize the cells and their movement. Although easy to
assemble as no micr ofabrication is involved (standard needles wer e used), it will be di ffi cult to fabricate
such devices r epeatedly due to di ffi culties in assembling these four wire electr odes in a repr oducible
manner , especially when precise cell manipulation is r equired.
Bahi et al. utilized an interdigitated (IDT) electrode design, wher e arrays of finger-shaped electr ode
structur es facing each other were utilized [
68
]. Her e the electrode material was platinum. This IDT
electr ode design is one of the most classical DEP electrode designs used for trapping [
77
], as the many
inter digitated finger structur e can generate arrays of high electric field regions, to which cells can
be attracted thr ough a positive DEP for ce (Figur e 5 b). As a large ar ea can be covered with such an
electr ode structure, this is a very e ffi cient way of trapping cells. In this application, marine microalga
Kar enia brevis was utilized.
W ang et al. also operated with such an IDT structur e [
73
] in a microfluidic flow cell to trap
Chlor ella vulgaris cells at a voltage of 10 V
pp
in a fr equency range of 0.1 to 5 MHz. Here the electr ode
material was gold.
Another planar electr ode design shown by Kumar et al. had an array of parallel electr odes
(modified IDT design) placed on both sides of a flow channel [
66
], as shown in Figur e 5 c. Here, cells
flowing thr ough the middle part could be attracted to both sides of the microfluidic channel under
the influence of dielectric for ce, trapping target cells to the side of the main flow str eam. Since the
micr ofluidic channel widened to both sides of the flow channel, the flow is weak on both sides, thus
trapping the cells was easier than having a straight microchannel design. The electrode material was
gold. This design was used to investigate the trapping of gr een microalga Coscinodiscus wailesii cells at
a voltage range of 1 to 10 V and a fr equency range of 1 kHz to 100 MHz.
The advantage of most of these designs is the extremely simple electr ode structur e and ease of
micr ofabrication since they often requir e only an electrode design and fabrication pr ocess on a printed
cir cuit board (PCB) or a single glass substrate. Additionally , compared to DEP-based cell separation
(to be described in the next section), these designs can accumulate and concentrate cells rather than
just separate cells, which is useful if concentrating cells fr om a solution is the main purpose.
However , the major disadvantages ar e that due to the relatively lar ge distance between the
electr odes, the trapping force is r elatively weak, limiting the flow rate that can be utilized. IDT electr ode
design over comes some of this limitation. However , the electric field and also the dielectr ophoretic
for ce, decreases with distance fr om the electrode. Since in all of these designs the electrodes ar e on
the bottom surface of the micr ofluidic channel, only cells flowing close to the bottom of a channel can
be trapped easily . Cells that ar e flowing close to the channel ceiling experience a much lower DEP
for ce. Therefor e, a relatively shallow micr ofluidic channel must be used for high trapping e ffi ciency ,
along with a r elatively slow flow rate, thus significantly limiting the overall throughput that can be
achieved. A shallow micr ofluidic channel can also be easily clogged by clumps of cells, posing another
practical challenge.
4.3. Micr opost Electrode for Cell T rapping
Gallo-V illanjueva et al. described a DEP trapping design based on arrays of micr o-post-electrodes
that can cr eate an asymmetric electric field [
69
], hence cr eating zones of high electric field gradients
which cells can be attracted to (Figur e 5 d). Here, the arrays of 20
µ
m tall posts ar e made of glass by
etching a glass substrate thr ough standard wet etching (20
µ
m channel depth). The electric field is
applied along the flow dir ection of the microfluidic channel. Thus, the array of insulating posts cr eates
an asymmetric electric field, consequently creating DEP for ce. This design was utilized to trap the cells
of Selenastrum capricornutum at an electric field of 250 V mm
− 1
DC potential. A r elatively high voltage
had to be applied as it is intr oduced via the in- and outlet of the flow channel.

Microor ganisms 2020 , 8 , 540 11 of 19
4.4. Sharp-T ip Electr ode Design for Cell T rapping
This design, where a sharp electr ode is used to cr eate a high electric field at the tip of the electrode
is one of the oldest designs and methods to cr eate an asymmetric electric field for DEP experiments [
78
].
Her e, a simple needle-like sharp electrode can be inserted into a solution, and when an electric field is
applied, cells can be attracted to this electr ode. This can be transferred easily into a micr ofabricated
version, as has been shown by Michael et al. (2014). In this study , the planar sharp-tip electr ode made
of gold was utilized to trap Chlamydomonas reinhardtii cells using high-fr equency DEP ( > 20 MHz). Due
to the simplicity , this design is ideal for quickly characterizing the DEP responses of cells, but beyond
that, it cannot really be applied for any cell trapping or cell separation applications due to its low
thr oughput nature.
4.5. Flow-Thr ough Deflection Structures for Cell Separation
In these designs, electr odes integrated into a micr ofluidic channel either apply a pDEP or nDEP
for ce depending on frequency . In the overwhelming majority of studies, the micr ofluidic flow exhibit
low Reynold’s numbers that can safely be characterized as laminar . The electrodes then deflect the
tar get cells to one or the other side of the microfluidic channel, resulting in a lateral shift in cell positions
inside the flow , thus separating target cells fr om the initial flow path.
4.6. Sharp-T ip Electr ode for Cell Separation
As discussed in the pr evious section, a sharp-tip electrode is one of the earliest forms of electr odes
that have been used to excite the inhomogeneous electric field needed to generate the DEP for ce.
Song et al. designed a microfluidic channel structur e that has a region wher e the exterior channel
shape r esembles a needle [
57
]. Since the voltage was applied thr ough the channel, this created a lar ge
inhomogeneous electric field ar ound the channel tip r egion. This is similar to having a sharp-tip
electr ode near the flow channel, without the need to having to create such an electr ode. This design
was used to deflect Chlorella vulgaris , Pseudokir cheriella subcapitata , and Dunaliella salina cells at an
applied DC voltage of several hundr ed volts.
The work fr om W ang et al. (2018) showed a microfluidic channel wher e on one side was an
array of sharp electr odes and on the other side, a flat planar electrode was positioned. By applying a
voltage between the electr odes, this design created an array of inhomogeneous electric field r egions
on one side of the micr ofluidic channel. The micr ofluidic device was composed of a multi-layer
polydimethylsiloxane (PDMS) channel with gold electr odes patterned on the glass slide. The electr odes
wer e insulated from the substrate via an underlying silicon nitride layer . Cells that experience a pDEP
for ce (in this case wild type Chlorella vulgaris ) r emained attracted towards the bottom electr ode and
came out of the lower outlet channel. However , cells that experience a nDEP force (in this case Chlor ella
vulgaris with higher Sr biomineral competence cells) wer e r epelled away from the electr ode and exited
the upper outlet channel. Based on these two di ff erent characteristics, this system was successfully
utilized to separate micr oalgae that show higher radionuclide bio-decontamination activity [ 79 ].
4.7. Angled Electr odes for Cell Separation
The second category of designs ar e those where the electr odes are inclined at an angle to the flow
dir ection. Deng et al. (2014) used a device wher e a pair of top-bottom electrodes wer e positioned at
an angle to the flow direction, wher e three such electr ode pairs were positioned in a zig-zag position
along the micr ochannel. The electrodes wer e made from gold, and the top / bottom substrates wer e
aligned and bonded together to form the top-bottom electr ode structure. The first two pairs were
utilized to first align all cells along the electr odes by applying a voltage at 5 MHz to apply strong
nDEP to all cells. The third electr ode was then utilized to separate cells based on their intracellular
lipid content by incr easing the frequency to 10 MHz. Her e, microalgae with 24% lipid content passed
thr ough the electrodes, while those with 35% lipid content wer e repelled by the nDEP for ce. Thus, in

Microor ganisms 2020 , 8 , 540 12 of 19
this device, the fr equency-dependent response of cells to experience either a pDEP for ce or nDEP force
was utilized for selective cell manipulation [ 80 ].
The most basic device design for the separation of micr oalgae would encompass a microfluidic
channel at the walls of which electrodes ar e integrated (Figure 5 e). The latter would be subjected
to oscillating electrical fields to act on particles as carried by the str eaming fluid. Figure 2 displays
the scheme of a 50
µ
m high channel with so-called deflector electr odes on the top and at the bottom
that was used in a finite-element model to simulate the DEP e ff ect. The DEP for ce was calculated for
Chlamydomonas r einhardtii [
63
] with geometrical and electrical parameters given in the literature [
64
].
For a flow velocity of
v <
1.9
µ
L / min, a fr equency of f = 1.85 MHz and a voltage of 12 V
pp
a separation
e ff ect could be shown causing the high-lipid fraction (blue) to enter the upper channel and the low-lipid
fraction (r ed) to enter the lower channel.
Hadady et al. (2016) also utilized arrays of angled electrodes to separate micr oalgal cells based
on their pr operties. Here, arrays of inter digitated angled gold electrodes (45
◦
angle, 50
µ
m width,
50 µ m gap) positioned at the bottom of the flow channel cr eated a sideway DEP force. Consequently ,
Chlamydomonas r einhardtii cells with high lipid content were minimally influenced by the DEP for ce
and went straight out thr ough the lower outlet channel. However , Chlamydomonas r einhardtii cells with
low lipid content wer e influenced by the DEP force the most, and, ther efor e, moved laterally to the
upper side of the micr ofluidic channel, and thus went out thr ough the upper outlet channel. Here,
a fairly high fr equency of 40 MHz and 60 MHz were utilized. A flow rate of 3
µ
L / min was utilized
thr oughout this experiment [ 59 ].
Overall, the angled DEP electr odes that attract or deflect cells based on their intrinsic properties,
especially depending on their intracellular lipid content, provides not only high accuracy in such
separation but also significantly higher thr oughput compar ed to static capture-type DEP designs.
Ther efore, these deflector-type devices ar e probably the most pr omising DEP microfluidic device
design for micr oalgae manipulation and separation.
5. DEP Microfluidic Devices Categorized Based on Their Applications
The various DEP-based micr ofluidic designs and devices wer e utilized for the identification of
micr oalgal species, analysis of various properties and r esponses of microalgae, separation based on
their size and intracellular lipid content, as well as for scr eening applications.
5.1. Cell T rapping and Concentration
Suscillon et al. and Siebman et al. fr om the same group simply demonstrated that micr oalgal
cells can be trapped using DEP [
56
,
60
]. In the first study [
60
], Chlamydomonas r einhardtii was trapped
between two electr odes under diverse media conditions, voltages, and frequencies, and showed
“chaining” behavior . In the second paper [
56
], this study was expanded to two other microalgal species,
Synechocystis sp. and Cyclotella meneghiniana . However , beyond the observation of this phenomenon,
no further specifics wer e discussed on any specific technical applications where such phenomena may
be utilized.
Bahi et al. showed a DEP device to trap marine microalga Kar enia br evis for lysis and downstr eam
RNA extraction and amplification purposes [
68
]. The main benefit of the DEP-based microfluidic
device was the capability of cr eating a high concentration of cells, which allowed RNA extraction and
purification much easier compar ed to using low concentration cell samples.
Siebman et al. studied how Chlamydomonas r einhardtii cells are impacted by envir onmental
contaminants [
61
,
62
]. When cells wer e exposed to contaminants such as mercury , methylmer cury ,
copper , copper oxide nanoparticles, and the herbicide diuron, reactive oxygen species pr oduction
and oxidative str ess increased in Chlamydomonas , which wer e measured by detecting the changes in
chlor ophyll autofluorescence. In this application, the DEP trapping structures wer e utilized to hold
onto cells while the cells ar e exposed to the environmental contaminants and while taking fluor escent
images, allowing easy micr oscopy .

Microor ganisms 2020 , 8 , 540 13 of 19
5.2. Cell Separation Based on Intracellular Lipid Content
Since lipid pr oduction by micr oalgal cells is one of the major reasons why micr oalgal resear ch is
of high inter est, several publications have described the use of DEP to separate microalgal cells based
on their intracellular lipid content.
Deng et al. pr esented two such studies [
58
,
80
]. In the first work, Chlorella vulgaris cells with
di ff er ent lipid content were successfully separated using a DEP device having arrays of parallel
electr odes. Here, the DEP micr ofluidic system demonstrated that Chlorella with 11 wt% and 45 wt%
lipid content showed very di ff er ent dielectric properties that could be successfully separated. This
DEP separation was performed at a r elatively high frequency of 20 MHz. In the gr oup’s second paper ,
Chlor ella cells with lipid content of 13% and 21% were separated, and cells with lipid content of 24%
and 30-35% wer e successfully separated. In this work, slightly lower frequencies of 7 MHz and 10 MHz
wer e utilized for cell separation.
In another work, Michael et al. used a relatively high fr equency (over 20 MHz) to show that
the upper cr ossover frequency of micr oalgal cells is reduced with incr easing intracellular lipid [
81
].
This phenomenon was successfully measur ed on Chlamydomonas reinha rdtii cells using a sharp-tip
electr ode device. Later , the same group utilized this phenomenon and applied it to a flow-thr ough
DEP device [
59
], wher e 74% of the high-lipid population and 75% of the low-lipid population could
be successfully separated. In this particular device, again a relatively high fr equency of 50 MHz was
utilized at a voltage of 30 V pp .
5.3. Cell Separation Based on Their Sizes or Fr om Other Particles
Song et al. presented a device that they used to separate marine micr oalgal species known
to have di ff er ent volumes from 5
µ
m diameter polystyr ene (PS) particles [
57
]. The justification for
this work was for analyzing bioparticles for water quality monitoring. These were Chlor ella vulgaris ,
Pseudokir cheriella subcapitata , and Dunaliella salina . P . subcapitata and D. Salina had a similar size to the
5
µ
m diameter PS particles, while C. vulgaris was in the range of 2–4
µ
m in diameter . Here, C. vulgaris
and P . subcapitata wer e successfully separated into two streams using their DEP micr ofluidic device.
Additionally , the DEP separation of P . subcapitata fr om 5
µ
m diameter PS beads and the DEP separation
of D. Salina fr om 5
µ
m PS beads wer e also demonstrated. These separations were all based on size
di ff er ences between the cells and the PS beads.
W ang et al. also demonstrated the separation of Platymonas and Closterium from micr oplastic [
73
].
Her e, the application was to select microalgae fr om ballast water in a ship, as biological contamination
of ballast water is a global problem as well as highly r egulated, where ballast water must be inactivated
for any biological materials befor e docking into a port. Thus, rapid detection of ballast water for any
micr oalgae is needed and has the potential to overcome the challenges of using filtration systems and
fluor escence measurement. Here, the gr oup demonstrated that both Platymonas and Closterium can
be successfully separated fr om Polystyrene particles (size not mentioned in the manuscript), using
a voltage range of 5–15 V at a fr equency of several tens of MHz. The system was also tested under
various flow rates, with the maximum being 0.03 mL / min. Overall, separation e ffi ciencies of around
90% wer e achieved.
5.4. Micr oalgae Analysis
Kumar et al. utilized a DEP device to measure the dielectric pr operties of a green alga Coscinodiscus
wailesii [
66
]. Her e, the lateral displacement, dielectrophor etic force, and translational dielectr ophoretic
velocity wer e measured. The authors concluded that thor oughly measuring the dielectric properties of
a given cell pr ovides the futur e capability in label-free manipulation of diatoms and for rapid scr eening
for envir onmental e ff ects on the dielectric pr operties of algal cells (but not yet conducted in their paper).
Gallo-V illanueva et al. utilized a DEP device to measur e the viability of Selenastrum
capricornutum [
69
]. The experimental r esult showed that live cells exhibit a stronger DEP r esponse

Microor ganisms 2020 , 8 , 540 14 of 19
compar ed to dead cells. This allowed rapid label-free sensing of the viability of the microalgal cells.
The authors pr esented that within a relatively short period of time (35 s), enrichment of about 10 times
could be achieved for each cell population.
5.5. Strain Selection Thr ough Screening
Selecting micr oalgal strains with specific phenotypes is of high interest in many micr oalgae
scr eening applications. Many such applications have been on selecting and obtaining
high-lipid-pr oducing strains, as micr oalgae-based lipid production is an important ar ea towards
r enewable bioenergy applications [
19
,
65
]. However , there ar e other examples of high-throughput
scr eening than just lipid production.
An example of such an application is identifying strains that show high e ffi ciency in
decontaminating hazar dous waste, such as radionuclide. W ang et al. used a microfluidic device to first
test the capability of DEP for their capability in separating Chlor ella vulgaris KMMCC9 , a strain known
for its high capacity of r emoving strontium (Sr), fr om Chlorella vulgaris KCTC AG10002 strain that has
only a weak capacity of r emoving str ontium. This separation was successfully conducted using the
developed DEP device [
79
]. Following this success, sub-populations of Chlor ella vulgaris KMMCC9
strains with higher Sr biomineral competence wer e successfully separated, and the selected strain’s
capability confirmed in lar ge-scale cultivation experiments.
6. Discussion
Fr om the analysis of DEP-based microfluidic platforms for micr oalgae resear ch, it can be concluded
that DEP in micr oalgae r esearch is pr omising but still not a mainstream technology . The latter fact is
caused by several r easons. First, the manufacturing complexity of micr ofluidic DEP devices is still
not standar d technology , especially to non-experts in microfabrication. Several works feature the
need for highly specialized micr ofabrication equipment or non-standardized / non-comparable setups.
Some of these r easons can be contributed to the fact that microfluidics devices have no standar dized
guidance, which has been a fundamental limitation for microfluidic systems in general. This, in
general, has hindered many excellent micr ofluidic systems from being adopted by the br oader life
science community . The functional part of a microfluidic DEP setup, in general, has in most cases not
exceeded technology r eadiness level 4, which stands for a principle demonstration in a laboratory
setup. Despite these limitations, micr ofluidic systems have been pr oliferating thanks to their powerful
and unique capabilities, as well as better availability of such devices through commer cial vendors that
pr ovide not only certain pre-designed micr ofluidic chips, but also custom microfluidic chips designed
by r esearchers and fabricated thr ough foundry services.
Second, and closely related to the first r eason, is the rather high operational complexity of
micr ofluidic systems. In addition to the external instruments r equired to drive the micr ofluidic chips
that may not be r eadily available in many life science laboratories, methods and protocols ar e developed
within individual r esear ch groups and rar ely adapted or proven by others. However , this is also
becoming less of a bottleneck as many external instruments that drive micr ofluidic devices (e.g., syringe
pumps) ar e becoming significantly less expensive, more standar d operation procedur es are becoming
available thr ough the large number of micr ofluidics-based papers that are being published, as well as
detailed pr otocol-type papers being published (e.g., through the Journal of V isualized Experiments),
and the lar ge number of micr ofluidics-based papers being published that ar e specifically focused on
simple device structur es or ease of operation for non-microfluidic experts in mind.
Thir d, more specifically to DEP micr ofluidic devices for microalgae applications, various physical
limitation needs to be consider ed. The local electric field gradient is crucial for the DEP for ce. However ,
high voltage r esults in the heating of cells that can negatively impact the cells or bubble generation on
electr odes that make the DEP microfluidic device not function pr operly . Additionally , since the DEP
for ce relies on di ff er ences in dielectric properties of tar get cells versus that of the surrounding media,
most applications so far have utilized low-conductivity media to incr ease the DEP for ce. This makes

Microor ganisms 2020 , 8 , 540 15 of 19
it di ffi cult to apply DEP microfluidic systems for various applications wher e in situ measurements
ar e desir ed or even requir ed. As electrical field responses of biological cells ar e dependent on the
applied fr equency , the capability of accurately measuring DEP responses over large fr equency ranges
ar e gaining importance for applying DEP microfluidic to r ecent microalgae r esearch.
Fourth, biological parameters also need consideration. Unlike many mammalian cells, wher e DEP
micr ofluidic devices have been extensively developed and utilized, many microor ganisms, including
types of micr oalgae, have non-spherical shapes. This adds complexity to not only their DEP responses
but also di ffi culties in accurately predicting and simulating the movement of such cells under the
influence of the DEP for ce. V arious sub-cellular structur es, such as intracellular lipid droplets within
these micr oalgae or external flagella, can also pose significant challenges. Cells that move actively due
to flagella could especially pose challenges for DEP-based cell manipulation and separation.
Finally , another category of biological parameters to consider is the fact that microalgal cell sizes
can vary sever ely depending on their physiological state. As can be seen in Equation (1), both the
cell size, as well as the dielectric properties of the cell, influence the degr ee of the DEP force. Some
micr oalgae, such as Chlamydomonas r einhardtii , show a rather constant cell size r egardless of their
physiological state e.g., exponential growth phase vs. stationary phase, lipid induction state vs. gr owth
phase. However , some micr oalgae cells such as Crypthecodinium cohnii can exhibit significantly di ff erent
cell sizes depending on their gr owth phase or levels of intracellular lipid content. Thus, having
to decouple cell size-dependent DEP e ff ect fr om other phenotypes of interest such as intracellular
lipid-dependent DEP e ff ect becomes important. As most cells have heter ogeneous subpopulations,
such heter ogeneity further adds to the challenges.
Despite these challenges, the unique capability of DEP-based micr ofluidic systems in enabling
label-fr ee manipulation of cells depending on their intrinsic properties r emains an extremely attractive
method for micr oalgal r esearch. Thus, we expect to see significantly more development in DEP
micr ofluidic systems specifically targeting micr oalgae in the near future.
7. Conclusions
Micr oalgae-based bioproduction of high-value biomolecules, including those for transportation
fuel, is a pr omising avenue towards achieving a higher degr ee of bioeconomy , however , it is curr ently
struggling with pr ofitability and, therefor e, commercialization. Conventional methods utilized
thr oughout the microalgae-based biopr oduct development pipeline have failed so far to deliver
economically viable pr oducts in most cases [
82
]. T o achieve the necessary yields and e ffi ciencies, new
or impr oved microalgal strains ar e needed. Many published works on DEP-based microfluidic devices
applied to micr oalgae have shown high potentials for applications in the fields of strain development,
pr ocess analysis, and characterization. Here, we have pr ovided a comprehensive r eview and analysis
of DEP-based micr ofluidic devices utilized for microalgae r esearch, with an in-depth analysis of the
various device categories and application areas of such works. W e have also provided a critical analysis
of the advantages and disadvantages of each method, with a concluding r emark on the perspective
and futur e of DEP-based microfluidic devices for micr oalgae resear ch.
Author Contributions:
Conceptualization, V .A., M.B.; methodology and formal analysis, M.B., P .N., A.H.;
investigation, F .G., A.H.; resour ces, P .N.; writing—original draft preparation, V .A.; writing—r eview and editing,
V .A., A.H., M.B.; visualization, V .A., M.B.; supervision, M.B.; project administration, M.B.; funding acquisition,
M.B. All authors have read and agr eed to the published version of the manuscript.
Funding:
This resear ch was funded by the German Federal Ministry for Education and Resear ch (BMBF) within
the program “Neue Pr odukte für die Bioökonomie“ as part of the “National resear ch strategy bio-economy 2030”
administrated by ptJ Projektträger Jülich, funding number 031B0381.
Acknowledgments: The authors thank Laura Niehaus for providing the micr oalgae picture used in Figur e 3 .
Conflicts of Interest:
The authors declar e no conflict of inter est. The funders had no role in the design of the
study; in the collection, analyses, or interpr etation of data; in the writing of the manuscript, or in the decision to
publish the result.

Microor ganisms 2020 , 8 , 540 16 of 19
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