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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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:r �->- J y:4-0 .J:ljUl �;'U.bll � w.r �I J (} yJI .) � � � yiJl ��I rl�1 M jl

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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

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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.