microalgae cell quantification using electrical parameters€¦ · microalgae cell quantification...
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United Arab Emirates UniversityScholarworks@UAEU
Theses Electronic Theses and Dissertations
5-2016
Microalgae Cell Quantification Using ElectricalParametersLeena Osama Fahmi Saqer
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Recommended CitationFahmi Saqer, Leena Osama, "Microalgae Cell Quantification Using Electrical Parameters" (2016). Theses. 278.https://scholarworks.uaeu.ac.ae/all_theses/278
lmEU (( t )) o� I � jJU I l.:'IIJ La � I Ci.st.o l? '\:J' United Arab Emirates University
United Arab Emirate Uni ersity
ollege of Engineering
Department of lectrical Engineering
MICROALGAE CELL QUANTIFICATION USING ELECTRICAL PARAMETERS
Leena Osama Fahmi Saqer
Thi thesi is submitted in part ia l ful fi l lment of the requirements for the degree of
Master of Science i n E lectrical Engineering
U nder the upervi sion of Dr. Mahmoud Al Ahmad
May 2016
Declarat ion of Origin al Work
L Leena 0 ama Fahmi aqec the under igned, a graduate tudent at the ni ted Arab
Emirate l�n i \ ersit) ( EU), and the author o[ th i the is entit led "lUicroalgae Cell
QII(/l 1t�f;ca{iol7 C\'ing Electrical Parameter " , hereby. olemnly d c lare that thi
thesi i' 111) own original r search \\ ork that ha been done and prepared by me under
the uper\' i ion of Dr. Mahmoud I hmad, in the o l lege of Engineering at A
rhi \\�orh. ha not pre\'iou ly been pre ented or publ i shed, or fomled the basis for
the a\'\-ard of an a ademic degree, diploma or a imi lar t it le at th is or any other
univer it) . Any material borrowed from other sources ( whether publ i shed or
unpllb l i bed) and rel ied upon or inc luded in my the i ha e been properly c i ted and
ach.n \\ ledged in accordance with appropriate academic convention . I further
dec lare that there is no potential conflict of interest with respect to the re earch. data
col lecti n, authorship, pre entat ion and/or publ ication of thi s thesis.
tlld en l' i gnahu'e: __ ---==,..:.:-----.:::::d::-::::...,==---____ _ Date : _�5"-L/_· �---,-' I_L_o_, Co_
i i
Copyright.£; 20 1 6 Leena Osama Fahmi Sager
A l l Rights Reserved
i i i
iv
Appro a] of t he Ma ter The i
Thi Ma ter The i i approved b) the f, l lowing amining Committ e Member :
1 ) d\'i or ( Committee hai r ) : Dr Mahmoud I hmad
Tit le : ciate Profe or
Department of Ie trical Engin ermg
liege of Engineering
ignature �. 2) 1 ember ( Internal Examin r ) : Dr. Falah Awwad
Title: Associate Profes or
Department of E lectrical Engineering
Coli g of Engineering
") 1ember ( External Examiner) : Prof. Mona Zaghoul
Tit le : Profes or
Department of E lectrical and Computer Engineering
Date 2j 6j 2-D I -b
Institution: The George Washington niversity, USA.
Signature -r+r-f--+--r�'-b""'::">';�1--- Date Lfjllf I 20 f b I /
Thi 1a ter Th accepted b} :
Dean of the Col lege of Engineering: Professor abah lka s
ignature _� __ �_�_edz) __ .f" ___ :-=--__ _ Date ---------
Dean of the Col lege of the raduate tudie : Profe or Nagi T. Wakim
igna[ure � Date s=, � , 2 I G
Copy � of�
v
vi
Ab t ract
Electrical pr pert ie of l i ving cel l s have b en prov n to playa sign ificant role
in under tand ing and characterizing the di fferent bi logical act iv i t ies of th cel l . The
obje t iye of this work. is to devel p an lectri al ba ed technique for d termin ing and
e t imating the number of m icroalgae cel l i n a u pen ion without the need for any
ample treatment or pre-proce ing.
The propo ed techn ique i based on the di rect u e of electrical capacit ive
model . fhe ba ic premi e beh ind thi id a is the electrical polarizat ion of microalgae
particles that get charged due to the appl ication of an electric field . Thi wouldn't cau e
an) di tort ion or d i o lv ing/di ffu ion of the content of the cel l s i nside the medium .
Th electrical mea urements of the capacitance - voltage concept i s emplo ed to
determine microalgae cel l count . The microalgae cel l s are considered as dopant
embedded inside a relevant medium. The cel l s count i s then estimated by subtract ing
the i ntrin ic impuri ties of the medium from the effecti e ensemble impuri t ies of the
suspension i nside a defi ned volume. Three strains of m icroalgae. namely
XOl1nochlorop i . Tetra. and Scenedesmu were cult ivated and examined under the
proposed methodology. For validation. samples with unknown cel l counts were
quanti fied u ing the proposed method and compared to other techniques used for
val idation.
Results of the study revealed that cel l count determined with the propo ed
electrical based methodology was done with in few minutes, which is significantly
shorter than al l other reported techniques. The enumeration of microalgae cel l s count
is i mportant for their growth optimization. A real t ime. rapid and efficient technique is
vii
needed � r uch purp e. The propo ed method pro"ided a better combination of high
'en. it i \ it) , quick re pon e, rev. minute . low co t. high throughput. and ea e o[ u e .
[he outcome f thi \-\- ork al low the development of a rapid technique [or the
determination of cel l count o[ microalgae or it could be further extended to detemline
the cel l count o[ other typ of su pended cel l s of comparable size. In addit ion. the
propo ed methodolog) could be upgraded to b appl ied in-situ with a feedback loop
that cou ld al low for cont inuous monitoring of the growth conditions and rapid
detemlination of microalga cel l s count .
Keyword : Capacitance - Vol tage measurement . cel l count, e lectrical parameter,
electrical characterization. microalgae.
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ix
:r �->- J y:4-0 .J:ljUl �;'U.bll � w.r �I J (} yJI .) � � � yiJl ��I rl�1 M jl
.�\.jA.1I �I wJy:, J �l:....hll tly' j....;:,!. I.J� I j.o �..ill�..).c. �� �I J �l:....hll
Ackn owledgemen t
Thi journe) \, ouldn't ha been achievable wi thout the upport of my
belo\ ed hu band. [ami I ) . profe or , and friend . Fir t and foremo 1, 1 thank Hah.
the almight) merc i fu l . for giving me the trength and poyver to carry on thi project
and for ble ing m with man) great people who have been my greate t upport in
m) l i fe.
x
r \\ ould l i k to grateful I and incerely thank my advi or Dr. Mahmoud AI
hmad for hi cont inuous gu idance and under tanding. His patience, support, and
mentor h ip \va of paramount importance in enr iching my educational experience. I
\\ u ld l ike to thank a l l member of the Electrical Engineering Department at Uni ted
rab Emirates University for a sist ing me a l l over m studies and research. M special
thank are extended to Dr. ulaiman Al -Zuhair for his support and assi stance dur ing
the \\ ork i n h is lab, Affi fa and haima Raji who helped me in the experimental work.
pecial thanks go to my husband. parent . s ib l ings. and fami ly who helped
me along the way. I an1 sure they u pected i t wa endless. My thanks are extended
to a l l my friends and col leagues for their support, encouragement, and friendship.
xi
Dedication
To my beloved husband, parents andjanzil)'
xii
Table of Con ten t
fitk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Dc larat ion of riginal v, ork . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 oP) right . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III ppro\ al oCthe Ma ter The i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...................... IV
\bstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . \ I Tit le and b tra t ( in rabic ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vlli
Ack.no\\ ledgm nt . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .
Ded i ation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xl
fable or Cont nts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xl1
Li t or Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XUI
Li ,t or Figure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIV
Li t of bbre\ iation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XVI
Chapter 1 : I ntroduct ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1 . 1 Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1 .2 Research Objective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1 .3 Th i s Significance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1 .-+ The i s Organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Chapter 2 : L i terature Re\' ie\ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
::U Microalgae B ioma s as an Energy ource . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2 . 2 Previou Work on M icroalgae Cel l Count ing . . . . . . . . . . . . . . . . . . . . . . . . . 6
2 . 3 Theory of E lectrical Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2
Chapter 3 : 1aterials and Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5
3 . 1 M icroalgae Cul tivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5
3 .2 M icroalgae E lectrical Polarization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 6
3 .3 Principle of Operat ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 9
3 .4 Experi mental Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Chapter -l: Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Chapter 5 : Conclusions and Future Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
5 . 1 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 3 9
5 . 2 Future Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Bib l iography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . 4 1
Li t of Pub l ications . . . . .. . .. . .. .. .. .. . .. .. . .. .. .. .. .. .. .. .. .. .. .. .. .. .. . .. .. . .. .. .. . .... 46 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
xiii
Li t of Table
Table 4 . 1 : De cription of the microalgae sample te ted and their results u ing the
ql\1 icro too l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Table 4 .2 : Compari on of performance between the di fferent couting technique . . . 3 8
xiv
Li t of Figu re
Figure 2 . 1 : ch matic diagram of th coulter count r working principle . . . . . . . . . . . . . . . . . . . . 8
Figur 2 .2 : qMicro de\ ice u ed to mea ure cel l count . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Figure 2 . 3 : The micropor moni tor the current flow through it and stretches
d pending on the particle pa ing thr uoh it . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Figure 2 .4 : An i l l u trati n of the working principle of the spectrophotometer
nlethodolog) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0
figure 2 .5 : l Iemoc)10meter l id . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II
Figure 2 .6 : Th count ing grid f the hemoc 1.ometer and visible and are visible u ing
the nl icro cope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1
F igur 2 . 7 : Polarization e1Tect of the material when subject to electric field . . . . . . . . . . . . 1 3
Figure 2 . 8 : para l l I-plate capaci tor \ \-i th a dielectric material i n between causes a
charge eparati n i n the internal electric field . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 4
F igure"'.I: Photo-biorcactor used for microalgae cul tivat ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 6
Figure 3 . 2 : ( a ) Random distribution of microalgae cel l s inside the medium. ( b)
Redi tribution of cel l due t o the appl ication of electric field . . . . . . . . . . . . . . . . . 1 7
Figure 3 . 3 : Charging pro fi le of a sample of micro algae suspension . . . . . . . . . . . . . . . . . . . . . . . . . . 1 8
F igure 3 .4 : Polarized microalgae cel l s inside an alt mating current electric field . . . . 1 8
Figure 3 . - : chematic of the charge distribution on the cel l tructure . . . . . . . . . . . . . . . . . . . . . . . 1 9
F igure 3 .6 : Experimental etup of the proposed electrical teclmique . . . . . . . . . . . . . . . . . . . . . . . . 2 1
F igure 4 . 1 : E lectrical measurements of onnochloropsis magni tude measurements
versu frequenc _ of microalgae particles compared to the medium pro file
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Figure 4 .2 : E lectrical measurement of onnochloropsis impedance phase versus
frequency of nucroalgae particles compared to the medium profile . . . . . . . . 24
F igure 4 . 3 : I mpedance mea urements : (a ) phase versus frequency and (b ) magnitude
versus frequency over di fferent periods of t ime . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
F igure 4 .4 : E lectrical measurements of "gonnochloropsi Current-Voltage ( rV) versus
bias profi le . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Figure 4 .5 : E lectrical mea urements of onnochloropsis Capacitance-Voltage (CV)
versus bias profi le . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
F igure 4 .6 : cenedesmus p. microalgae cel l count distribution over their
corresponding diameter using the conventional qMicro equipment. The
i nset is enlargement from 7 to 1 5 /Jm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
F igure 4 . 7 : Current-vol tage (IV ) measurement curves for the 1 6 samples presented i n
Table 1 conducted at 1 0 Hz. The y-axis ( logarithmic scale) unit i s amperes
and the x-axis ( l i near ) unit is volts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3
F igure 4 . 8: Capacitance-voltage (CV ) measurement curves for the 1 6 sanlples
presented in Table 1 conducted at 1 0Hz. The y-axi s ( logarithmic scale )
unit is Farad and the x-axis ( l i near) wlit i s volts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
xv
Figure 4 .9 . omparat i \ e anal) i of the microalgal cel l count perfonned by q�l icro
and electrical method : ( a ) qMicro \ er es Gamr) . ( b )Test of the sen i t i \ ity
of det ction of the electrical method using Gamry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Figure 4 . 1 0 : Comparat i \ e of the cel l count using di fferent method . . . . . . . . . . . . . . . . . . . . . . . . . 37
Li t of Abbreviation
C02 Carb n dioxide
a onductivi�
E Permitti\ it)
V Capaci tance - \ ol tage
{) umber of microalgae ce l l s
Ns Doping concentrat ion of microalgae su pen ion
N M Doping concentration of medi um
Ds Deby \ ol ume of microalgae suspension
DM Debye volume of medium
N Doping concentrat ion
D Debye yolume
q E lectron charge = 1 .602 1 7661 x 1 0- 1 9 C
Es Dielectric con tant of the material
A Overlapping area of a two plates capacitor
C Capacitance
V Voltage
Geometrical length of the host coaxial capacitance
to Debye length
K Boltzmann constant = 1 .38064852 x 1 0-23 m2_kg_s-2. K-1
T Room temperature ( Kelvin)
10 Leakage current
I) I deality factor
xvi
xvii
Is aturat ion current
k Voltage coefficient
r Voltage coe fficient
Eo Vacuum dielectric permittivity = 8 . 85 1 0- 1 2 F.m-I
Er E ffe ti\ 'e permitti\ ity of the u pen JOn
a I nner radiu of the coaxial cable
b Outer radiu of the coaxial cable
¢ Bulk pot ntial
'Jo.,' Depletion v" idth
Chapte r 1: In troduction
1.1 M oth-ation
\1icr algae are on idered a promi ing sour e for biodie el production and
hme the potential to replac petro fuel [ 1 -3]. Microalgae has received an increa ing
focu a a rene\'\ able energ) urce if product ion of microalgae biodiesel and fuels are
to be fea ible and econom ical l ) u tainable [4 ] . Therefore, i t is required to have a deep
under tand ing of the chemical compo i t ion of algae biomass, and opt imization of
cul ture c ndit ion i s needed. The e t imation of algae population is important when
tud) ing th growth kinetic that is highly affecting the biofuels production cycle. in
addit ion to the biochemical constitu nts [ 5 .6] .
Ce l l count detemlination is not an ea y task due to the microscopic size of
algal cel l . Genera l l . convent ional protocols are avai lable and sufficient for cel l
counting. Microalgae ce l l are enumerated with l i ght ab orption [ 7 ] , and by the
detemlination of wet/dry bioma s [ 8 ] . Mo t conventional techniques are in most cases
t ime con wll ing, tedious. indirect. require excessi e sample treatments, and prone to
error. Despite the development of modem techniques, some of the latest
methodologie proposed require sophisticated and expensive equipment such as the
i nverted m icroscope and the chamber [9 ] . In addi tion, i t is t ime consuming. as
ometimes 24 hours are needed for a smal l nwnber of algae cel l s exist ing in a sample
to settle dO'WTI in the chamber. The most common technique for cel l s count is the
hemOC)10meter [ 1 0] . However. in spite of its usage. on- l ine measurement of real t ime
cel l counts i s not possible using this method.
Therefore. i t is desired to have a rapid based monitoring system for cel l
quantification that could b e a leading edge to b e appl ied i n - i tu, which ould be then
integrated in continuou fe dback control loop to adju t the grov, ing condition
\\ i th ut the need for an) time con uming and expensive ample preparat ion . To
achieve thi effecti vely, a rapid method for cel l count ing within less than a minute
rc pon e, equipped with c ntinuou monitoring capabi l i t) i necessary in contro l l ing
and adj u t ing the cult ivation cond it ion to achieve the desi red object ive of the grov\'1h.
1.2 Re earch Objective
The main advantage of thi research is th generat ion of electrical based
methodolog) \\ hich ha the abi l i ty to quant i fy cel l s count. Due to their free label ing
capabi l i t ie electrical characterization technique have received increasing attentions
in the last decade [ 1 L 1 2 ] . FUlihermore, they are noninvasive characterization tools
that can detect and quantify unl imi ted type of materials without the need for any
ample treatments or preparations. Therefore the main research objectives of this thesis
are :
1 ) Develop a rapid electrical based technique for microalgae cel l cOlmting.
2) Pre ent a veri fied correlation to relate the electrical parameters with cel l s count .
3 ) Compare the efficiency and sensit ivity of the presented approach \ ith other
methodologies proposed in earl ier research work.
1 .3 Thesis Significance
Although UAE i s one of the largest o i l exporters. the production of biodie e l
fal l s "\" i th in the country' s vision to sat isfy the coming generation's need for c lean
energy. The reshaping of other energy resources is needed for a sustainable fuel
production. M icroalgae have been uti l i zed worldwide for biodiesel production. The
UAE enjoys abundant and continuous sunshi ne throughout the year, combined with
avai l ab i l i ty of seawater, which makes it ideal for microalgae cul t ivation. These
, J
features d.::arl} ho\.\ the potent ial of cult ivat ing microalgae in large cale in the E .
j 'c\ erthelc , an economical biodie el production from microalgae require high
bioma producti\.it) .
Thc outcome o[thi re earch \\ uld a l lo\\. the development of a rapid teclu1ique
for the enumerat ion of cel l count! concentrat ion, which i s v i tal for the effect ive
manipu lation f the cul t ivation condit ions to achieve the de i red objective. Thi
certain ly enables the adj u tment of the groV\ih condit ions in real t ime environment.
Be ide microalgae, thi approach could be appl ied to other t) pe of organi m as wel l .
The l ipid monitoring and quanti fication a t the cel l Ie e l could be effectively used i n
the opti m izat ion of the gro\\ih rate and l ip ids productivity .
lA Thesis Organization
The thesi is organi zed as fol lows :
Chapter 1 ( I ntroduction) : This chapter presents the motivation and objectives
of this ,,;ork. I t starts with explaining the demand for microalgae cel l counting and the
technologies used. The chapter end by stating the objective and the sign ificance of
this research .
Chapter 2 (Literature Review) : This chapter reviews the pre iOlIS research
work related to the topic of the thesi s . It discusses the different techniques used to
determine cel l s concentrat ion. It also presents the theory of characterization technique
proposed i n this work.
Chapter 3 (Materia ls and Methods) : This chapter demonstrates the materials
and too ls used. i n addit ion to the methodology and experimental procedures conducted
in order to perform the desired measurements and analysis.
4
Chapter 4 ( Re ul t and Oi cu sion ) : Thi chapter hows the resul t gained from
performing the xp riment . the proce ing and analy is of data, fol lowed by a
di cu ion of the re ul t and the alldation u ing a different technique.
hapter 5 (Conc lu ion and Future Works): Thi chapter is the last one in the
thesi that \\Tap up all the re earch findings, and relat s them to the object i \ 'e
pre entcd i n the first chapter.
5
Chapter 2: Literature Review
In thi hapter, \\ e pre ent an O\ erVleV, of the importance of microalgae
bioma a an energy ource. and di fferent technologie developed to enumerate its
cel l COllnt. and then highl ight their di advantage and l imi tations. This chapter al 0
intr duce the theory of the electrical characterization technique presented in this
"\ork.
2.1 Microalgae Bioma a an Energy Sou rce
1icroalgae are a diver e group of photos nthet ic microorganisms that can
gro\\ and l i\e in fre h or marine water nvi roru11ents that conveli inorganic carbon l ike
C02 into algae b iomass i n the presence of water. l ight and nutrient [ 1 3 ] . They grow
ei ther autotrophical l y or heterotrophical ly . The autotraphic growth require C02,
l ight. and nutrients to grow. whereas heterotrophic algae needs organic carbon source
l i ke g lucose which i a food source, in addit ion to nutrients [ 1 4] . They can grow rapidly
and l ive in harsh condition due to thei r imp le unicel l ular or multicel l ular structure.
They exist as colonies. fi laments or ameoboids. They convert solar energy i nto
chemical energy completing a ful l growth c cle ever few days [ 1 5 ] . M icroalgae stores
energy in a form of it main compon nts of l ipids, carbohydrates, and proteins [ 1 6, 1 7 ] .
This chemical composition d iffers depending on the microalgae spec ies which depends
on the temperature. l i ght i ntensity_ sal in i ty, PH of the med ium, and the growth phase,
which al lows using the microalgae in d ifferent appl ications ranging from food
products to biodiesel product ion. The global c l imate change and the rise of
transportation fuels has resul ted in focused interest on generating power from
renewable sources to meet the i ncreasing energy demand [ 1 8 ] .
6 Man re arch reports dem n trated the ad, antage of using microalgae for
biodie el production 0\ er ther avai lable fe d tock [ 1 . 1 9-23 ] . From a practical point
of , iew. microalgae are a ) to cult ivate and gro\\ extremely rapidly and they are rich
in o i l [ 1 ] . The) can gro\\ any\.\ h re \\ i th l i tt le anl unt of simple nutrient and un l ight.
and the grO\\ 1h proc s can be accelerated by adding more nutrients and needed
aerat ion [ J �] . nother advantage o f microaJgae biofuel i s that i t can replace petro
fuel a they are expensive source and they stat1ed exploit ing at the expense of
em ironm nt. variet; of micro a lgae specie are adapted to grov\ and l ive in di fferent
em i ronmental condit ion , which i not easy to find with other biodie el feedstocks.
l icroalgae are a great ource for different type of fuels including biodiese l . methane,
ethanol . and h, drogen. The performance of algae biodiesel i s imi lar to the petroleum
diesel and does not contain any sulfur [ 1 5 ] .
l though m icroalgae lipids and carbohydrate are the most a luable
component in the context of biodie e l production process, the other principal
biological components i nc luding proteins of the algae biomass i s i mportant to be
estimated. The complete chemical composition is needed to determine the economics
of the production process and the measurements of each component separately are
important for cost detem1 ination [ 1 7] .
2.2 Previous Work on Microalgae Cell Counting
The b iovol ume method is a set of mathematical equations ba ed on geometric
models that calculates the microalgae b iovolume for more than 850 types of pelagic
and benthic marine and freshwater microalgae. The rule of this technique is only
appl icable to i ndividual cel ls . The mathemat ical model min imizes the eITor,
inexpensive, and easy to apply . It also provides a h ighly systematic reso lut ion of the
7
identi fied cel l . On the other hand. thi method doe not cover a l l t) pe of algae hape :
re-examination of the equation u ed i s needed. Becau e of the variation in the
microalgae l i fe Cy c le that affect i t cel l size. the method has problems in accuraC) .
1 herefore. depending on the average biovol ume mea urement is not enough . I t ha to
be calculated for e\ cr) ample tak ing into consideration the di fferent depths in the
ame medium at each cxperiment. in e the gen ral rul of this method cover
individual cel l n l ) and thi is d i fficult to appl) in some ca es. the shape can be
appl ied to th colon) \\ i th each ingle cel l mea ured . Th biovol ume t chnique may
ovcre timate the size oflarge algal cel l s with a higher relative vacuole volume [24.25 ] .
The electronic part ic le counter technique provides pruiicle sizing distribution
of cel l . I t is a lso referred to i t as a coulter counter. The idea of this method is the use
of an aperture that a l low the pa sage of an electri c CUlTent through it given that partic les
are uspended in an electrolyte sol ution. A change in resistance i a result of passing
the CUITent t l u'ough the aperture which results in a change in the CUITent and voltage
cOITespondingly . This variation in resistance can be converted to a part ic le count
electronical ly and i t j proportional to the size of the part ic le itself. A fu l l diagram
pre enting the mechanism is shown in Figure 2 . 1 . Different ranges of partic les size can
be counted and this is l im ited by the size of the pore used in the experiment . The sizing
i s independent of the part ic le shape and it orientation i n the sol ution. Up to 500 cel l s
can be counted and sized per second. This methodology i s straightforward and fast;
however. it is vulnerabl e to elTors due to the presence of ce l l c lusters [26,2 7 ] .
External Electrode Internal Electrode JI
R R .?' Sensing Zon.
•
• Particles In Electrode
Transducer Chamber
Enemal Electrode (+) -...,.... .......
8100d�1I Suspension -�-
-E-- Constant DC Current
- Source .. :
, -'-
'ntom.1 ElectTod.(.)
Apertur.
Aperture Tube
Figure 2. 1 : chematic diagram o f the coul ter counter working principle
8
RFPower Soun:e
The q hero i one of ueh exist ing tools as i t appears in Figure 2 .2 . The qM icro
apparatus al lows one to measure the ize of biolooical or synthetic part icles in a size
range between 1 -300 jll11 i n a \'ol ume of 1 jl l to 1 ml ( IZON. ew Zealand) . The
principle of re istive pulse-sensing ( RP ) is adapted by the qMicro device. which
monitor the cunent flow through the pore ( Figure 2 . 3 ) . The aperture size i s adjusted
al lowing a l imi ted range size of particles to be regulated by the passage of ionic cunent
through the pore [ 28 ] .
Figure 2 . 2 : q 1icro device used to mea ure cel l count
Current
• •• • ••
If t
�/·qrrf!" lT 1V · "'TTII--r-"I'IIrr-1 --rlllr-T"I t ( Olocnad <;
9
Figure 2 . 3 : The micro pore monitors the current flow through it and stretches
depending on the part ic les passing through it
pectrophotometry i a method used to relate the algal density to the scattered
l ight absorbed by p31iicIes in suspension at pec ific wavelengths ( see Figure 2 .4 ) . The
\\ avelength range of the e lectromagnetic spectrum in the visible range from 400 to 700
nm is used to determine the algae biomass. It is an accurate technique but sometimes
used to find relative estimates of cel l density . S ince cel l s are d isorganized and
randomly distributed. if the cel l density increases, less l ight wi l l pass through the
cuvette. pectrophotometers are not considered to be a rel i able method for cel l density
est imation as they do not actual ly count ce l ls but rather measure absorbance. that i s
1 0
affected by \ ariable omponent of cel l su p n ion . The u e of l ight ab orption i
more adequate for the e t imation of the population ize of microalgae rather than the
d t rminati n of the a tual numb r of individual cel l . Thi would not al low
distingui h cel l type , and the a e ment of c 1 1 viabi l i ty [ 7, 29 ] .
LIght Source
Collimator
Monochromator
(Prism of Grating) I I
Cuvette
(Sample Solution)
Slit
(Wavelength Selector)
11 U
Detector
(Photocell)
Figure 2 .4 : n i l l ustrat ion of the \',;orking pri nciple of the spectrophotometer
methodology
Di gital Display
Another technolog, presented in a device cal led counting chamber that
determines the number of cel ls per unit volume of a suspension. The most widely used
typ of thi chamber i the hemocy10meter. I t i a special type of m icro cope chamber
s l ide i l l u trated in F igure 2 . 5 that is divided into squares of a defined area over which
the volume of a uspension is d istributed ( Figure 2 .6) . Using the hemocytometer, the
number of cel l s can be determined in a cu lture after tain ing . Some calculations need
to be appl ied after to find the total number of cel l s per ml. The counting procedure
require a l ight or phase contrast microscope. the hemocytometer itself, and a tracking
device, uch a a handheld . This cost of th is setup can range from a few hundred dol l ars
to everal thousand dol l ars. Proper washing and loading of the device are needed to
start the operation. Analyzing the results is not t ime consuming and requires a few
manual calculations. The measurement process i s tedious and subject to errors due to
I I the cel l c l umping or heterog neit)' of the cel l ize. I t i al a su cept ibl to error due
to the mal l numb r of ce l l c unted if Ie than 1 00 [ :'0.3 1 ) .
F igure 2 . : : I l em c)iometer l ide
Small sqUAre = 11400 sq mm 1/25 sq mm
� CouIIllng gnd (cenna! area)
Figure 2 .6 : The count ing grids of the hemocytometer and vis ible and are visible
using the m icroscope
Most conventional techn iques for the microalgae cel l s enumeration are t ime
consuming and i n most cases are require tedious steps and requ i re sample treatments
and preparations steps. In addit ion to this, the avai l abi l i ty of sophisticated equipment
1 2
in the pre\ iou I ) ment ion d te hnique adds m re co t and decrea e the opportunity
of u ing them . Furthermore. on-line mea urement of real time cel l count i not
po ible u ing uch method .
2.3 Theory of Electrical Characterization
Electrical characterization and pec i fical l) die lectric mea urements is the most
intere t ing technique becau i t " a traightfonvard. measure cel l counts directl .
cont inuou 1) . and in real -t ime. A material that has the abi l i ty to charge \ i thout
conduct ing it to a ign ificant degree is dielectric materia l . The d ifferent materials
\ \ uld have \ ariat ion in their capacitance that upports the induced charge [ 32 ] .
Ever) material has t\VO types o f charge that are posi t ive and negat ive particles.
nder n011l1al condit ions. the total charge in any area of the material i s equal to zero
becau e of the trong bond and mutual attraction that keep these d ifferent charges
together. When the material is exposed to the appl ication of electric fie ld. charges
experience electrical force that drives the charges to move freely in the material [ 3 3 ] .
The movement of charges i s basica l ly dependent o n the trength o f the bonds between
the two part ic le . I f it was weak, then they are free to move through the materia l . and
the material i cal led electric conductor. However. for an electric insulator, charges are
a l lowed to move s l ight ly from their posi t ions as their bonds are strong. As a result . the
dielectri c material can be conductive as long as i t polarizes [ 34 ] .
The polarization of an material i possible provided that electric dipoles exist
in i t . When the materia l i s placed between two electrodes. and an e lectric field is
appl ied, one surface of the electrodes develops a net negat ive charge whi l e the other
1 3 urface ac umulaled the po iti \'e charge. Thi i i l l u trated in F igure 2 . 7 below.
N o e lectr ic fi e l d App l i ed e l ectr ic fie l d
Figur 2 . 7 : Polarizat ion effi ct of the material when subject to electric field
1 1 electric d ipole or impurity i a re ult of the presence of two opposite charges
separated b) ome distance [ 3 5 ] . When the die lectric material i placed in an electric
field, the d ipole of the material a l ign in the electric field as shown in Figure 2 . 8 . So
within the material . the electric dipoles wi l l cancel each other but at the surface the
dielectric wi l l attain the net posi ti ve charge (+Q) on the negat ive electrode and the net
negative charge (-Q ) on the po i t ive lectrode [ 36] .
The polarizat ion mechanism is dependent on the nature of the material under
test. General ly . material polarization doesn ' t occur instantaneou ly once the material
i s subject to electri c field appl ication; i t takes some t ime to react to changes [ 3 7] . And
due to tlus, conduct ivi ty , permitt i ity, and other factors are frequency dependent [ 3 8 ] .
1 4
D i e l ec t r i c mate r i a l
•
Figure 2 . 8 : paral lel-plate capacitor with a dielectric material in bet"\\. een causes a
charge eparation in the internal electric field
The electrical pr pert ie of material are a correspondence on ho the material
react ,,,, hen it i exposed to electric field appl ication. The response of the material is
described by d ifferent e lectrical propert ies such as the conductivity ( (1 ) . and
permitt ivity (E) [ 39] . The conducti ity parameter measures the level of charge
conduction through the materia l . and pennitti i ty parameter measure the charge
storage level due to the polarizat ion. It is impOltant to note that both parameters are
i rrespect ive of the materia l ' s d imension [40.4 1 ] .
I n order to measure these parameters, the material should be placed 1 11 a
capaci tor. A capacitor is described as any device that stores charge, but usual ly it
consists of two conducting plates with a dielectric material in between [34 ,4 1 ] .
Parameters can be determined by conductance and capacitance measurements.
Conductance i measured from the magni tude of the electric current pa s ing through
the material as a function of the voltage appl ied, whereas capacitance is defined by the
amount of charge stored on the plates as a function of the voltage appl ied .
1 5
C h a p t e r 3 : M a te r i a l a n d M et h od
Thi chapter d m n trate the material u ed and the experimental proc dures
conducted in thi the i \\ rk in ord r to determine the cel l count [or d ifferent
micr algae trains.
3.1 Microalgae Cultivation
The cul t i \ ation approach \.V a done for di fferent strain of microalgae \ hich
are XOl7l7och!orop. is. 7>,,. I and L cel 7ede mils p. The were al lov,'ed to grow in
nutrient - rich med ium ( F/2 ) and ( 3 - - BBM ) hown in appendix A [ 42.43 .44]
re pecti\ e ly for tv. o w ek in a 5L bubble column bioreactor to enhance the biomass
productiyit) . The prepared med ium was purified in an autoc lave ( Hi rayama HV -50.
Japan) for 1 5 min at 1 2 1 °C and cooled to room temperature before using i t [ 45 ] .
The 5 L bubble colunID photobioreactor has an outer d iameter of 1 0 cm , an
imler diameter of 5 cm. and 40 cm height. I t was i l luminated with one 50 cm. 60 watts
\\ h i te fluore cent l ight at an inten i ty f LO �mol m-2 S- l . A l l the cul t ivation i n this
\\-ark were autotrophic, mean ing that C02 is the sole carbon source i n the system and
natural ly presented in a ir bubbled through the ystem [46 ] . A photograph of the
bioreactor is i l lustrated in F igure 3 . 1 .
1 6
Figure 3 . 1 : Photo-bioreactor u ed for microalgae cul t ivation
3.2 Microalgae Electrical Polarization
The \\ ork in thi the is demonstrate the use of semiconductor theory and its
ba ics and principles to achieve the main objecti e which is the enwneration
microalgae cel ls . The principle behind the idea was the polarization of the microalgae
cel l as a resul t of exposing i t to the appl ication of electric field [47] . Microalgae cel l is
basica l ly compo ed of protein . carbohydrates. l ipids. nucleic acids and other minor
constituents [47. -+8 ] . The polarization depends on the composition of the cel l itse lf
and i t interaction \\'ith the cult ivation medium polarity [ 49 ] . Genera l ly, m icroalgae
cel l are randomly distributed in the medium as shown in F igure 3 .2 (a) . The cel l s
could be redistributed according to their polarizat ion; the negat ively charged cel l s w i l l
be attached or get c loser to the posit ive e lectrode surface. whi le the posit ively charged
cel l s ",; i l l be attached or get c loser to the negat ive electrode surface; Figure 3 . 2 (b )
demonstrates this .
(a )
M icroa lga ce l l med i u m
( b ) +ve
e l ectrod�
- ve e lectrodes
Figure 3 .2 : ( a) Random di tribution of micro algae cel l s inside the medium. ( b )
Redistribution of cel l due t o the application of e l ctric field
1 7
To demon trate the polarization of microalgae cel l s, a sample of microalgae
LIspen ion wa loaded into an electrical analyzer ( Gamry Reference 3000) and
mea ured it charging profi le . The re ul t is shown in F igure 3 . 3 . Based on the e results,
the microalgae part ic le could be presented as an e lectrical dipole that has two pairs of
electrical charges of equal magnitude but opposite s ign, separated by some d istance
( d ) [50] as depicted in Figure 3 .4 . Therefore, i t is sugge ted to consider a single
m icroalgae cel l a an " impurity" or "dopant" that ex i sts in a non-intrinsic
emiconductor materia l . A schematic of the charge distribution inside the cel l is
depicted i n Figure 3 . 5 .
1 4
1 2 -2 (5 G 1 0 Q) OJ � (5 0 8 >
0 6
o 50 100 1 50 200 Time (Seconds)
250 300
Figure 3 . "' : Charging pr fi le of a ample of microalgae suspension
+Q
+Q
F igure 3 .4 : Polarized microalgae cel l inside an alternat ing CUITent electric field
1 8
Figure 3 . 5 : chematic o f the charge d i tribution on the cell structure
3.3 Principle of Operation
1 9
The principle of the propo ed electrical approach i s to calculate the effective
dopant concentrat ion of microalgae suspension, which represents the microalgae
dopant ummed with the intrin ic dopants of the cult ivation medium. The cultivation
medium intrin ic dopants are embedded and can be found by subtracting i t from the
effecti\ e dopant concentration value. The num ber of cel l s in a suspension (B ) is
e t imated u ing Eq. ( 3 . 1 ) [ 5 1 ]
( 3 . 1 )
\',-here. ( Ns ) and ( NM ) are the doping concentrations of the suspension containing the
microalgae particles and that of the cel l s free medium, respect ively, and Ds and DM are
the Debye volumes of both suspension and medium, respect i e ly . The doping
concentrations ( N ) and the Debye volumes ( D ) are determined from the capaci tance-
voltage ( C V ) measurements, as given in Eqs. ( 3 . 2 ) [ 52 ] and ( 3 . 3 ), respectively :
( 3 .2 )
D = Lb ( 3 . 3 )
20
\\ here. q i e lectron charge. lOs i the dielectric on tant of the u pen ion (or m dium),
A i the o\ crlapping area. ' i the mea ured capaci tance. � ' is the appl ied vol tage. and
Lv i Debye electrical length. determi ned b ' Eq . ( 3 .4 ) [ 53 ]
( 3 .4 )
\-" here. K i Bol tzmann con tant ( 1 . 38 x 1 0 -23 J/K) . and T i the kelvin temperature
[ 54 ] .
3 .... E x perimental Setup
To c nduct the experiment. d ifferent microalgae trains were used and
anal) zed in this study. The fir t tep in thi approach is to have the microalgae strain
and cul t ivate it in a photo-bioreactor with its corresponding medium ba ed on the type
of algae u ed under pec ific condit ion of l ight and nutrients. To perform the e lectrical
mea urement . ample of m icroalgae suspen ion were col lected from the bioreactor
O\ er predetennined periods of t ime. a wel l as amples from the control medium . They
were loaded into an open-ended coa. ial capac itance structure, and modeled as a
dielectric materia l . The coax ial capacitance adaptor was used as a the host structure
and it ha an i nner and outer conductors with dimension of 2 mm and 5 mm ,
respect ively. and a length of 7mm.
The main advantages behind using this topology i s that the radio frequency
signal propagations are protected from the outside i nterferences that could lead to noise
results and the signal s do not escape between the inner and outer conductors [ 55 ] . The
host structure is d irectly connected to the e lectrical analyzer ( Gamry Reference 3000).
The different set of e lectrical measurements for both the microalgae
suspenslOns and their relative media were perfonned including impedance.
capacitance-vol tage (CV), and current-voltage ( IV ) measurements. The ful l diagram
2 1
o f the experimental etllp of the prop ed electrical ba ed technique i depicted in
figure 3 .6 .
Figure '" .6 : Experin1ental setup of the proposed electrical techn ique
The mam component of thi s experiment \ hich i s the electrical analyzer
( Gamr)' Reference 3000) that is used to make measurements on electrochemical cel l s
that has the conduct iv i ty property . The electrical analyzer ha a current USB
potentiostat \\i'ith 1 1 current ranges from 3 amps to 300 picoamps of high performance.
and a voltage that reaches up to 32 \'olts . The measurements are conducted over a range
of frequency from 1 0 IlHz to 1 MHz using an electrochemical impedance
spectroscop . Performing impedance measurements was done over a range of 1 00
mHz to 1 00 KHz with an osc i l lation appl ied at a voltage of 1 5 m V pp. Frequency range
selection was based on measurements conducted earl ier for d ifferent m icroalgae
strains at h igh frequency from 1 MHz to 1 3 . 5 GHz. A better ident i fication of
microalgae frequency responses was found on the range 1 00 mHz to 1 00 KHz. This
wi l l make identification and characterization of microalgae much easier using i ts
peci fic . . ignature" ' . a w 1 1 a e'{ploring it viabi l i t) for t i ter quantification. Power
signals are pr duced at a range of frequenc ie u ing the radio frequency generators.
The de\ ice i al 0 u ed to mea ure CUlT nt-voltage, capacitance-\ oltage,
p larilation a wel l as charging/di charging pro fi l e with the abi l i ty to change ome
parameter ba ed on the mea urement requirements. The ystem was cal ibrated before
the tart ing of the experiment u ing the provided manufacturer cal ibrat ion kit to
guarantee that the mea urements actual ly present the samples under test . An losses
or pha e h ifts are excluded in order to avoid adding noise to the measured signaL
hence: the reference \va moved to the te t cables ends [ 56 ] .
C h a p t e r 4 : Re u l t a n d D i c u io n
To d mon trate the concept and operat ional principle. di fferent microalgae
train \\ re u ed and t be te ted and analyzed in thi tudy. namely Xal1l1ochloropsi
sp and Telra. vy hich are a maline train . and cenedes17111s sp . . which i a fre h water
train . Thi chapter di u es the re ults obtained from the conducted experiments on
microalgae r r cel l quant i fication.
et of electrical mea urement were p rformed on the microalgae samples
and r lati\ e media. The) \\ ere col lected at predetermined periods of time and loaded
into a coax ial capaci tance cable to perfom1 th exp riment. The setup was cal ibrated
before mea urement to n ure that it actual ly pre ents the ample under test. The
cal ibration excludes the effect of losses and phase shifts due to the cables and host
tructure ,,"hich could add noi e to the measured signal .
The impedance magnitude and phase of the al l the tested microalgae
uspen ions and their COITe ponding blank medium were measured at mid-band
frequency of the capaci t ive region to en ure the capaci t ive beha ior of the sample
under te 1 . A sample of Nannochloropsis uspension and control medium
mea urements conducted i depicted in Figures 4 . l and 4.2. These graphs represent
the phase and the magnitude of the impedance of the control medium and the
u pension. re pecti ely. This show that the DC field polarize the algae due to the
i nduced charging effect. F igure 4.3 (a ) and (b ) how how the impedance magnitude
and phase change over time for measurements on the same type of strain Scenedes177us
over a period of t ime. The microalgae cel l exhibits a capac it i e behavior and i t
increases with t ime. This is shown from the impedance phase degree that ranges from
1 0k ......... (f) E
.c 0 '-"
(1) 1 k -0 :::J C 0> co 2 1 00
1 0 �������=L����=L�U=��W 1 0m 1 00m 1 1 0 1 00 1 k 1 0k 1 00k
Frequency (Hz)
Figure 4 . 1 : E lectrical mea urements of jI/annochlorop i magnitude measurements
\ ,ersu frequency of micro algae part ic le compared to the medium profile
0
- 1 0
- -20 (1) (1) L.. 0> -30 (1)
-0 (1) -40 (f) co
.c -50 0....
-60
-70
1 0m 1 00m 1 1 0 1 0 0
Frequency (Hz ) 1 k 1 0k 1 00k
Figure 4 .2 : E lectrical measurements of Nannochloropsis impedance phase versus
frequency of microalgae part ic les compared to the medium profile
24
2 5
( a ) 0
- 1 5 ... . ....
....- wIth t i m e <l> <l> � 0> <l>
"'0 -..-
<l> U') co -60 .c. 0...
- 7 5
- 90 1 0.1 1 0c 1 0 1 1 0� 1 02 1 0! 1 0=
Frequency (Hz)
(b )
1 0=
....-(J) E with tim e
.c. 1 0· 0 --
<l> "'0 :::J 1 O�
.� C 0> co �
1 O�
1 01
L-�������������-L��L-��
1 0.1 1 O� 1 0' 1 0=
Frequency (Hz)
F igure 4 . 3 : I mpedance measurements : (a ) phase versus frequency and ( b) magnitude
versus frequency over d ifferent periods of t ime
Th I and
26
mea ur ment are repre ented in Figure 4 .4 and 4. �
rcspect i \ c l) . The J pro fi le xhibited a chottky- l ike diode performance of typical p-
'emiconductor mal rial ince the " tum on" vol tage is of posi t ive alue . The CV
mea urement revealed that the microalgae part ic le exhibited a h igher dielectric
con 'tant than their re lati \ e blank medium. Th host tru ture used in these DC voltage
mea uremcnt \\ a an open-ended coaxial cable that form a capaci tance wherein the
microalgae u pen ion act a it di lectric material . The CV profi le displays a smooth
frequen ) beha\ ior with a higher capaci tance of the u pension compared to the
cul t i , alion medium.
1 m �------------------------------------�
1 00�
-CJ) L. (l) 1 0� 0.. E <{ '-' � 1 � c (l) L. L. ::J U
1 00n
•
1 0n - 1 .0 -0 . 5 0 . 0 0 . 5 1 .0
Bias (volts )
Figure 4 . 4 : E lectrical measurements o f annochloropsis Current-Voltage C I V ) ersus
bias profile
----LL '-""
Q) (,) c
� ro 'u ro Q. ro 0
5� ,---------------------------�
4�
mlcroalge
3�
2�
m e dium
1 � /
O �--�--�--�----L---�--�--�--� - 1 .0 -0 . 5 0.0
Bias (vol ts) 0.5 1 .0
27
Figure 4 .5 : E lectrical measurement of rannoch/orop is Capacitance-Voltage (CV)
yer u bia profile
The difference between the measured capac i tance i due to the volume of
microalgae partic les suspended in the medium and their intrinsic prope11ies. As a
re ult the microalgae demon trate a semiconductor behavior, which could help i n
deteml in ing the cel l s count of the microalgae in a medium. Along wi th the electrical
mea urements conducted. the cel l count and the size distribution of m icroalgae
part ic les were detemlined using the qMicro .
The qMicro tool al lows one to measure the size of biological or synthetic
part ic les in a ize range between 1 -300 �m in a volume of 1 �l to 1 ml ( IZO , e\
Zealand ), The cel l concentration versus size d istribution i s depicted in F igure 4 .6 . The
basic premise of the qMicro tool i s the principle of resi stive pulse sensing ( RPS) which
monitors the cun'ent flow through a pore, a l lowing the ionic current passing through
the pore and part ic les to be regulated by adj usting the pore size. The cost of this device
2 8
i high. and require qual i fied and \\- e l l trained people to hand I the e periment . and
understand the anal) i f the resu lt \ .. hich i s done automatica l ly by the oftwar .
q hero pro\' idc the di tribution of cel l s sizes and count directly. I t measures the
indi\ idual micropa11icle izes in a olution. and counts the number of part icle in the
loaded anal)' i 'v ol ume to calculate the cel l concentrat ion. There is a l i near
relation hip exi t between the change in the electric resi stance that re ults from the
pa age of th ionic current . and this a lue is correspondent to the d i fferent part icles
\ o lume. l i near cal ibrat ion curve to a series of cal ibration part ic les of various
d iameter is created and then appl ied to calculate the size of "unknown" micropartic les
The procedure is a bit lengthy; it requires around 30 minutes for each sampJe
to be measmed becau e at each experiment. the device need to be cal ibrated with the
conducti ve sol ution and then measure the suspension sample. The real t ime scanning
of the pore conductivi ty at di fferent tretches enables the detect ion and di crimination
of micropart ic les in a mixed mult imodal suspension. Ho\ ever. when using a single
micropore in an experiment. the ize d istribution cell detection is l im i ted b the pore
ize. As sho\\TI in F igme 4.6, the total number of microalgae part ic les , which is the
sum of a l l exist ing d ifferent sized part ic les, is of l Ox 1 06 part ic les per 1 m! . The
m icropore fi l ter size used \vas of 25 )..tm size which only al lowed the size in the range
of 1 to 50 )..tm to pass [28 , 57 ] .
1 . 5x 1 06
E -Q) () :p ro 1 . 0x 1 06 0... C o :;:::; ro .b � 5.0x 1 05 () C o o
0 . 0 5
E OJ u
1 5x10'
� 1 Ox10 0.. �
c o � � 5 Ox 10' � c o ()
0 0 1 0 15 Diameter distribution(mlcrons)
1 0 1 5 20 25 30 35 40 45 Dia mete r d i stri bution (microns )
29
Figure 4 .6 : cCl7ede mils p. microalgae cel l count di tribution 0 er their
corresponding diameter u ing the conventional qMicro equipment . The inset is
enlargement from 7 to 1 5 I-lm
The e lectrical measured data \ as then used \vith the hel p of equations 3 . 1 - 3 .4
to detennine the m icroalgae cel l count. Before this tep. the measurement conducted
on each sample were processed with a developed program in MATLAB ba ed on the
theory explained earl ier. A mentioned previously. the proposed methodology uti l izes
the use of semiconductor model . The extractions of the semiconductor parameter set
associated with each measurement are carried out through fitting the resul ted Current-
Voltage ( IV ) and Capacitance-Voltage (CV) curves . The fitt ing procedure is based on
construct ing a curve using a mathemat ical fomlUla that best fi ts the set of plotted data .
The simple re ul t of IV measurements can be used to provide a large amount
of u eful information. By fitting the forward characteristics of the IV curve. Two types
of fitt ing were used in order to extract the first set of parameters; with a third degree
polynomial represented by Eq. (4. 1 ) the leakage current ( 10 ) and conduct ivity ( (J ) are
3 0
e tractcd \\ hi le fi tt ing the I mea urement \\ i th exponential fi tt ing i repre ented by
Eq . (4 . 2 ) [ 58 ] . re pect i\ el ) . th id a l i t} parameter ( ? ) i determined. The mobi l ity
could be d termined c rrespondingl) u ing Eq. ( 4 . 3 ) [ 59 ] .
I = 1 0 + or + k T ' ? + yr '
1 = lo exp(q 1 - 1 17K T )
( 4 . 1 )
( 4 . 2 )
(4 . 3 )
\\ here I , i the aturation cunent. K i Boltzmann constant ofl . 3 806488x 1 0-23 J/K. T
i the te t temperature of 300K. and q i the Jectron charge of ] .602 1 7657 x 1 0- 19 C .
k and y are \ ol tage coeffic ients.
C mea urements are performed in forward bias with a l imited DC voltage
bia from -] V to + 1 V. Rather than plott ing dC/dV. i t i s sometime desirable to view
the data a l /C2 vs. vol tage becau e some parameters are related to thi type of data.
For example. the doping density (N ) was derived from the slope of the l inear region of
the CV curve. and \\" ith the he lp of Eqs. ( 4 .4 - 4 .6 ) [ 53.60 ] , the doping concentration
( ) dielectric constant ( c, ) are then computed .
x = hc,/f [d(l l C"2 ) 1 dV ] = 2 /(qc, A"2 ( slope» )
C = 27fc} / ln (b l a )
(4 .4 )
(4 . 5 )
( 4 .6 )
\\"here A i s the capac itor area (94 .2478 nunl ) , and C is the represented coaxial cable
capaci tance. f O i s the vacuum dielectric permitt i i ty ( 8 . 85 x l 0- 1 1 ) • Er i s the effective
permitt ivity of the suspension, I is the cable length, a and b are the i nner and outer
rad iuses. respectively. ext, the Debye Length (LD) , bulk potential (¢) and the
3 1
depiction v, idth ( 11 ) are computed u ing Eq . ( 4 .6 and 4 . 7) [54 ] and Eq. (4 .9 ) [ 6 1 ] .
L / K'T ' \' = \ E:, q - .
¢ = KT ln ( \ / r; )/ q
TV = 4c,¢ / .\ q
(4 . 7 )
(4 . 8 )
(4 .9)
For the data pre ented in Figure 4 . 1 - 4.2 . and 4.4 - 4 .5 . the corre pond ing
cel l count i found to be o f 8 . 7 x 1 06 cel l in a olume ofone mi l l i l i ter. This di fference
between the qMicro and our electric teclmique is most l ikely due to the fact that any
" impuritie . ,
comparable to electron izes (� 2 .82 x l 0- 1 3 cm) were calculated by our
method. \\ h i le qMicro detection has a ize l imitation of the micropore fi l ter used.
The average t ime taken for microalgae cel l count ing usmg the electrical
technique ,va about 5 minutes. This is potent ial ly less than a l l other conventional
technique . and was attained without any t ime consuming sample preparat ion or
treatment tep . It is wOlthwhi le to mention that the t ime needed to perform the qMicro
was about 30 minutes as it needs to fi r t perfonn a cal ibrat ion on each sample and then
the sample measurements, whi le with the current propo ed teclmique. there was no
need for sample preparation.
The accuracy and reproducibi l ity of the presented method was val idated by
repeating the electrical measurements both the current-vol tage and capac itance-
voltage for mul t iple m icroalgae strains prepared at di fferent t imes. For further
validation and statistical anal is. a total of 1 6 samples were considered in this study.
Table 1 shows the types of m icrolagal cel l s tested along with their properties. The cel l
SIze range, size average, and physical count were measured using the qMicro
equipment. ext. the corresponding IV and CV measurements for each sample were
conducted u:ing the Gamr) 3000. Figure 4 . 7 and 4 .8 ho\\ a et of current-\"ol tage
( l ) and capac itance-\ ol tage ( ) profile for tb 1 6 microalgae ample ,
rc pecti \·c l ) . A can bc een, the mea memen( for each ample di ffered from the
others and th e re ult trongl_ dependent on the intrin ic property of the
material te ted . This led to di fferent IV and V ample profi l s.
Table -+ . 1 : De cription or the microalgae ample te ted and their results u ing the
q M · t I lcro 00 Sam ple Microalgae
# train Tested
1 .\"o17ochloropsi
J Xanochlorop. 'is
" Xonochloropsi
4 XUl7ochlorop i
5 Tetra elll7i
6 Tetraselmi
7 Scenede 1111 1
8 Sce/1edesl7111
9 Scenede 17711
1 0 Nanochlorop. i
1 1 Nanochloropsis
1 2 Nanochloropsi
1 3 Nanochloropsis
1 4 l\'anochloropsi
1 5 Nanochloropsis
1 6 Nanochloropsis
Size Range
( /-l m ) 7 .85 - 36. 1 1
2 .05 - 24. 1 1
2 . 99 - 23 .0 1
2 .77 - 1 7 .32
2 . 1 6 - 1 8 . 32
3 . 96 - 27 . 1 4
2 .47 - 1 4 .02
2 .29 - 2 1 .67
3 . 87 - 36.42
1 .44 - 1 5 .24
6 .54 - 1 4 .49
7 . 73 - 45 . 1 3
7 .78 - 3 3 . 3 7
7 . 5 3 - 23 .45
7 .78 - 49.52
8 . 8 1 - 57 .6 1
Size Average Electrical q Micro
( /-l m ) Cell Count Cell Count
2 l .98 426 1 707
1 3 .06 4043 968
1 3 .00 1 7 1 6 1 303
1 0 .05 1 0 1 33 3273
1 0 .24 1 1 43 792
1 5 . 55 1 723 4 1 0
8 .25 1 723 758
1 1 .98 1 723 83 1 4
20. 1 5 1 723 3 764
7 .84 1 806 1 00 1 8
1 0 . 52 1 806 863
26.43 8453 8289
20. 58 30063 4 1 324
1 5 .49 873 200
28 .65 1 063 3654
33 . 2 1 3 3 3 1 76
.--.: ______ -,0 0 l() l() o 0
.-....--
_____ --,0
•
("')
l("J o
o o
(/) 0
r-.r-----------,O
�
l() o
o o
o L..8-:::l. ....... 0-:::l.--��-g�-�----'2 �
.. .-_____ ---,0 ..-
l("J o
o o
(f) 0 c:: C. o ..-
:::l. :::l. :::l. 8 � ..-..-
_. 0 o
.-..----__________ ---,0
•
r--
l() o
o o
l() o
(/) 0 c:: c:: c. 8 � �
nr ____________ -,O
:::l. :::l. :::l. c:: c:: 8 � g � .,-
l() o
o o
........ ______ ---,0
..-
-=---.
l() o
o o
r--.--_____ � O
1.0 o
o o l("J
N q .-
(/) 0 .-_-.--____ -,0
:::l. :::l. :::l. o 0 o ...-
l("J o
o o
l() o ,
(/) 0 c:: c:: c. 8 � .-
..-
,--___ ._---.0
o .-
l() o
o o
o , (/) 0
c:: c:: o 0 o ..-
�r_------------.O
.....
O'l
l() o
o o
(/) 0
..--______ -,0
l() o
o o
,......,----_____ -,0 l() o
o o
l() o l(", , .-
(/) 0 �r_----------�O
.-
l() o
o o
u") o ,
./') 0 :::l. :::l. :::l. c:: c:: 8 � g � ..-
..... ____________ --.0
:::l. :::l. :::l. o 0 .-o .
.-
l("J o
o o
l() o \"1 '
...-
;f) 0 c:: c:: c. 8 0 ..-
.., ... J J
Figure 4 .7 : Current-voltage ( IV ) measurement curves for the 1 6 samples presented in
Table 1 conducted at 1 0 Hz. The y-axis ( logari thmic scale) uni t is amperes and the x
axi s ( l inear) uni t i s volts
3 4
0 0 0 0 \ � J U") I U") U") U")
J 0 J 0 0 0 0 0 0 0 0 0 0 0 Ii"J Ii"J Ll") Ll") q q 0 q N cD JJ � \ (j) .-0 0 0 (fl 0
::l. ::l. g-: ::l. ::l. g-: ::l. ::l. g-: ::l. ::l. g-: 0 .- 0 0 0 .- 0 .- 0 0 ..-- 0 0 0 0 0
, ..-- ) .- � Ll") U") U") Ll") 0 0 0 0 0 0 0 0 0 0 0 0 U") Ll") U") Ll") q q q Lf) q � r- ..- I ..-0 (j) 0 " (j) 0 (j) 0
::l. ::l. 8- ::l. ::l. c-;- ::l. ::l. 8- ::l. ::l. c:"";" 0 .- 0 8 0 0 .- 0 .- 0 .-0 0 0
U") U") I.{') 0 0 0
0 0 0 0 0 0 Ii"J U") Ll") q q 0 "'1" N 65 .--(j) 0 0 (j) 0 •
::l. ::l. g: ::l. ::l. g: ::l. ::l. g: ::l. ::l. g: 0 0 .- 0 0 0 0 .- 0 0 ..-- ..--0 0 0 0
� I U") I.{') Ii"J Ll") 0 ) 0 0 0
0 0 0 0 0 0 0 0 Ii"J Ii"J I.() Ii"J
c? q 0 q ('-J
\ .- i.I. en ..-if' 0 (j) 0 (j) 0 (j) 0
::l. ::l. 8- ::l. ::l. c-;- ::l. ::l. c-;- ::l. ::l. 50 0 0 .- 8 0 8 0 ..-- ..-- 0 .- .- .- ..--
Figure 4 . 8 : Capacitance- o ltage (CV) measurement curves for the 1 6 samples
presented in Table 1 conducted at 1 0 Hz . The y-axis ( logari tlU11 ic scale) unit is Farad
and the x-axi s ( l i near) unit is volts
3 5
A c mpari o n o f the extracted cel l count \" i th the electrical method and th
q 1 icro arc sho\\1l in F igure 4 .9 (a ) . To determine the l imi t of detect ion of the proposed
method. a kno\\ n concentrat ion o[ th .\'onnochloropsi microalgae cel l s were di luted
to d i fferent concentration in thei r re i vant cul t i \ at ion control medium. The d i luted
'ample \\ ere mea ured u ing both the qMicro and the proposed method. Figure 4 .9(b)
h \\ that the l im i t of detection using the Gamry 3000 was excel lent with 20 cel l s per
m i l l i l iter \ l ume. The sen i t i \ i ty and the l im i t of detection could be further improved
b) using microOuidic channel s v .. hich would al low one to quantify a single cel l .
o..n c::>
� C> <..> � c:r
[J
('\J <..>
· c U � UJ
o
. ,
C> c::>
. ',",�; . " .-. " . .... . ' ""'" . . '. " �
L· . . " . " . • . " . . ", .'" . "
. . . " . . . � . �- . -�� , . � -. ��. � . . ' . . " . . "
'! lj t ��/�'-- ,,:' >< • • " • . � . .;.... �.-v • ...;.v...;. ., �
'" . .. . . . ", - " .. " .. .. � . .. ",'" � .. "
<.0
00
c: . 0 ---co ::::l -0 co . ;:::: (l)
(f)
(f) (l) c.... E co
(f)
" 6
Figure 4 .9 : Comparative analysis o f the microalgal cel l count performed by qMicro
and electrical methods : ( a) qMicro vs. Gamry ( b ) Test of the sensit ivity of detection of
the electrical method using Gamry
3 7 ' J 0 further , al idate the pre ented approach. the cel l count o f the
\ anllochlorop s'i microalgae uspen ion reported earl ier \va counted also u ing the
l ight spectrophotometer and the "", e l l -known hemoc)iometer method. The obtained
results from the d ifferent technique . qMicro. current approach. pectrophotometer
and hemocytom ter for the aJne u pension are pre ented in F igure 4 . 1 0 . A summary
f the ompari on b t" een the performance of the mentioned technique i pre ented
in Table 4 ._ .
1 0
C 5 :::J o <..:>
<3 3
o
q M i c r o E lectri c a l
Photos p ec .
Meth od
F igure 4 . 1 0 : Comparative of the cel l count using d ifferent methods
H e m oc yto
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5.1 Conclu ion
The aim of thi w rk de cribed in the the i wa to explor and demonstrate
the u e of an electrical ba ed technique ba ed on capacitanc -voltage mea urements
to enumerate the microalgae cel l count m a u pen ion. The methodology
characteriLes microalgae cel l s using the diele tric p ctroscopy and semiconductor
theol) .
Th ba ic idea of the pre ented approach is that microalgae uspen ion that
includes cel l and the medium get polarized when they are exposed to the electric field
appl ication. The trength of polarizat ion is mainly dependent on the composition
medium a wel l a the composition of the cel ls : the chemical constituents of the
microalgae part ic le . Each of the microalgae suspen ions under test were exposed to
three 1) pe of measurements which are impedance spectroscopy. Current-Voltage
( IV) . and Capacitance-Voltage (CV) . The appl ication of voltage to the uspension
would lead to the alteration of the electric field in both magnitude and phase. The cel l s
count was then estimated by calculating the dopant concentrat ion of the suspension.
and de-embedding the contribution of the cul t ivation medium. The difference between
both dopant concentrations was found and mult ip l ied by the different of the
corre ponding volumes for both the suspension and medium.
This technique provides a better combination of high sensit ivity and quick
response and overcomes the problems of the previous technologies. When compared
".,,-i th other con entional counting techniques. the developed approach was found to be
the fastest and cheapest. The characterization experiment potent ia l ly gives extra
information about each single part ic le of microalgae which are d ifficul t to get in real
.to
t ime with other method . Thi would inc l ude infomlation about the l ipids.
carboh) drate . and protein content in the cel l . In addit ion to that, mea urement can
be conducted \\ ith no preproce sing r pr treatments for the samples under te 1.
The appl i ed technique i ugge ted to be appl icable for a l l type of microalgae
and other t) pes of biological cel l a long as the cel l get polarized after the appl ication
of an electric field . The ensi t i vit) of the method "ya demon trated by di lut ing a
knO\\11 concentration of cel l with the corre ponding cul t i ation medium solution,
reveal ing a detected l i mit of <W cel l per mi l l i l i ter. Hence, thi reveal that the
proposed technique is ufficiently sensit ive to al low the detection of ce l ls present
during the early stage of growth cycle. Mult ip le experiments demonstrated good
accuracy and predict ion.
OYeral L the project was successfu l and has been hown that cel l count ing
mea urements using the electrical characterization technique is done rapidly and the
proce sing \\ a achie ed using relat ively simple mathematical relat ions.
5.2 F u t u re Work
E lectrical characterization technique of microalgae cel l s count i s sol id ly based
on theory. There are se eral potential appl ications that could be suggested as a future
work for this project . The recommendat ions are as fol lows and i nc lude:
• Upgrade the implementat ion of the system to be appl ied in-situ with a feedback
control loop which wi l l not only pave the way for d i rect and rapid cel l counting,
but also enable the continuous monitoring and opt imizat ion of the microalgae
gro\\th process.
• Implement the proposed methodology to determine l ipids, proteins. and
carbohydrates content .
4 1
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46
L i t o f P u b l i c a t i o n
• L. aqer, 1 . hmad, H . Taher. . I -Zuhair and aqbi, "Monitoring of
microalgae l ipid accumulation ystem overv iew" . in GCC Conference and
Exhihition (GCCCE). _ 0 1 5 IEEE 8th, Mu cat. �0 1 5 , pp. 1 -5 .
• 1 . . ager. I hmad, H . Taher. . l -Zuhair and . A I aqbL " ov el E lectrical
Ba ed Technique for icroalgae L ipid ontent Quanti fication", in 'A E Gradllate
Research COl�lerel1ce ( L A E GSRC) 2015. bu Dhabi , AE .
• L. aqer. . Al lU11ad, A E A l1nual R e carch al1d Inno\'CI/ion Conference 2 0 1 5 tAb tract #8" ).
• 1 . I AlU11ad, L . aqel', . A I -Zuhair. F. Mustafa, " E lectrical Quanti fication of
icroaJga Cel l " , cience Direct [ nder Reviev. ] .
A p p e n d i x
M icroalgae Strain and Culture M edium
.+7
B th fr hv" ater and mari ne strain of micro algae were te ted in this study. The
ere. h \\ ater train Scel7edeslI7l1 ' p. cultur \Va obtained fr m Algal Oi l Company.
Phi l ippines. and cult i \ ated in a modi fied Ba sel medium ( +3 -BBM ) compos d of
( m 1 ) 0. 1 7 CaCb·2H20. 0 .3 Ig O.J · 7 H20. 1 . 29 KH2PO.J. 0 .43 K2HPO.J. 0.43 aC I . 1
m l . L- 1 of Vi tamine B 1 2, and 6 m l . L- 1 of P- IV olut ion that con i ted of 2
a2EDT ' 2 H20. 0 . "6 FeC b ' 6H20. 0 .2 1 MnCb-4H20. 0 . 37 ZnCh. 0 .0084
o h · 6H20 and O.0 1 7 a2MoO.J · 2 H20.
The marine train Nal7l1oc/oropsis and Tetra were obtained from a local
marine environment mm AI -Quawain Marine Research Center. UAE. They were
grO\\11 and obtained i n the F/2 medium ( 3 2 ppt a l ini ty ) consist ing of the fol lowing
major nutrient ( in 11M ) ; 880 aN03. 36 aH2PO,J . H20. 1 06 Na2Si03 .9H20. 1 (m l L
I ) of: y i tan1 i n B 1 2. biotin vitamin. and thiamine vi tamin sol utions, and 1 ( m l L- 1 ) of
trace metals solut ion that con isted of ( 11M) : 0 .08 Zn 04 .7H20. 0 .9 MnSO,J . H20. 0.03
a2MoO.J . 2H20. 0.05 CoSO.J . 7H20, 0.04 CuCh.2H20. 1 1 .7Fe(N H4h( S04 h ,6H20,
and 1 1 . 7 a:!EDTA.2H20. The prepared media were steri l i zed in an autocla e
( Hi rayan1a H V -50. Japan) at 1 2 1 C for 1 5 min and cooled to room temperature prior to
use.