Oxygen Accumulation in Photobioreactors

Oxygen accumulation
in photobioreactors
Cláudia Sousa
Oxygen accumulation in
photobioreactors
C lá u d ia A le x a n d ra da F onseca e Sousa
Thesis committee
Promotor
Prof. dr. ir. R. H. W ijffe ls
Professor a t Bioprocess Engineering
W ageningen U nive rsity
Co-promotor
Dr. ir. M .H. V e rm u ë
A ssistant p ro fesso r a t Bioprocess Engineering
W ageningen U nive rsity
Other members
Prof. dr. ir. G. Zeem an, W ageningen U niversity, The N etherlands
Prof. dr. G.J.W. Euverink, U nive rsity o f G roningen, The N etherlands
Prof. dr. E. M o lin a G rim a, U nive rsity o f A lm eria, Spain
Dr. ir. A.J.B. van Boxtel, W ageningen U niversity, The N etherlands
This research was co nd ucte d u n d e r th e auspices o f th e G raduate School VLAG

(Advanced studies in Food Technology, A g ro b io te c h n o lo g y , N u tritio n and Health
Sciences).
Oxygen accumulation in
photobioreactors
Cláudia Alexandra da Fonseca e Sousa
Thesis
s u b m itte d in fu lfillm e n t o f th e re q u ire m e n ts fo r th e degree o f d o c to r
at W ageningen U nive rsity
by th e a u th o rity o f th e R ector M agnificus
Prof. dr. M.J. Kropff,
in th e presence o f th e
Thesis C o m m itte e a p p o in te d by th e A cadem ic Board
to be d e fe nd e d in public
on Tuesday M ay 21, 2013
a t 4 p.m . in th e Aula.
Cláudia Sousa
Oxygen a ccu m u la tio n in p h o to b io re a c to rs
136 pages
PhD thesis W ageningen U niversity, W ageningen, NL (2013)

W ith references, w ith sum m aries in English, D utch and Portuguese
ISBN: 978-94-6173-554-6
Table o f co nte nts
Table o f con ten ts
T a ble of c o n ten ts
Chapter 1. General introduction & thesis outline 1
1.1. M icroalgae 1
1.2. Large-scale o u td o o r c u ltiv a tio n 2
1.3. P hotosynthesis, p h o to re s p ira tio n & p h o to in h ib itio n 3
1.4. Thesis o u tlin e 7
1.5. References 9
Chapter 2. Growth of the microalgaeNeochloris oleoabundans at high

partial oxygen pressures and sub-saturating light intensity 15
2.1. In tro d u c tio n 16
2.2. M a te ria l and m eth od s 17
2.2.1. Cultures and medium 17
2.2.2. Photobioreactor 18
2.2.3. Dry weight concentration 19
2.2.4. Specific absorption coefficient 20
2.2.5. Photosynthesis-irradiance curve (PI- curve) 21
2.3. Results and discussion 21
2.3.1. Light regime 21
2.3.2. Growth and productivity 23
2.3.3. Implications on reactor design 26
2.4. C onclusions 28
2.5. A ckn ow le dg em en ts 28
2.6. References 28
A p pe n dix 2A. D is trib u tio n o f in cid e n t p h o to n flu x d en sity o ve r p h o to b io re a c to r

surface 32
A p pe n dix 2B. C alculation o f lig h t g ra d ie n t in p h o to b io re a c to r 33
A p pe n dix 2C. Specific a b so rp tio n sp ectrum N. oleoabundans 34
Chapter 3. Effect of Oxygen at low and high light intensity on the

growth of Neocholoris oleoabundans 37
3.2. M a te ria l and m eth od s 40
3.2.4. Chlorophyll and carotenoids 41
3.3.1. Controlled cultivation o f algae at high and low light intensity 42
3.3.2. Oxygen effects o f microalgal growth at high and low light intensity 43
3.3.3. Oxygen effects o f pigm ent content at high and low light intensity 44
3.6. References 49
Chapter 4. Effect of Dynamic Oxygen Concentrations on the growth of

Neocholoris oleoabundans at sub-saturating light conditions 55
4.2. M ate ria ls and m eth od s 57
4.2.5. Chlorophyll and Carotenoid determination 59

4.3.1. Effect o f the applied light-regime and the dynamically changing 0 2 on

algal growth 60
4.3.2. Effect on biomass yield on photons 63
4.3.3. Chlorophyll and carotenoid content 64
4.3.4. Final remarks 66
4.6. References 68
Chapter 5. Effect of Dynamic Oxygen Concentrations on the growth of

Neochloris oleoabundans at high light conditions 73
5.2. M a te ria l and m e th o d 75
5.2.1. Culture and photobioreactor system 75
5.2.3. Off-line analysis o f samples 76
5.3.1. Effect o f dynamic 0 2 and light regime on algal growth 77
5.3.2. Chlorophyll and carotenoid content 80
5.6. References 82
Chapter 6. Oxygen production in photobioreactors A look to the

economics 85
6.1.1. Effects o f oxygen on microaigai growth 86

6.1.2. Effects o f (dynamic) accumulating oxygen in closed photobioreactors 89
6.2. Reducing th e costs fo r degassing w ill reduce th e o verall costs 90
6.2.1. Biomass productivity costs - base case 90
6.2.2. Effect o f increasing the C02/ 0 2 on biomass production costs 90
6.2.3. Biomass productivity costs - increase the length o f the tubes / decrease
the velocity in the tubes. 92
6.4. References 93
Summary 99
Samenvatting 103
Sumário 109
Acknowledgements 115
About the Author 119
Overview of completed training activities. 121

C hapter 1
G eneral in tro d u c tio n & thesis o u tlin e
Chapter 1. G e n e r a l in t r o d u c t io n & th esis o u t lin e
1.1. Microalgae
P h o to tro p h ic m icroalgae are p ro k a ry o tic o r e uk a ry o tic m icroscopic organism s th a t
are able to u tilize lig h t energy and use it to in c o rp o ra te ino rg a nic carbon in th e
fo rm o f dissolved carbon d io xide (C 0 2) and b ica rb o n a te (H C 0 3 ) in to th e ir
biom ass, w h ile p ro du cing 0 2. A long w ith th e p h o to a u to tro p h ic species th e re are
som e m icroalgae th a t are capable o f u tilizin g organic carbon fro m com p ou nd s
such as glucose and glycerol via re sp ira tio n (h e te ro tro p h s ). The e no rm o u s v a rie ty
in algae m e ta b o lism makes m icroalgae ve ry in te re s tin g fro m a b io te chn olo gical
p o in t o f vie w as th e y can be used fo r fo o d , fe ed , hea lthca re c o n stitu e n ts,
chem icals, energy and w a te r tre a tm e n t a pp lica tion .
N owadays, a lo t o f research is done on large-scale m icroalgal p ro d u c tio n using

p h o to tro p ic algae as th e y are regarded as th e m o st p ro m ising fe e d sto ck fo r
sustainable biodiesel p ro d u ctio n , as th e y can use n atu ra l s u n lig h t as lig h t source
and are able to u tilize C 0 2 fro m flu e gases and n u trie n ts (P, N) fro m w aste
stream s (Boelee e t al., 2011; V unja k-N o vako vic e t al., 2005) (Figure 1). C om pared
w ith co n ve n tio n a l te rre s tria l plants th a t are c u rre n tly used fo r biodiesel
p ro d u c tio n , m icroalgae sh ow high p h o to s y n th e tic conversion e fficiencies and
hig he r areal p ro d u c tiv itie s (M a ta e t al., 2010). They can p ro spe r in d iffe re n t
ecosystem s and do n o t c o m p e te fo r land w ith crops, n e ith e r w ith th e fo o d m a rke t
(Zeng e t al., 2011).
1
C hapter 1
® V “ \/
Figure 1 - Schematic representation o f the environmental factors influencing microalgae

growth
N eochloris o le oabundans is m e n tio n e d as one o f th e m o st p ro m ising strains o f

oleaginous green algae (C hlorophyceae) fo r th e p ro d u c tio n o f b io fu els (Li e t al.,
2008b). It is a fre s h w a te r m icroalga th a t can also th riv e a t saline m edium
co n d itio n s. N eochloris o le oabundans is able to accum ula te lipids in th e fo rm o f
tria cylg lyce ro ls (also called trig ly c e rid e o r TAG) and p a rtic u la rly a t n itro g e n
d e fic ie n t co n d itio n s, lipid c o n te n ts up to 56.0 % o f d ry w e ig h t o f biom ass w e re
re p o rte d w ith lipid p ro d u c tiv itie s up to 133 m g .L ^.d '1, (Gouveia e t al., 2009;
G ouveia & O liveira, 2009; Li e t al., 2008a; Pruvost e t al., 2009; Pruvost e t al.,
2011 ).
1.2. Large-scale outdoor cultivation

To m ake b io fu els fro m m icroalgae a viable s u b s titu te fo r fossil fuels, th e
p ro d u c tio n should be sustainable, w h ich m eans th a t th e p ro d u c tio n should have a
lo w e n v iro n m e n ta l fo o tp rin t, is e con om ically c o m p e titiv e and th e algal fe e d sto ck
should be available in s u ffic ie n t a m o un ts to have an im p o rta n t im p a ct in th e
energy supply chain (A m aro e t al., 2012). A lth o u g h th e p ro d u c tio n o f b io fu els
fro m m icroalgae is te ch n o lo g ica lly feasible, th e o u td o o r large-scale p ro d u c tio n
still presents issues conce rn ing th e e con om ic fe a s ib ility and th e energy re q u ire d
2
(Cheng & T im ilsina, 2011; N orsker e t al., 2011; Stephens e t al., 2010a; Stephens e t
al., 2010b).
For th e o u td o o r p ro d u c tio n th e re are tw o m ain algae c u ltiv a tio n system s (open

and closed systems) c u rre n tly being used. Open racew ay ponds are th e m ost
w o rld w id e used system s as th ese system s are re la tiv e ly cheap. H ow ever, low
biom ass densities are achieved and th e areal p ro d u c tiv itie s and yields are
re la tiv e ly lo w (N orsker e t al., 2011) and th e re is a high risk fo r c o n ta m in a tio n by
bacteria and sudden collapse o f th e c u ltu re caused by p ro tozo a and o th e r
p re d a to rs (Carvalho e t al., 2006; Pulz, 2001; Richm ond, 1992). W ith closed
system s (PBR) higher biom ass densities can be achieved and th e re is sm aller risk
fo r c o n ta m in a tio n b u t th e re are o th e r b o ttle n e cks to o vercom e to m ake large-
scale p ro d u c tio n in th ese p h o to b io re a c to r system s e con om ically feasible. The
m ain b o ttle n e c k in closed PBRs is th e high energy in p u t th a t is re q u ire d fo r m ixing
to p ro vid e th e algae w ith s u ffic ie n t lig h t and carbon d io xide and o th e r n u trie n ts
and to rem ove th e oxygen th a t is produced d u rin g p h o tosyn th esis (Dism ukes e t
al., 2008; N orsker e t al., 2011; W ijffe ls e t al., 2010). This oxygen needs to be
rem ove d to p re ve n t adverse e ffe cts via p h o to re s p ira tio n and p h o to in h ib itio n on
g ro w th and p ro d u c tiv ity o f th e algae.
1.3. Photosynthesis, photorespiration & photoinhibition

In th e p h o to s y n th e tic process, w a te r is sp lit in to oxygen and e le ctron s w ith lig h t
as th e d rivin g fo rce . The e le ctron s are used to fix and reduce carbon d io xide to th e
level o f sugar (trióse) in th e Calvin cycle fro m w h ich new biom ass is fo rm e d . In th is
Calvin cycle th e enzym e Rubisco is invo lved in th e fix a tio n o f C 0 2. Analogous to
any p h o to s y n th e tic p la n t cell, m icroalgae gen erate oxygen d u rin g th e
p h o tosyn th esis (Figure 2).
In closed p h o to b io re a c to rs , p h o tosyn th esis causes th e e v o lu tio n o f dissolved

oxygen levels e q u iva le n t to m any tim e s th e a ir sa tu ra tio n . The a ccu m u la tio n o f
p h o to s y n th e tic a lly p roduced 0 2, and conse qu en tly, th e high p artial 0 2 pressures
results in in h ib itio n of p ho tosyn th esis, due to p h o to re s p ira tio n and
3
C hapter 1
p h o to in h ib itio n (Becker, 1994). This m akes dissolved oxygen a crucial p a ra m e te r

to c o n tro l in th e m icroalgae p ro d u c tio n systems (Aiba, 1982).
Photosynthesis
Photosynthesis can be subdivided into tw o types of reactions: dark reactions, which are not directly
influenced by light and reactions directly influenced by light. The chloroplast o f a green alga contains
thylakoid membranes in which tw o types o f photosystems (PS I and PS II) are fixed and connected by
an electron transport chain. In the photosynthesis, the only driving force comes from the light
excitation o f the special electron carriers fixed in the thylakoid membrane aided by particular
protein complexes. The electrons flow through the electron transport system, from Photosystem II
to Photosystem I and reduce the low energy NADP+ to high energy NADPH, and transform the light
energy into ATP molecules (Vacha, 1995). The latter occurs via electron transport associated w ith
pumping o f protons across the thylakoid membrane, which develops a gradient of pH th a t is used to
the production o f ATP by ATP synthase. Next, the energy and reducing power o f NADPH and ATP is
utilized to fix C 02 via the action of Rubisco in the Calvin Cycle, or to the synthesis o f saccharides in
the carbon metabolism, from which new biomass is form ed. ADP, Pi and NADP+ are released and
enter again in photosynthesis (Janssen, 2002; Vacha, 1995).
ADP
NADP-
Light
Dark reaction
reaction
NADPH
ATP
Figure 2 - Simplified scheme o f photosynthesis. Adapted fro m Bosma (2010)
P h o to re sp ira tio n is a process characterized by th e oxygenase a c tiv ity o f Ribulose-

1 ,5-bisphosphate carboxylase oxygenase (Rubisco), th e princip al enzym e involved
in th e C 0 2 assim ila tio n in th e Calvin Cycle (Figure 2) (Raven & Larkum , 2007;
Vacha, 1995). 0 2 in h ib its C 0 2 fix a tio n in vivo th ro u g h c o m p e titiv e in h ib itio n o f th e
Rubisco. The oxygenase and carboxylase s e le c tiv ity o f Rubisco is closely related to
4
th e CO 2 / O 2 ra tio in its vic in ity . This ra tio decreases in c o n d itio n s o f high levels o f
oxygen, leading to th e re d u c tio n o f th e carboxylase a c tiv ity and th e increase o f
th e oxygenase a c tiv ity (O sm ond, 1981; Raso e t al., 2011). The enhanced
oxygenase a c tiv ity results in a decrease o f th e m icroalgae p ro d u c tiv ity . To
o vercom e th e oxygenase/carboxylase d u a lity o f Rubisco, and its lo w a ffin ity fo r
C 0 2, m any algae acquired C 0 2-co n ce n tra tin g m echanism s (CCM), w h ich are
e v o lu tio n a ry m echanism s th a t use energy to increase C 0 2 co n c e n tra tio n s in th e
p ro x im ity o f Rubisco (G iordano e t al., 2005).
Photorespiration
When Rubisco fixes C 02 w ith o u t photorespiration per one molecule Ribulose 1,5 biphosphate
(RuBP) tw o molecules of 1,3-biphosphateglycerate (l,3bPGA) are formed. One of the l,3bPGA is
used to generate ATP in the Calvin cycle, while the other can be used as building block fo r sugars to
be used to form biomass components. On the other hand, if 0 2 is fixed, only one molecule o f 3-
phosphoglycerate is formed and one molecule o f Glycolate 2-phosphate (G2P). G2P is converted in
glycoxylate, and finally in l,3bPGA, at the cost o f C 02 and ammonia (NH4+). All these processes
require ATP and NADPH, which are generated in the light reaction o f photosynthesis. Consequently,
when photorespiration occurs, less energy is available fo r microalgal growth, decreasing the yield of
microalgal biomass on light energy (Kliphuis et al., 2010).
P h o to in h ib itio n is a n o th e r m echanism w h ich evokes algal g ro w th in h ib itio n , b u t

th is process o n ly happens a t high lig h t co n d itio n s. A t th ese high lig h t co n d itio n s
an excessive a m o u n t o f e le ctron s is g e n erated a t P hotosystem II and these
e le ctron s rea ct w ith p h o to s y n th e tic a lly pro du ced oxygen, fo rm in g oxygen radicals
(Figure 3) (M u ra ta e t al., 2007).
5
C hapter 1
W a te r-w a te r cycle
IN A D P H NA D P -
In the w ater-w ater cycle the
photoreduction o f oxygen to w ater
takes place in PS I by the electrons
generated in PS II (Asada, 1999). In STROMA C u Z n -S O D '
the w ater-w ater cycle, the

electrons are used again to reduce
oxygen, via the reactive oxygen
species superoxide and hydrogen
peroxide, w ith the help of the
enzymes, superoxide dismutase
(CuZn-SOD) and peroxidase (APX).
These reduced and reactive oxygen
species are converted to water,
hence the name w ater-w ater cycle.
This cycle is also known as the
M ehler reaction or pseudo-cyclic Photons
electron transport and serves to

scavenge the reactive oxygen Figure 3 - Formation o f oxygen radicals in the water-w ater
cycle during photosynthesis. Adapted fro m Asada (2006)
radiais and toxic H20 2. This process
occurs only at high (i.e over-saturating) light intensities an d/or nutrient lim itations, leading to an
over-reduction o f the photosynthetic system (Asada, 1999; Ledford & Niyogi, 2005).
These oxygen radicals are usually d e a lt w ith in th e w a te r-w a te r cycle, b u t w he n

to o m any e le ctron s are g en erated , th e enzym es in th e w a te r-w a te r cycle are no
lon ge r capable o f dealing w ith th e surplus o f e le ctron s and oxygen radicals and
o th e r rea ctive oxygen species (ROS) such as H20 2 are a ccum ula ting and th is has a
d e stru ctive e ffe c t on biological system s (Asada, 2006; Asada, 1999; Endo & Asada,
2008).
6
Photoactivation
STROMA Photons captured by P680 cause excitation o f P680

and when it falls back to the ground state the energy
is used to reduce w ater resulting in form ation of
oxygen and 4H+ ions. In case o f high light conditions a
surplus o f light falls at P680 and the surplus o f energy
is transferred to other pigments (Pheo, Q a - Q b ) and
used fo r form ation of trip le t state P680 (SP680). The
energy released when SP680 returns to the ground
singlet state (1P680) the energy is partly used to
transfer trip le t state oxygen (s0 2) into the highly
reactive singlet oxygen (10 2).
Figure 4 - Generation o f singlet oxygen. Adapted

Photons fro m Asada (2006)
F u rth e rm o re , th e fo rm a tio n o f singlet oxygen is bound to occur a t high ligh t

inten sitie s. This hig hly reactive co m p o u n d is pro du ced p h o to ch e m ica lly, via
p h o to a c tiv a tio n (T rian ta ph ylid es e t al., 2008) (Figure 4). The sing let oxygen can
"a tta c k " p h o to s y n th e tic pig m e n ts in PSII, causing p h o to -o x id a tiv e dam age.
A lth o u g h th e in h ib itin g e ffe cts o f oxygen have been described in d e ta il, in o nly a
fe w studies th e e ffe c t o f 0 2 was m easured as p a ra m e te r, in d e p e n d e n t o f th e lig h t
(Kliphuis e t al., 2011; M o lin a e t al., 2001; Ogren, 1984; Raso e t al., 2011). The
e ffe cts on g ro w th th u s o fte n re fle c t a co m b in e d e ffe c t o f ligh t, pH and oxygen on
p ho tosyn th esis (Torzillo e t al., 1998; Ugwu e t al., 2007).
1.4. Thesis outline

In th is thesis th e e ffe c t o f a ccum ula ting oxygen on th e g ro w th o f N eochloris
o le oabundans is stu die d a t sub- and n e a r-sa tu ra tin g lig h t co n d itio n s in a fu lly
c o n tro lle d p h o to b io re a c to r o p e ra te d in tu rb id o s ta t m ode (Figure 5) to reveal to
w h a t e x te n t th e oxygen in h ib its th e g ro w th o f th e algae and w h a t oxygen
c o n ce n tra tio n s w o u ld still lead to acceptable g ro w th rates o f th e algae. In
a d d itio n , th e oxygen a ccu m u la tio n w h ich m ay occur in closed p h o to b io re a c to rs
was m im icked and th e e ffe cts o f th ese d yna m ica lly changing oxygen co n d itio n s on
th e algal g ro w th w e re exam ined. W ith th e gen erated kn ow led ge it has been
possible to p re d ic t th e m in im u m a m o u n t o f energy needed to keep th e oxygen
7
C hapter 1
level s u ffic ie n tly low and calculate th e energy savings th a t are possible w h e n an
o u t-d o o r tu b u la r p h o to b io re a c to r system is o p e ra te d a t large scale using these
m in im ize d m ixing and degassing co n d itio n s.
Figure 5 - Lab-scale CSTR photobioreactor used in the experiments.
C hapter 2 o f th is thesis describes th e e ffe c t o f p a rtia l oxygen pressure on g ro w th

o f N eochloris oleoabundans a t su b -sa tu ra tin g lig h t in te n s ity in a fu lly -c o n tro lle d
s tirre d ta n k p h o to b io re a c to r. In th is w o rk w e stu die d 3 d iffe re n t p a rtia l oxygen
pressures (Pq2) and e valua te its e ffe c t on specific g ro w th rates. 2 d iffe re n t p artial
carbon d io xide pressures ( P Co2) a t th e highest PO2=0.84 bar w e re considered and

used to co n firm th e presence o f p h o to re s p ira tio n p henom ena and to o vercom e it.
In c h a p te r 3 a c o n tin u a tio n o f th e stu dy o f c h a p te r 2 was p e rfo rm e d a t near-
s a tu ra tin g lig h t inten sitie s. The e ffe c t o f p a rtia l oxygen pressure on g ro w th o f
N eochloris o le oabundans was e valuated a t 4 d iffe re n t p a rtia l oxygen pressures
(Po2= 0.24; 0.42; 0.63; 0.84 bar) as w e ll as an increase o f th e PC02 fro m 0.007 to
0.02 bar a t Po2 o f 0.84. The specific g ro w th rates and p ig m e n t c o n te n t o f th e

m icroalgae w e re used to assess th e presence o f pheno m e na like p h o to a c lim a tio n ,
p h o to in h ib itio n and p h o to o x id a tiv e dam age.
In ch a p te r 4 and 5 th e e ffe cts o f th e increase o f th e oxygen c o n c e n tra tio n

fo llo w e d by a decrease o f th e oxygen in th e degasser w e re sim u la te d a t lo w and
8
high lig h t in te n s ity and th e e ffe c t o f a 10 tim e s e lo n g a tio n o f th e residence tim e at

in th e solar receiver was inve stiga te d. The lig h t regim es used w e re : c o n tin u o u s
lig h t ON; 30 m in u te s o f lig h t ON fo llo w e d by 6 m in utes lights OFF and 300 m in u te s
o f lig h t ON fo llo w e d by 6 m in utes lights OFF. The e ffe c t o f d yna m ica lly changing
oxygen co n ce n tra tio n s fro m PO2=0.21 bar to PO2=0.63 bar fo llo w e d by subsequent
degassing to PO2=0.21 bar d u rin g th e d ark p erio d resulted in s im ila r specific
g ro w th rates. The decrease o f th e algae specific g ro w th observed w h e n applying

d iffe re n t lig h t regim es, show s th a t th e exposure o f th e algae cells to d ark periods
in th e degasser has bigger negative im p a c t th a n th e te m p o ra ry exposure to
a ccum ula ting oxygen co n ce n tra tio n s in th e solar receiver. In c h a p te r 5 th ese
results th e same results w e re fo u n d in d ica tin g th a t th e algae do n o t experience
th e expected p h o to -o x id a tiv e in h ib itio n caused by high oxygen c o n c e n tra tio n in
c o m b in a tio n w ith high ligh t, as long as th e oxygen is rem ove d via regular
degassing.
In ch a p te r 6 a m odel w h ich was deve lo pe d to calculate th e energy and costs

associated to m icroalgae biom ass p ro d u c tio n in th e N etherlands fo r th re e
d iffe re n t system s a t 100 ha scale was used to e valuate th e im p le m e n ta tio n o f th e
fin d in g s described on th e previous chapters. C hapter 6 is a general discussion
a b o u t th e e ffe c t o f th e re d u c tio n o f th e costs fo r degassing and its influ en ce on
th e overall costs and n e t energy balance.
1.5. References
Aiba, S. 1982. G ro w th kinetics o f p h o to s y n th e tic m icroorganism s. Advances in
Biochem ical Engineering, 23, 85-156.
A m aro, Fl.M., M acedo, A.C., M alcata, F.X. 2012. M icroalgae: An a lte rn a tiv e as
sustainable source o f biofuels? Energy, 44(1), 158-166.
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m icroorganism s. A p plie d M ic ro b io lo g y and B iotechnology, 57, 287-293.
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281-286.
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b iofuels. N ature B iotechnology, 28(2), 126-128.
Stephens, E., Ross, I.L., M ussgnug, J.H., W agner, L.D., B orow itzka, M .A., Posten, C.,
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T orzillo , G., B ernardini, P., M asojidek, J. 1998. O n-line m o n ito rin g o f c h lo ro p h yll
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high oxygen co n c e n tra tio n and low te m p e ra tu re and its e ffe c t on th e p ro d u c tiv ity
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5 0 4 -5 1 0
T ria ntap hylide s, C., Krischke, M., H oeberichts, F.A., Ksas, B., Gresser, G., Havaux,
M ., Van Breusegem, F., M u e lle r, M.J. 2008. Singlet oxygen is th e m a jo r reactive
12
oxygen species involved in p h o to o x id a tiv e dam age to plants. Plant Physiology,

148(2), 960-968.
Ugwu, C.U., Aoyagi, H., U chiyam a, H. 2007. In flue nce o f irradiance, dissolved
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P h otosyn th etica , 45(2), 309-311.
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334.
V unjak-N ovakovic, G., Kim, Y., W u, X.X., Berzin, I., M erchuk, J.C. 2005. A ir-lift
b io re a cto rs fo r algal g ro w th on flu e gas: M a th e m a tic a l m o d e lin g and p ilo t-p la n t
studies. In du stria l & Engineering C hem istry Research, 44(16), 6154-6163.
W ijffe ls , R.H., Barbosa, M.J., Eppink, M .H .M . 2010. M icroalgae fo r th e p ro d u c tio n

o f b ulk chem icals and biofuels. Biofuels, B io prod ucts and B io re finin g, 4(3), 287-
295.
Zeng, X.H., Danquah, M .K., Chen, X.D., Lu, Y.H. 2011. M icro alga e bioen gin ee rin g:
From C 0 2 fix a tio n to b io fu e l p ro d u c tio n . R enew able & Sustainable Energy
Reviews, 15(6), 3252-3260.
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C hapter 2
14
G ro w th o f the m icro a lg a e N eochloris o le oabundans a t hig h p a rtia l oxygen
pressures a n d s u b -s a tu ra tin g lig h t in te n s ity
Chapter 2. G r o w t h of th e m ic r o a lg a e N e o c h lo r is
OLEOABUNDANS AT HIGH PARTIAL OXYGEN PRESSURES AND SUB-
SATURATING LIGHT INTENSITY
Claudia Sousa1,2, Lenneke de W i n t e r 2, M arcel Janssen2, M arian H. V e rm u ë 2 and

Rene H. W ijffe ls 2
1 W etsus, P.O. Box 1113, 8900 CC Leeuwarden, The N etherlands
2 Bioprocess Engineering, W ageningen University, P.O. Box 8129, 6700 EV, W ageningen,
The Netherlands
A b s tra c t - The e ffe c t o f p a rtia l oxygen pressure on g ro w th o f N eochloris

o le oabundans was stu die d a t su b-satu ratin g lig h t in te n s ity in a fu lly -c o n tro lle d
s tirre d ta n k p h o to b io re a c to r. A t th e th re e p artial oxygen pressures te s te d (Po2=
0.24; 0.63; 0.84 bar), th e specific g ro w th rate was 1.38; 1.36 and 1.06 day"1,
respectively. An increase o f th e PC02 fro m 0.007 to 0.02 bar a t Pq2 o f 0.84 bar
resu lted in an increase in th e g ro w th ra te fro m 1.06 to 1.36 day"1. These results

c o n firm th a t th e re d u c tio n o f algal g ro w th a t high oxygen co n c e n tra tio n s a t sub-
s a tu ra tin g lig h t c o n d itio n s is m ainly caused by c o m p e titiv e in h ib itio n o f Rubisco.
This negative e ffe c t on g ro w th can be o vercom e by re s to rin g th e 0 2/ C 0 2 ra tio by
an increase in th e p a rtia l carbon d io xide pressure. In com p arison to general
pra ctice (Pq2=0.42 bar), w o rk in g a t p a rtia l 0 2 pressure o f 0.84 bar could reduce
th e energy re q u ire m e n t fo r degassing by a fa c to r o f 3 to 4.
Key w o rd s : N eochloris oleoabundans, pho tosyn th esis, oxygen in h ib itio n ,

p h o to re s p ira tio n , p h o to b io re a c to r
Sousa, C., de Winter, L., Janssen, M., Vermuë, M.H., Wijffels, R.H. 2012. Growth o f the
microalgae Neochloris oleoabundans at high partial oxygen pressures and sub-saturating
light intensity. Bioresource Technology, 104, 565-570.
15
C hapter 2
2.1. Introduction
Lipid-rich p h o to a u to tro p h ic m icroalgae such as N eochloris oleoabundans are
p ro m ising ren ew a ble resources fo r biodiesel p ro d u c tio n , because o f th e ir high
p ro d u c tiv ity and because th e ir p ro d u c tio n does n o t have to c o m p e te w ith fo o d
(Chisti, 2007; Schenk e t al., 2008; W ijffe ls and Barbosa, 2010). H ow ever, large-
scale o u td o o r p ro d u c tio n o f m icroalgae is n o t y e t e con om ically feasible. High
energy in p u ts are re q u ire d fo r m ixing to p ro v id e th e algae w ith lig h t and carbon
d io xide and to rem ove th e p h o to s y n th e tic a lly pro du ced oxygen (Dism ukes e t al.,
2008; N orsker e t al., 2010; W ijffe ls e t al., 2010). W h e n oxygen accum ulates in th e
c u ltu re m ed iu m , p h o to in h ib itio n and p h o to re s p ira tio n ta ke place, leading to a
decrease in biom ass yield on lig h t energy (Torzillo e t al., 1998). P h o to in h ib itio n
occurs m a in ly a t high and o v e r-sa tu ra tin g lig h t inten sitie s. A t th o s e c o n d itio n s an
excess o f e le ctron s is g e n erated in P hotosystem II and th ese w ill react w ith th e
p h o to s y n th e tic a lly pro du ced oxygen, leading to th e fo rm a tio n o f oxygen radicals
(M u ra ta e t al., 2007, Pospísil, 2011). In a d d itio n , lig h t s tim u la te s th e fo rm a tio n o f
sing let oxygen via p h o to -a c tiv a tio n (T rian ta ph ylid es e t al., 2008) w hich dam ages
th e p ho tosystem s o f th e algal cells.
P h o to re sp ira tio n is associated w ith th e oxygenase a c tiv ity o f th e enzym e Rubisco.

The a ccu m u la tio n o f oxygen ( 0 2) w ill lead to an increase o f th e local 0 2/ C 0 2 ra tio
and, consequently, to reduced carboxylase a c tiv ity and increased oxygenase
a ctivity. O verall th e p ro d u c tiv ity o f th e m icroalgae c u ltu re w ill decrease (O sm ond,
1981). P h o to re sp ira tio n o n ly occurs in th e d ark rea ction o f p h o tosyn th esis and is
th u s n o t re la te d to fo rm a tio n o f rea ctive oxygen species o ccu rrin g a t high- and
o ve r-sa tu ra tin g lig h t co n d itio n s. A t su b-satu ratin g lig h t in te n sitie s p h o to in h ib itio n
is n egligible sm all, w h ich m akes p h o to re s p ira tio n th e d o m in a n t process leading to
reduced p h o to s y n th e tic yield u n d e r oxygen a ccum ula tion .
D uring p h o to re s p ira tio n , C 0 2 and a m m o n iu m (NH4+) are lost and th e ir re -fix a tio n
requires a d d itio n a l ATP and NADPH. This m eans th a t less energy is available fo r
g ro w th and th e biom ass yield on lig h t energy w ill decrease w he n p h o to re s p ira tio n
occurs (Kliphuis e t al., 2010). The p h o to re s p ira to ry p a th w a y th u s has an influ en ce
on th e p h o to s y n th e tic yield w h ich can be d e fin e d as th e a m o u n t o f C 0 2 fixe d per
a m o u n t o f lig h t energy absorbed and, as such, w ill d ire c tly influ en ce th e
p ro d u c tiv ity o f m icroalgae cultures.
16
A lth o u g h th e in h ib itin g e ffe cts o f oxygen have been described in d e ta il, in h ardly
any o f th e re p o rte d studies th e e ffe c t o f 0 2 was m easured as in d e p e n d e n t
p a ra m e te r (Kliphuis e t al., 2011; M o lin a e t al., 2001; Ogren, 1984; Raso e t al.,
2011). The e ffe cts on g ro w th o fte n re fle c t a co m b in e d e ffe c t o f ligh t, pH and
oxygen on p ho tosyn th esis (Torzillo e t al., 1998; Ugwu e t al., 2007). In th e pre sen t
study, th e e ffe c t o f p a rtia l oxygen pressures on th e g ro w th o f N eochloris
o le oabundans at su b-satu ratin g lig h t c o n d itio n s in a fu lly c o n tro lle d
p h o to b io re a c to r o p e ra te d in tu rb id o s ta t m od e was d e te rm in e d . The specific
g ro w th rate as w e ll as th e biom ass yield on p h o to n s u n d e r th re e d iffe re n t p artial
oxygen pressures (Pq2 = 0.24; 0.63; 0.84 bar) w e re m easured. A t th e highest
p a rtia l oxygen pressure (0.84 bar) th e e ffe c t o f increasing th e p a rtia l carbon
d io xide pressure was d e te rm in e d to assess w h e th e r p h o to re s p ira tio n could be
reduced by decreasing th e 0 2/ C 0 2 ra tio in th e m icroalgae cu ltu re .
2.2. Material and methods
2.2.1. Cultures and medium

N eochloris oleoabundans (UTEX 1185) cu ltu re s w e re m a in ta in e d in 100 m l liqu id
cu ltu re s in 250 ml E rlenm eyer flasks closed w ith p orous stoppers (Bio-silico,
H irschm ann Laborgeräte GmbH & Co.KG, G erm any). The flasks w e re placed in an
in c u b a to r w ith o rb ita l shaker (Innova 44R, New B runsw ick S cientific, USA) u nd er
flu o re s c e n t lig h t (40 p m o l m"2 s"1) a t 25 °C and 120 rpm . The air inside th e
in c u b a to r was enrich e d w ith 2% carbon dioxide.
A d ap te d f/ 2 m ed iu m (G uillard and Ryther, 1962) was used to g ro w and m ain ta in

N eochloris o le oabundans cu ltures. The m edium was com posed o f a rtific ia l sea
w a te r (in m M ): NaCI, 419; M gCI2.6 H 20 , 48.2; CaCI2.2 H 20 , 3.6; N a2S 0 4, 22.5;
K2S 04, 4.9. The a rtific ia l sea w a te r was enriched w ith th e fo llo w in g n u trie n ts (in
m M ): N aH 2P 0 4.2H 20 , 2.50; N a N 0 3, 32; tra c e ele m e n ts (in p M ): EDTA-FeNa, 29.3
CuS04.5H 20 , 0.10; N a2M o 0 4.2 H 20 , 0.07; ZnS 04.7 H 20 , 0.19; CoCI2.6 H 20 , 0.19;
M nC I2.4 H 20 , 2.27; v ita m in s (pg L"1): th ia m in e , 200; b io tin e , 1.00;
cyanocobalam ine, 1.00. The pH was adjusted to 7.8 w ith 0.5 M NaOH and th e
m ed iu m was sterilized via filtra tio n th ro u g h 0.22 pm filte rs . For th e re a c to r
17
C hapter 2
e xp e rim e n ts th e c u ltu re m edia was enrich e d w ith N aH C 03 to a fin a l c o n c e n tra tio n

o f 10 m M .
2.2.2. Photobioreactor
C ontinuous tu rb id o s ta t e xp e rim e n ts w e re p e rfo rm e d in a 3 L ja cke te d b io re a c to r
(A p plikon B iotechnology, The N etherlands) (Fig. 1). The in te rn a l d ia m e te r was
12.5 cm and th e liq u id v o lu m e was 2 L resu ltin g in an illu m in a te d surface o f 0.061
m 2 (Ar). The re a cto r was e qu ip pe d w ith a m arine im p e lle r. All sensors and
reg ula tors w e re co nnected to an E z-controller e qu ip pe d w ith Bioexpert® so ftw a re
(A p plikon B iotechnology, The N etherlands).
The m easured and c o n tro lle d process p aram eters w e re : pH; te m p e ra tu re ; oxygen
and carbon d io xide p artial pressure in th e liq u id phase (Po 2 and PCo2); liq u id level;
s tirre r speed and o ptica l d e n sity (OD). The pH was m a in ta in e d in th e range 7.8 ±
0.15 by a u to m a tic a d d itio n o f gaseous carbon d io xid e (C 0 2). T e m p e ra tu re was
m a in ta in e d a t 25°C. The p a rtia l oxygen pressure increased as a re su lt o f
p ho tosyn th esis and was m a in ta in e d a t th e desired level by a u to m a tic a d d itio n o f
gaseous d in itro g e n (N 2). The speed o f th e m arine im p e lle r was ke pt c o n s ta n t at
250 rpm . The o p tica l d e n sity was c o n tro lle d a t 0.55 CU using a tu rb id ity sensor
(ASD19-N, O ptek, G erm any) co nn ecte d to a p e ris ta ltic p um p a u to m a tic a lly adding
fresh m ed iu m to th e re a c to r w h e n req u ire d . The liq u id level was m a in ta in e d by a
level sensor c o n tro llin g a n o th e r p e ris ta ltic p um p to rem ove excess cu ltu re .
The Clark ty p e Pq2 sensor (L o w D rift sensor, Applisens, The N etherlands) was
ca lib ra te d inside th e p h o to b io re a c to r w ith g ro w th m ed iu m using pure 0 2 giving a
Po 2 o f 1 bar. The PC02 sensor was based on a pH sensor equ ip pe d w ith a C 0 2
selective m em b ra n e (InPro 5000, M e ttle r T oledo, S w itzerland) and was c a lib ra te d

s im ila rly b u t w ith 4% v /v C 0 2 enrich e d a ir giving a PC02 o f 0.04 bar.
18
S tirrer
C02
EZ-controller
N2
MFC
OD
Figure 1 - Experimental set-up (not on scale).

S = gas distributor, M = marine impeller, P = peristaltic pumps, Po2 = partial oxygen
pressure, OD = optical density or turbidity, Ez- controller = reactor control unit, MFC = mass
flo w controllers fo r carbon dioxide (C02) and nitrogen (N2)
The c u ltu re was c o n tin u o u sly illu m in a te d w ith tw o lig h t panels (20x20 cm ) w ith
red (627 nm ) LED lights (SL3500, Photon Systems In strum en ts, Czech Republic).
U nder o p e ra tin g co n d itio n s th e em ission peak sh ifts to hig he r w ave len gth s due to
lam p hea ting and a peak in te n s ity o f 635 nm was used in th e ca lcu latio n o f th e
lig h t g ra d ie n t. The panels w e re placed a t b o th sides o f th e re a c to r and a p la te o f
opal glass was placed in fr o n t o f th e lig h t panels to ensure b e tte r ligh t
d is trib u tio n . R eflective m a te ria l was placed on th e screens su rro u n d in g th e
re a c to r to hom ogenize th e lig h t fu rth e r (A p pe nd ix 2A). The in c id e n t lig h t in te n s ity
was m easured w ith a PAR q u a n tu m sensor (m odel SA-190, LiCor Biosciences, USA)
b e fo re th e s ta rt o f each e xp e rim e n ta l run. The m ea surem e nts w e re d on e at
d iffe re n t heights and radial p osition s to d e te rm in e th e average in c id e n t p h o to n
flu x d e n sity (PFD avg). The average value fo r th e d iffe re n t e x p e rim e n ts was always
b e tw e e n 187 and 210 p m o l m"2s-1. (A ppendix 2A)
2.2.3. Dry weight concentration

The d e te rm in a tio n o f th e d ry w e ig h t c o n c e n tra tio n o f re a c to r sam ples was done
in trip lic a te . Samples o f 5 mL w e re d ilu te d w ith 10 m l a m m o n iu m fo rm a te (0.5 M ).
19
C hapter 2
The d ilu te d sam ples w e re filte re d o ve r p re -w e ig he d glass fib e r-filte rs (W h atm a n

GF/F) and w ashed w ith an a d d itio n a l 40 ml o f a m m o n iu m fo rm a te (0.5 M ). The
filte rs w e re d rie d a t 95 °C fo r 24 hours in a lu m in u m trays, a llo w e d to cool d o w n in
a desiccator fo r a t least tw o hours, and w eighed (ME235P-SD, Sartorius,
G erm any).
2.2.4. Specific absorption coefficient

Light a b so rp tio n by th e m icroalgae cells was m easured in a specialized
s p e c tro p h o to m e te r set-up to m in im ize th e e ffe c t o f lig h t sca tte ring on th e
a b so rp tio n m ea surem e nt. A sam ple w ith e x tin c tio n a t 680 nm (ch lo ro p h yll
a b so rp tio n peak) b e tw e e n 1.8 and 2.2 was used, as m easured in a 1 cm c u v e tte in
a s p e c tro p h o to m e te r (Beckman DU®640, Beckman C oulter, USA). The absorbance
was th e n m easured w ith a fib e r o p tic CCD based s p e c tro p h o to m e te r (Avantes,
The N etherlands). The sam ple was placed in 2 m m lig h t path cu v e tte (Hellm a,
100.099-OS, 2 m m lig h t p ath ) and illu m in a te d w ith an AvaLight-H al lig h t source via
a FC-IR600-1-M fib e r e qu ip pe d w ith a co llim a tin g lens. An in te g ra tin g sphere
(AvaSphere-50) was d ire c tly placed beh in d th e c u v e tte and connected to th e
Avantes Avaspec-2048 d e te c to r via a n o th e r FC-IR600-1-M fib e r. The resu ltin g
absorbance was m easured fro m 400 nm to 750 nm . The average absorbance fro m
740 nm - 750 nm was su b tra cte d fro m th e absorbance b e tw e e n 400 nm and 700
nm , th u s co rre ctin g fo r residual sca tte ring (D ubinsky et al., 1986). The
w a ve le n g th -d e p e n d e n t d ry w e ig h t specific a b s o rp tio n c o e ffic ie n t (a¿ m 2 g"1) was
calculated based on th e absorbance (ABS) a t w a ve le n g th A, th e d ry w e ig h t (Cx, g
m"3), th e lig h t path o f th e cu ve tte (/, m) (e q u a tio n 1):
2 .3 0 3 - A B S Ä
(D
The lig h t g ra d ie n t inside th e b io re a c to r has been e stim a te d using Beers' law and
th e g e o m e trica l re la tio n sh ip d erived fo r cylind rica l vessels (Evers, 1991) w ith a
m o d ific a tio n to a ccou nt fo r th e use o f a fla t cosine receiver as lig h t sensor
(A p pe nd ix 2B.). The biom ass-specific a b so rp tio n c o e ffic ie n t was used fo r th is
ca lcu latio n. The fu ll a b so rp tio n sp ectrum o f a d ilu te d N eochloris oleoabundans
c u ltu re g ro w n a t 200 p m o l m"2 s"1 can be fo u n d in A p pe n dix 2C.
20
2.2.5. Photosynthesis-irradiance curve (PI- curve)

In o rd e r to co n firm th a t th e (average) PFD inside th e p h o to b io re a c to r indeed
im poses su b-satu ratin g lig h t co n d itio n s, a p h o tosyn th esis irra dian ce (PI) curve was
d e te rm in e d fo r N eochloris oleoabundans. The sam ple fo r th e PI curve
m e a su re m e n t was ta ke n fro m a batch c u ltu re in a fla t panel p h o to b io re a c to r w ith
2 m m lig h t path. Light in te n s ity on th e surface was set to 200 p m o l m"2 s"1,
te m p e ra tu re was 30 °C, pH was 7.5 and th e air flo w ra te was 0.70 L L"1 m in"1
enrich e d w ith 2% C 0 2. The sam ple was ta ke n w he n th e biom ass d en sity was 1.5
gD W L"1 and th e algae w e re exposed to re la tiv e ly lo w lig h t inten sitie s. The specific
oxygen p ro d u c tio n rate was m easured w ith a Biological Oxygen M o n ito r (BOM )
(Hansatech In stru m e n ts Lim ited, N o rfo lk England). The DW3 e le c tro d e ch am be r o f
Hansatech had a lig h t path o f 2 cm w h ich was illu m in a te d w ith a LH36/2R red LED
lig h t source (peak w a ve le n g th 655 nm ). The e le c tro d e ch am be r was fille d w ith 9
m l o f b u ffe re d m ed iu m w ith o u t any carbon. The m ed iu m was th e n flu she d w ith
p ure d in itro g e n fo r 10 m in utes. S ubsequently, 3 ml o f sam ple and 80 pL o f 0.75 M
N aH C 03 s o lu tio n w e re added. The rate o f dark re s p ira tio n was fo llo w e d fo r
several m in u te s a fte r w hich th e n e t specific oxygen p ro d u c tio n rate was fo llo w e d
fo r 3 m in u te s at 10 d iffe re n t PFD levels. C orresponding gross rates of
p h o tosyn th esis w e re th e n calculated by adding th e m easured d ark re s p ira tio n to
th e m easured n e t rates o f oxygen e vo lu tio n .
2.3. Results and discussion
2.3.1. Light regime

P h o to re sp ira tio n is expected to be th e d o m in a n t process leading to g ro w th
in h ib itio n because o f oxygen a ccu m u la tio n u n d e r su b -sa tu ra tin g lig h t co n d itio n s.
21
Chapter
(O
2.0
o
c max
0)
D)
>
X
o
ö
E
=L
(0
'w
0)
c
>*
W
5
o
x:
Q_
(0
</)
o
1—
o
0.0 ê -
0 200 400 600 800 1000
PFD (|j,mol m "2 s"1)
Figure 2 - Gross rate o f photosynthesis o f Neochloris oleoabundans versus irradiance cells

in pm ol oxygen g D W 1 s'1 a t different photon flu x densities (PFD)
To ensure th a t th e average lig h t in te n s ity experienced by th e algae inside th e

p h o to b io re a c to r was indeed sub-satu ratin g, p h o tosyn th esis irradiance (PI) curve
was d e te rm in e d fo r N eochloris oleoabundans. Figure 2 show s th e PI curve o f
N eochloris oleoabundans acclim ated to lo w lig h t co n d itio n s. A t lo w irradiance
levels, th e gross p h o to s y n th e tic rate o f th e algae is lin e a rly p ro p o rtio n a l to th e
irradiance. The graph show s th a t th e cells experienced su b-satu ratin g lig h t b e lo w
218 pm o l m"2 s-1.The PI curve th u s co n firm s th a t th e algae g re w u nd e r lig h t
lim itin g co n d itio n s at th e p h o to n flu x d en sity a pplied, w hich ranged fro m an
average o f 210 pm o l m"2 s 1 at th e re a cto r surface to values as lo w as 25 pm o l m"2
s 1 in th e re a cto r ce ntre (A ppendix B). A lth o u g h th e lig h t levels at th e b io re a c to r
surface w ere above 200 p m ol m"2 s"1, th e algae w e re o nly s h o rtly exposed to these
sa tu ra tin g lig h t levels considering th e m ixing o f th e liqu id . This te m p o ra l exposure
a p p a re n tly did n o t resu lt in sig n ifica n t p h o to in h ib itio n co nsidering th e high
g ro w th rate m easured. In a d d itio n , th e high biom ass yields on p ho ton s are
com p arab le to g ro w th rates fo u n d by Pruvost e t al. (2009). A p p a re n tly th e
te m p o ra l exposure to high lig h t locally at th e re a c to r surface did n o t neg ative ly
a ffe c t g ro w th .
22
G row th o f th e m icro a lg a e N eochloris oleoabundans a t high p a rtia l oxygen
pressures a nd s u b -s a tu ra tin g lig h t in te n s ity
2.3.2. Growth and productivity

Figure 3 shows a typ ical run at 0.21 bar p artial oxygen pressure. The b io re a c to r
was run nin g batch-w ise u n til th e desired o ptica l d e n sity o f 0.55 CU was reached
and th en th e tu rb id o s ta t c o n tro l was a ctivate d. The optical d e n s ity (0.55 CU)
corresponds to a biom ass c o n ce n tra tio n of 0.40 gDW L"1 fo r N eochloris
oleoabundans. The biom ass d ensity was ke p t c o n s ta n t and th e specific g ro w th
rate was d e te rm in e d w hen th e cells w ere acclim ated to th e c u ltu re c o n d itio n s and
th e d ilu tio n rate did n o t change fo r 4 consecutive days.
2.0
OD (CU)
PO (bar)
0.8 -
l-i (day )
- 1.5
Ü * 0 .6 -
CM
CL
O - 1.0
Û
o 0.4 -
- 0.5
0.2 -
0.0 0.0
0 2 4 6 8
Time (days)
Figure 3 - G raphical representation o f th e tu rb id o s ta t experim ent w ith Neochloris

oleoabundans a t an p a rtia l oxygen pressure o f 0.21 bar.
Presented are the p a rtia l oxygen pressure (Po2), optical density (OD), d ry w eight
concentration (Cx) and the specific g ro w th ra te (p.).
A t a dissolved oxygen c o n ce n tra tio n o f 100% air s a tu ra tio n (P q2 = 0.21 bar), a

specific g ro w th rate o f 1.38 day"1 was m easured (Fig. 3). This co rresp on ds to a
daily v o lu m e tric biom ass p ro d u c tiv ity o f 0.55 gDW L"1 day"1 and a biom ass y ie ld on
p h o to n s o f 1.04 gD W m ol-ph "1. Using th e daily v o lu m e tric biom ass p ro d u c tiv ity fo r
23
C hapter 2
N eochloris oleoabundans m easured by Pruvost e t al. (2009) and calcu latin g th e

p h o to n d aily v o lu m e tric p ro d u c tiv ity w ith th e p ro vid e d data o f in c id e n t lig h t flu x
and illu m in a te d surface to vo lu m e ra tio o f th e rea ctor, a biom ass yield on p ho ton s
o f 0.71 gDW m ol-ph "1 was calculated. This com p arison show s th a t N eochloris
o le oabundans show s high biom ass yields g ro w n on a m arine salt w a te r m ed iu m or
on a fre s h w a te r BBM m ed iu m as used by Pruvost and cow orkers. This o u tc o m e is
a ctu a lly n o t surprising since N eochloris o le oabundans (UTEX 1185) has been
isolated fro m an a rid soil (Guiry, 2011).
Table 1 show s th e c o m p ila tio n o f th e results o b ta in e d fo r th e d iffe re n t Pq2 te ste d.
The specific g ro w th rate and biom ass co n ce n tra tio n w e re calculated. Going fro m
a ir sa tu ra tio n levels (P o2= 0.21 bar) to th re e tim e s a ir s a tu ra tio n (Po 2 = 0.63 bar),
th e specific g ro w th ra te rem ain ed nearly th e same b u t reaching fo u r tim e s air
s a tu ra tio n (P 02 = 0.84 bar), a decrease in g ro w th rate was observed. W h e n te s tin g
N eochloris o le oabundans u n d e r a Pq2 o f 0.84 bar, th e specific g ro w th rate
decreased fro m 1.36 to 1.06 day"1.
Table 1 - Specific growth rate, biomass concentration o f the microalgae Neochloris

oleoabundans at different oxygen and carbon dioxide partial pressures.
N aH C 03(m M ) Po 2 (bar) Pco 2 (bar) p (d a y 1) iS td e v Cx (g Kg"1) ±Stdev
10 0.21 0.007 1.38 ± 0 .1 7 0.40 ± 0 .0 0 2
10 0.63 0.007 1.36 ± 0 .1 8 0.41 ±0.04

10 0.84 0.007 1.06 ± 0 .0 2 0.39 ± 0 .0 0 4
30 0.84 0.020 1.36 ± 0 .0 0 2 0.39 ± 0 .0 0 4
M o lin a et al. (2001) observed a decrease in p h o to s y n th e tic a c tiv ity of

P haeodactylum tric o rn u tu m a t Po2 hig he r th a n 0.21 bar. A lth o u g h a decrease in
g ro w th rate upon an increase o f th e p a rtia l pressure o f oxygen fro m 0.63 to 0.84

bar was fo u n d in th e c u rre n t study, no decrease was observed upon changing its
p a rtia l pressure fro m 0.21 to 0.63 bar. This re su lt could be due to th e a c tiv ity o f a
carbon co n c e n tra tio n m echanism s (CCM) p re sen t in N eochloris oleoabundans.
CCMs are e v o lu tio n a ry m echanism s deve lo pe d to o vercom e th e low a ffin ity o f
Rubisco fo r C 0 2 and its oxygenase and carboxylase d u a lity (G iordano e t al., 2005;
24
G row th o f th e m icro a lg a e N eochloris oleoabundans a t h igh p a rtia l oxygen
pressures and su b -s a tu ra tin g lig h t in te n s ity
Raven et al., 2008). H ow ever, th e CCMs could n o t p re v e n t a decrease in g ro w th

w hen going fro m p artial oxygen pressure o f 0.63 bar to 0.84 bar. Also, a CCM has
a lim ite d and requires a su bstantial e nergy in p u t (Vance and Spalding, 2005).
2.0
OD (CU)
P02 (bar)
(.i (day )
X
O
CM
o
0-
Q
O
0.5
C 02
= 0.007 bar C02 = 0.02 bar
0.0
o 2 4 6 8 10
Time (days)
Figure 4 - Graphical representation o f the tu rb id o s ta t experim ent w ith Neochloris

oleoabundans a t a p a rtia l oxygen pressure o f 0.84 b a r and carbon dioxide p a rtia l pressures
o f 0.007 an d 0.02 bar.
Presented are the p a rtia l oxygen pressure (P0f), o ptical density (OD), dry w eight
concentration (Cx) and the specific g ro w th ra te (p.).
In o rd e r to overcom e th e in h ib ito ry e ffe ct o f oxygen a t high p artial pressures, th e

C 0 2 co n c e n tra tio n was increased by th e a d d itio n o f extra N aH C 03 to th e c u ltu re
m edium . The N aH C 03 co n c e n tra tio n in th e m edium was increased fro m 10 m M to
30 m M co rresp on din g to an increase in th e PCo2 o f 0.007 ± 0.0001 to 0.020 ± 0.001
bar (m easured value). The d ilu tio n rate and hence th e specific g ro w th rate,
sta rte d to increase w ith in th e firs t day and reached a level o f 1.36 day"1 (Fig. 4),
sim ila r to th e g ro w th rates th a t w ere reached a t Po2=0.21 bar and 0.63 bar. This
resu lt indicates th a t p h o to re s p ira tio n is indeed responsible fo r th e observed
decrease in g ro w th rate at higher oxygen c o n c e n tra tio n . The o xygenase/
carboxylase a ctiv ity o f th e enzym e Rubisco is associated w ith th e ra tio b etw e e n
0 2 and C 0 2 in th e m ed iu m and once th e b ica rb o na te (N aH C 03) c o n c e n tra tio n and
25
C hapter 2
th e co rresp on din g C 0 2 co n c e n tra tio n was increased, th e specific g ro w th rate o f

th e algae increased again. Kliphuis e t al. (2011) studied th e e ffe c t o f 0 2/ C 0 2 ra tio
on th e p rim a ry m eta bo lism o f C hlam ydom onas re in h a rd tii u n d e r sim u late d
p ro d u c tio n co n d itio n s and fo u n d a decrease in specific g ro w th rate w ith
increasing 0 2/ C 0 2 ratios. Using a m e ta b o lic flu x m odel, th e y q u a n tifie d
o xygenase/carboxylase reactions and th e fluxes th ro u g h th e p h o to re s p ira to ry
p a th w a y and show ed th a t th e decrease fo u n d can be explained by th e increased
oxygenase a ctivity. They fo u n d th is e ffe c t a lrea d y w h e n increasing Po2 in th e liqu id
phase fro m 0.11 to 0.26 bar a t a PC02 o f 0.014 bar a ltho ug h th e y w e re w o rk in g at
o ve r-sa tu ra tin g in cid e n t lig h t o f 600 p m o l m"2 s"1. To co nclude fro m b o th th e

presented stu dy as w e ll as fro m th e stu d y p resented by Kliphuis e t al. (2011), an
increase o f dissolved 0 2/ C 0 2 ratio s in p h o to b io re a c to rs w ill re s u lt in reduced
p ro d u c tiv ity a lth o u g h th e actual q u a n tita tiv e e ffe c t is species d ep en de nt.
Especially N eochloris oleoabundans appears to to le ra te high Po2 levels a t sub-
s a tu ra tin g light.
2.3.3. Implications on reactor design

Large-scale o u td o o r p ro d u c tio n o f m icroalgae is n o t y e t e co n o m ica lly feasible.
High e nergy inp uts are re q u ire d fo r m ixing to rem ove th e p h o to s y n th e tic a lly
p ro du ced oxygen ( 0 2). O ur results can help in reducing th e e nergy re q u ire m e n t
fo r c u ltiv a tio n system s w h e re spatial d ilu tio n o f lig h t is a pplied. W hen using
system s o f ve rtica l panel p h o to b io re a c to rs (R odolfi e t al., 2009), s u n lig h t in te n s ity
can be reduced and d is trib u te d o ve r a larger surface. In v e rtica l fla t panel reactors
th e p h o to s y n th e tic e fficie n cy is high because o f th e d ilu tio n o f lig h t (Cuaresma e t
al., 2011). A t th ese reduced lig h t levels in high d e n s ity m icroalgae cu ltu re s th e
e ffe c t o f p h o to in h ib itio n w ill be absent and th e negative e ffe c t o f high oxygen
c o n ce n tra tio n s on N eochloris o le oabundans can be o vercom e by w o rk in g a t
e levated C 0 2 co n ce n tra tio n s.
W h e n w o rk in g w ith co m b u stio n gasses w ith 10 to 20 % v /v C 0 2, it is a rea listic

scenario to m a in ta in C 0 2 p a rtia l pressures in th e o rd e r o f 0.02 bar w h ile still being
able to rem ove m ore th a n 80% o f th e C 0 2 fro m th e gas. The advantage o f w o rk in g
a t a hig he r p a rtia l C 0 2 pressure is th e fa c t th a t a hig he r p a rtia l 0 2 pressure can be
to le ra te d in th e c u ltu re as show n in th is stu dy and th e re is a hig he r d riv in g fo rc e
26
fo r oxygen tra n s fe r fro m th e liq u id to th e gas phase. C onsequently, th e rate o f

gassing can be reduced.
As an e xam ple th e re d u c tio n in gas flo w rate, Fg (m 3 s'1) was calculated fo r a case
w h e re a fla t panel p h o to b io re a c to r was ru n nin g a t a dissolved 0 2 c o n c e n tra tio n
e q u iv a le n t to a Po2 o f 0.84 bar instead o f PO2=0.42 bar (assumed to be c u rre n t
p ractice). In th is ca lcu latio n, w e assum ed a 1.5 m high panel re a c to r w ith a d e p th
o f 0.07 m. The oxygen tra n s fe r re q u ire m e n t (OTR) fo r such a system was
calculated considering a v o lu m e tric oxygen p ro d u c tio n rate o f 1.98 x IO"4 m ol m"3
s'1. This oxygen p ro d u c tio n rate was e stim a te d based on a q u a n tu m re q u ire m e n t
w h ich was calculated fro m th e biom ass yield on p h o to n s observed in th is study
(1.04 gD W m ol-ph "1) and a lig h t energy in p u t o f 200 p m o l m"2 s 1 on th e ve rtica l
side o f th e panel (one side only).
The oxygen tra n s fe r c o e ffic ie n t K¡.a (s'1) needed to rem ove th e oxygen produced
fro m th e p h o to b io re a c to r w h ile m a in ta in in g a dissolved oxygen c o n c e n tra tio n in
th e liqu id phase, Coh o f 0.537 m ol m"3 (e q u iva le n t to Pq2=0.42 bar) o r 1.073 m ol m"
3 (e q u iva le n t to PO2=0.84 bar). The c o n c e n tra tio n o f 0 2 in th e liq u id a t th e gas-

liq u id in te rfa ce was assum ed to be in e q u ilib riu m w ith th e oxygen level in air, so
C 0i= 0.268 m ol m"3. The d iffe re n ce b etw e e n C0¡ and C 0¡ represents th e d rivin g
fo rc e o f 0 2 tra n s fe r o ve r th e liq u id b o u n d a ry layer su rro u n d in g th e gas bubbles
(e q u a tio n 2).
(2 )
The gas flo w rate, Fg (m 3 s"1) was calculated using th e re la tio n fo r K|.a d e te rm in e d
by Sierra e t al., (2008) fo r fla t panel reactors (e q u a tio n 3).W h e re p (Kg m"3) stands
fo r th e d e n sity o f th e liq u id ; g (m"2) th e g ra v ita tio n a l accelera tion and A dg (m 2) th e
degassing area (equal to th e re a c to r h o rizo n ta l cross section).
K ,.a = 2 .3 9 -1 0 4 (3 )
Upon increasing th e dissolved oxygen co n c e n tra tio n w e fo u n d a decrease in

_A Q A _A Q A
re q u ire d Fg fro m 2.52 x 10" m s" to 0.70 x 10" m s" and th is m eans a decrease
by a fa c to r o f 3.6. Such a decrease could g re a tly reduce energy re q u ire m e n t fo r
27
C hapter 2
large-scale c u ltiv a tio n o f m icroalgae; h ow eve r, th is strateg y w o u ld o n ly be

possible th o u g h in case C 0 2-rich (flue ) gas can be used in c o m b in a tio n w ith a
re a c to r design aim ing a t reduced lig h t levels on th e light-exposed surface (spatial
d ilu tio n o f light).
2.4. Conclusions
N eochloris o le oabundans appears to to le ra te high Pq2 levels a t s u b-satu ratin g lig h t
since no decrease was observed upon changing Po2 fro m 0.21 to 0.63 bar. A t Po2
=0.84 bar a decrease o f g ro w th was fo u n d u n d e r th e co n d itio n s stu die d. A t sub-
s a tu ra tin g lig h t inten sitie s, p h o to re s p ira tio n appeared to be th e m ain cause o f
g ro w th in h ib itio n because re sto rin g th e 0 2/ C 0 2 ra tio th ro u g h increasing PCo2,
could o ffs e t th e negative im p a ct o f e levated Pq2. A s im ila r stra te g y can reduce th e
energy in p u t needed fo r oxygen rem oval. This approach could be used fo r

p ro d u c tio n in a closed system (using d ilu te d ligh t) w ith lo w e r energy
re q u ire m e n ts fo r degassing.
2.5. Acknowledgements
This w o rk was p e rfo rm e d in th e T T IW -co op eratio n fra m e w o rk o f W etsus, C entre
o f Excellence fo r Sustainable W a te r T echnology (w w w .w e ts u s .n l). W etsus is
fu n d e d by th e D utch M in is try o f Econom ic A ffairs, th e European U nion Regional
D e ve lo p m e n t Fund, th e Province o f Fryslân, th e C ity o f Leeuw arden and th e
EZ/Kompas p ro gram o f th e 'S am enw erkingsverband N o o rd -N e d e rla n d ". The
a u th o rs like to th a n k th e p a rticip a n ts o f th e research th e m e "A lga e" fo r th e
discussions and th e ir fin a n cia l su pp ort.
2.6. References
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Cuaresma, M ., Janssen, M ., Vilchez, C., W ijffe ls , R.H., 2011. H orizontal o r vertica l

p h o to b io re a cto rs? H ow to im p ro ve m icroalgae p h o to s y n th e tic e fficiency.
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28
Dismukes, G.C., C arrieri, D., B ennette, N., Ananyev, G .M ., Posewitz, M.C., 2008.
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p h y to p la n k to n . Plant and Cell Physiology, 27, 1335-1349.
Evers, E.G., 1991. A m odel fo r lig h t-lim ite d co n tin u o u s cu ltures: g ro w th , shading,
and m aintenance. B io tech n olog y and B ioengineering, 38, 254-259.
G iordano, M ., Beardall, J., Raven, J.A., 2005. C 0 2 C on cen tratin g M echanism s in

Algae: M echanism s, E nviro nm e nta l M o d u la tio n , and E volution. A nnual Review
Plant Biology, 56, 99-131.
G u illard, R.R.L., R yther, J.H., 1962. Studies o f m arine p la n k to n ic dia to m s. I.

C yclotella n an a h uste dt, and D eton ula confervagea (Cleve) gran. Canadian Journal
o f M icro b io lo g y, 8, 229-239.
G uiry, W ., 2011. In G uiry, M .D. and G uiry, G.M . AlgaeBase. W o rld -w id e e le ctro n ic
p u b lica tio n , N ational U nive rsity o f Ireland, Galway, h ttp ://w w w .a lg a e b a s e .o rg ;
searched on 10 O cto b e r 2011.
Kliphuis, A.M.J., de W in te r, L., Vej'razka, C., M arte ns, D.E., Janssen, M., W ijffe ls ,
R.H., 2010. P h o to syn th e tic e fficie n cy o f C hlorella s o ro kin ia n a in a tu rb u le n tly
m ixed s h o rt lig h t-p a th p h o to b io re a c to r. B io tech n olog y Progress, 26, 687-696.
Kliphuis, A.M.J., M arte ns, D.E., Janssen, M., W ijffe ls , R.H., 2011. Effect o f 0 2:C 0 2
ra tio on th e p rim a ry m e ta b o lism o f C hlam ydom onas re in h a rd tii. B iotechnology
and B ioengineering, 108 (10), 2390-2402.
M o lin a , E., Fernández, F.G.A., Chisti, M.Y., 2001. T ub ula r p h o to b io re a c to r design

M u ra ta , N., Takahashi, S., Nishiyam a, Y., A llakh ve rd ie v, S.I., 2007. P h o to in h ib itio n

o f p ho tosystem II u n d e r e n v iro n m e n ta l stress. Biochim ica e t Biophysica Acta,
1767, 414-421.
N orsker, N.H., Barbosa, M.J., V erm uë, M .H ., W ijffe ls , R.H., 2010. M icroalgal
p ro d u c tio n - A close loo k a t th e econom ics. B io tech n olog y Advances, 29, 24-27
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C hapter 2
O gren, W.L., 1984. P h o to re sp ira tio n : Pathways, reg ula tion , and m o d ific a tio n .
A nnual Review Plant Physiology, 35, 415-442.
O sm ond, C.B., 1981. P h o to re sp ira tio n and p h o to in h ib itio n . Some im p lica tio n s fo r
th e energetics o f pho tosyn th esis. Biochim ica Biophysica Acta, 639, 77-98.
Pospisil, P., 2011. M o le cu la r m echanism s o f p ro d u c tio n and scavenging o r reactive

oxygen species by p ho tosystem II. Biochim ica Biophysica Acta,
d oi:1 0 .1 0 1 6 /j.b b a b io .2 0 1 1 .0 5 .0 1 7 .
Pruvost, J., Van V ooren, G., Cogne, G., Legrand, J., 2009. In vestig atio n o f biom ass
B ioresource Technology, 100, 5988-5995.
Raso, S., V erm uë, M .H ., W ijffe ls, R.H.,. Effect o f oxygen c o n c e n tra tio n on th e
g ro w th o f N annochloropsis np. a t low lig h t in te n sity. Journal o f A pplie d Phycology,
1-9.
Raven, J.A., G iordano, M ., Beardall, J., 2008. Insights in to th e e v o lu tio n o f CCMs

fro m com parisons w ith o th e r resource a cqu isitio n and assim ila tio n processes.
Physiology P lantarum , 133, 4-14.
R odolfi, L., Z itte lli, G.C., Bassi, N., Padovani, G., Biondi, N., Bonini, G., Tredici, M.R.,
2009. M icro alga e fo r Oil: Strain Selection, In d u ctio n o f Lipid Synthesis and
O u td o o r Mass C u ltiva tio n in a Low-Cost P h o to b io re a c to r. B io tech n olog y and
B ioengineering, 102, 100-112.
Schenk, P.M., Thom as-H all, S.R., Stephens, E., M arx, LI., M ussgnug, J.H., Posten,
C., Kruse, O., H ankam er, B., 2008. Second G e ne ra tio n Biofuels: H igh-E fficiency
M icroalgae fo r Biodiesel P roduction. B ioenergy Research, 1, 20-43.
Sierra, E., Acién, F.G., Fernández, J.M ., García, J.L., González, C., M olina , E., 2008.
C ha ra cte riza tion o f a fla t p la te p h o to b io re a c to r fo r th e p ro d u c tio n o f m icroalgae.
Chem ical Engineering Journal, 138, 136-147.
T orzillo , G., B ernardini, P., M asojidek, J., 1998. O n-line m o n ito rin g o f c h lo ro p h yll
high oxygen co n ce n tra tio n and lo w te m p e ra tu re and its e ffe c t o f th e p ro d u c tiv ity
30
o f o u td o o r cu ltures o f S piru lin a p la te nsis (C yanobacteria). Journal o f Phycology,

34, 504-510.
M ., Van Breusegem, F., M u e lle r, M.J., 2008. Singlet oxygen is th e m a jo r reactive
148, 960-968.
Ugwu, C.U., Aoyagi, H., U chiyam a, H., 2007. Influence o f irradiance, dissolved
P h otosyn th etica , 45, 309-311.
Vance, P., Spalding, M .H ., 2005. G ro w th , pho tosyn th esis, and gene expression in
C hlam ydom onas o ve r a range o f C 0 2 co n c e n tra tio n s and C 0 2/ 0 2 ratios: C 0 2
regulates m u ltip le a cclim atio n states. Canadian Journal o f Botany, 83, 796-809.
W ijffe ls , R.H., Barbosa, M.J., 2010. An o u tlo o k on m icroalgal biofuels. Science,

330, 913-913.
W ijffe ls , R.H., Barbosa, M.J., Eppink, M .FI.M ., 2010. M icroalgae fo r th e p ro d u c tio n

o f b ulk chem icals and biofuels. Biofuels B ioproducts and B io re finin g, 4, 287-295.
31
C hapter 2
Appendix 2A. Distribution of incident photon flux density over

photobioreactor surface
In th e e xp e rim e n ta l set-up used p e rfo rm e d th e in c id e n t lig h t in te n s ity varied o ver
th e illu m in a te d surface. The p h o to n flu x d e n sity peaked a t 350 p m o l m"2 s"1 w he n
fa cing th e lig h t source; was as lo w as 100 p m o l m"2 s"1 w he n fa cing th e re fle c tiv e
screens. The average value fo r th e d iffe re n t e x p e rim e n ts was alw ays b e tw e e n 187
and 210 p m o l m"2 s"1.
Table A Í - Incident light measured at different radial positions and different heights o f the
photobioreactor prior to one o f the experiments. All numbers represent photon flu x
densities in ¿umol PAR photons m 2 s'1.
Radial Axis
H eight a b c d e f g h
1 (0 cm) 262 160 105 173 252 154 90 150

2 (4 cm) 334 167 112 177 288 161 92 186
3 (8 cm) 353 175 114 187 195 171 93 173
4(12cm ) 360 174 107 174 295 169 87 190
Average 187
_B_
iii
S lii
*!!
::: ;;;
iii ^
1 / iii
S
::: \ ' :::
C B C
Figure A l - Top view illustrating the illumination o f the bioreactor
A = LED panels, B = screens with reflective material, C = opal glass, a,b,c,d,e,fg,h = radial
positions fo r incident light measurement.
32
Table A2 - Photon flu x density fo r each experimental run.
Po2 (bar) Light in te n s ity (pm ol m"2 s"1) ± Stdev

0.21 189 ± 83
0.63 187 ± 78
0.84 210 ± 7 1
Appendix 2B. Calculation of light gradient in photobioreactor

The biom ass specific a b so rp tio n c o e ffic ie n t a t a w a v e le n g th o f 635 nm was used in
th e ca lcu latio n, since th is was th e d o m in a n t w a ve le n g th o f th e LED lig h t source
used. The lig h t g ra d ie n t inside th e b io re a c to r was e stim a ted using Beers' law and
th e g e o m e trica l re la tio n sh ip d erived fo r cylind rica l vessels (Evers, 1991) w ith a
sm all m o d ific a tio n to a ccou nt fo r th e use o f a fla t cosine rece ive r as lig h t sensor.
This re la tio n was m o d ifie d to o b ta in th e p h o to n flu x d en sity on a fla t (27i) cosine
rece ive r facing th e re a c to r w all (PFD(z)wan) and facing th e re a c to r ce n tre
(PFD(z)centre) e qu atio ns 1, 2 and 3. W h e re X (g m"3) stands fo r biomass
c o n c e n tra tio n ; 0 is th e angle o f lig h t path w ith line tro u g h vessel ce nte r; a (m 2g)
th e a b so rp tio n c o e ffic ie n t; r (m) th e vessel radius and s (m ) th e distance fro m
vessel surface.
PFDin r r r i
PFD(z)waII = — jc o s (0 + z).exp - a.X. (r - s).cos(0) + [r2 - (r - s)2. sin(0)2J° i
©
jc o s (0 + z)d®
r r p .
PFDin
PFD(z)center = - jcos(© + ;r).exp-ar.A' j>-s).cos(©) + [r2 - ( r - s ) 2.sin(©)2] i
©
jcos(© + jr)d®
PFD(z) = PFD(z)wall + PFD(z)center
33
Chapter
200
------------- P F D (z) W a ll
P F D (z) c e n te r
150 -
P F D (z)
E
Ö
100 -
Q
Li.
Q-
50 -
0.00 0.01 0.02 0.03 0.04 0.05 0.06
z (m)
Figure B Í - The PAR photon flu x density on a fla t (2n) cosine receiver facing the reactor
wall (PFD(z)waii) and facing the reactor centre (PFD(z)centre) as a function o f the depth z
inside the cylindrical photobioreactor used. PFD(z) is the sum o f both.
Appendix 2C. Specific absorption spectrum A

0.5
0.4
0.3
CM
TO 0.2
0.1
0.0
400 500 600 700
W avelength (nm)
Figure Cl - The wavelength dependent specific absorption coefficient o f N. oleoabundans

culture grown a t 2 0 0 p m o lm 2s 1.
34
35
C hapter 3
36
E ffect o f oxygen a t lo w a n d hig h lig h t in te n s ity on the g ro w th o f N eochloris
oleoabundans
Chapter 3 . E ffe c t o f O xyg en a t l o w a n d hig h l ig h t in te n s ity

ON THE GROWTH OF NEOCHOLORIS OLEOABUNDANS
Claudia Sousa1' 2, Ana C om pa d re 1, M arian H. V e rm u ë 2and Rene H. W ijffe ls 2
1Wetsus, P.O. Box 1113, 8900 CC Leeuwarden, the Netherlands
2 Bioprocess Engineering, Wageningen University, P.O. Box 8129, 6700 EV, Wageningen,
the Netherlands
A b s tra c t - The e ffe c t o f p a rtia l oxygen pressure on g ro w th o f N eochloris

o le oabundans was stu die d a t n e a r-sa tu ra tin g lig h t in te n s ity in a fu lly -c o n tro lle d
p h o to b io re a c to r. A t th e p a rtia l oxygen pressures te s te d (Po2= 0.24; 0.42; 0.63;
0.84 bar), th e specific g ro w th rate was 1.36; 1.16; 0.93 and 0.68 day"1,
respectively. An increase o f th e PCo2 fro m 0.007 to 0.02 bar a t Po2 o f 0.84 bar did
n o t show any p ositive e ffe c t on th e overall g ro w th o f th e algae, c o n tra ry to w h a t
happens a t su b-satu ratin g lig h t inten sitie s. These results in d ica te th a t a t near-
s a tu ra tin g lig h t in te n s ity th e in h ib ito ry e ffe c t o f oxygen by p h o to re s p ira tio n
c a n n o t be o vercom e. The ch lo ro p h yll c o n te n t o f N eochloris o le oabundans g ro w n
a t 200 p m o l m"2 s"1 is a b o u t 1.9 tim e s higher th a n w he n c u ltiv a te d a t 500 p m o l m"2
s"1, w hereas th e c a ro te n o id c o n te n t was a b o u t 1.5 low e r, b o th d e m o n s tra tin g
p h o to a c c lim a tio n e ffects. The e levated oxygen c o n c e n tra tio n in th e g ro w th
m ed iu m does n o t a ffe c t th e p ig m e n t c o n te n t b o th a t sub- and n ea r-sa tu ra tin g
lig h t c o n d itio n s. This indicates th a t e levated oxygen co n c e n tra tio n s in th e m edium
does n o t c o n trib u te to p h o to o xid a tive dam age a t th e lig h t co n d itio n s th a t are
p re d o m in a n tly experienced by algae in closed p h o to b io re a c to rs , b u t o nly in h ib it
th e g ro w th via p h o to re sp ira tio n effects.
Key w o rd s : N eochloris oleoabundans, oxygen in h ib itio n , p h o to o x id a tiv e dam age,

p h o to a cclim a tio n , p h o to re s p ira tio n , p h o to b io re a c to r
Sousa, C., Compadre, A., Vermuë, M.H., Wijffels, R.H. 2013. Effect o f oxygen at low and
high light intensities on the growth o f Neochloris oleabundans. Algal Research, 2 (2), 122-
126.
37
C hapter 3
3.1. Introduction
N eochloris o le oabundans is one o f th e algae w h ich com bines high specific g ro w th
rate a t o p tim a l g ro w th co n d itio n s (Gouveia e t al., 2009; Li e t al., 2008; Pruvost e t
al., 2009) w ith a ccu m u la tio n o f lipids w ith large c o n te n t o f sa tu ra te d fa tty acids
d u rin g n itro g e n sta rva tio n c o n d itio n s (P ruvost e t al., 2009; Santos e t al., 2012;
T ornabene e t al., 1983). These ch aracte ristics m ake th is alga species a p ro m ising
fe e d stock fo r b io fu e l p ro d u c tio n (Chisti, 2007; Schenk e t al., 2008; W ijffe ls e t al.,
2010). For large-scale o u td o o r p ro d u c tio n o f algae, closed p h o to -b io re a c to r
system s (PBR) have been proposed. To m ake th e p ro d u c tio n e con om ically
feasible, how eve r, b o ttle n e cks still need to be o vercom e. One o f th ese
b o ttle n e cks is th e high energy in p u t th a t is re q u ire d fo r m ixing to p ro vid e th e
algae w ith ligh t, carbon d io xide and to rem ove th e p h o to s y n th e tic a lly produced
oxygen (Dism ukes e t al., 2008; N orsker e t al., 2011; W ijffe ls e t al., 2010).
In p h o to -b io re a c to rs oxygen th a t is pro du ced d urin g pho tosyn th esis, accum ulates

and induces processes like p h o to re s p ira tio n and p h o to in h ib itio n , b o th leading to a
decrease in biom ass yield on lig h t energy o f th e m icroalgae (Torzillo e t al., 1998).
In p h o to re s p ira tio n , oxygen binds w ith th e enzym e Rubisco and com petes w ith
carbon d io xide needed fo r pho tosyn th esis. Hence, high oxygen levels lead to
lo w e r C 0 2 upta ke and reduced fix a tio n o f lig h t energy in to ca rb oh ydrates (Bader
e t al., 2000).
P h o to in h ib itio n occurs m ainly a t high and o v e r-s a tu ra tin g lig h t inten sitie s. A t
th o se c o n d itio n s an excess o f e le ctron s is gen erated in P hotosystem II and th ese
e le ctron s w ill react w ith th e p h o to s y n th e tic a lly p roduced oxygen, leading to th e
fo rm a tio n o f oxygen radicals and o th e r rea ctive oxygen species (ROS) such as
H20 2 (M u ra ta e t al., 2007). In a d d itio n , lig h t s tim u la te s th e fo rm a tio n o f th e h ighly
rea ctive sing let oxygen via p h o to -a c tiv a tio n (T rian ta ph ylid es e t al., 2008). The
sing let oxygen causes dam age o f th e w a te r-o x id iz in g c e n te r and deactivates th e
e le ctro n tra n s p o rt chain (Krieger-Liszkay e t al., 2008; Hakala e t al., 2005) and th is
results in loss o f p h o to s y n th e tic a c tiv ity and d eath o f cells.
To o vercom e th e p h o to -o x id a tiv e dam age caused by p h o to in h ib itio n a t high light,

th e m icroalgae have developed several p ro te c tio n m echanism s, g en erally re fe rre d
to as p h o to -a cclim a tio n . P h oto-acclim atio n can easily be recognized by changes in
38
oleoabundans
th e p ig m e n ta tio n o f th e algae, re su ltin g in lo w e r c h lo ro p h y ll c o n te n t and higher

ca ro te n o id c o n te n t o f th e algae w h e n exposed to h ig he r irradiance. The
ca ro ten o id s c o n te n t n o rm a lly increases to enable th e algae to dissipate energy o f
excited c h lo ro p h yll and e lim in a te ROS and to m a in ta in th e p ho tosystem s tru c tu re
(D em m ig-A dam s & Adam s, 2002). In a d d itio n , ca ro te n o id s scavenge tr ip le t
ch lo ro p h yll and quench singlet oxygen (Falkow ski & Raven, 2007). A t ve ry high
lig h t irra d ia tio n , h ow eve r, th e p ro te c tiv e m echanism s ca n n o t s u ffic ie n tly deal
w ith th e surplus of e le ctron s and fo rm a tio n of singlet oxygen and th e
a ccu m u la tio n o f ROS occurs, leading to cell dam age (T rian ta ph ylid es e t al., 2008).
A lth o u g h th e co m b in e d e ffe c t o f oxygen and lig h t have been described in d eta il,
th e e ffe c t o f a ccum ula ting oxygen on algal g ro w th is o n ly studied in d e p e n d e n tly
a t c o n tro lle d lo w lig h t co n d itio n s (Kliphuis e t al., 2011; Raso e t al., 2011; Sousa e t
al., 2012). U pon an increase in oxygen c o n c e n tra tio n s in th e algal cu ltu re s a
general decrease in specific g ro w th rates has been observed. This in h ib itio n a t low
lig h t co n d itio n s is related to th e ca rb o xy la tio n /o x y g e n a tio n ra tio o f th e enzym e
Rubisco and its a ffin ity fo r oxygen and th e oxygen in h ib itio n e ffe cts a t low lig h t
co n d itio n s can be co m pensated by an increase of th e carbon d io xide
c o n c e n tra tio n (Sousa e t al., 2012). In o u td o o r c u ltiv a tio n ; h ow eve r, algae w ill
experience d iffe re n t lig h t co n d itio n s. It is th u s im p o rta n t to k n o w h o w th e algae
respond on a ccum ula ted oxygen a t c o n tro lle d c u ltu re c o n d itio n s a t h ig he r lig h t
in te n sitie s and inve stiga te if a d d itio n o f carbon d io xide could be used to
o vercom e th e in h ib itin g e ffe cts o f oxygen a t hig he r lig h t co n d itio n s as w e ll. In th is
paper, th e e ffe c t o f oxygen p a rtia l pressure on th e g ro w th o f N eochloris
o le oabundans exposed to n e a r-sa tu ra tin g co n d itio n s was d e te rm in e d in a fu lly -
c o n tro lle d p h o to -b io re a c to r o p e ra te d in tu rb id o s ta t m ode and co m p ared w ith th e
in h ib itin g e ffe cts o f oxygen on g ro w th a t lo w lig h t co n d itio n s. The m a g n itu d e o f
th is e ffe c t on th e specific g ro w th rate as w e ll as on th e biom ass yield on ligh t
energy and p ig m e n t c o n te n t was d e te rm in e d . Finally, th e C 0 2/ 0 2 ra tio was
increased to see if th e in h ib itin g e ffe c t o f 0 2 in m icroalgae could be overcom e.
39
C hapter 3
3.2. Material and methods
3.2.1. Cultures and m edium

A d ap te d f /2 m ed iu m (G uillard & R yther, 1962) was used to g ro w and m ain ta in
N eochloris oleoabundans (UTEX 1185) cu ltures. The m ed iu m was com posed o f
a rtific ia l sea w a te r (in m M ): NaCI, 419; M gC I2.6 H 20 , 48.2; CaCI2.2 H 20 , 3.6;
N a2S 0 4, 22.5; K2S 0 4, 4.9. The a rtific ia l sea w a te r was enrich e d w ith th e fo llo w in g
n u trie n ts (in m M ): NaH2P 0 4.2H 20 , 2.50; N a N 0 3, 32; tra c e e le m e n ts (in p M ):
EDTA-FeNa, 29.3 CuS04.5 H 20 , 0.10; N a2M o 0 4.2 H 20 , 0.07; ZnS 04.7H 20 , 0.19;
CoCI2.6H 20 , 0.19; M nC I2.4 H 20 , 2.27; v ita m in s (pg L"1): th ia m in e , 200; b io tin e ,
1.00; cyanocobalam ine, l.OO.The pH was a djusted to 7.8 w ith 0.5 M NaOH.
N eochloris o le oabundans was p re -cu ltu re d in an in c u b a to r w ith o rb ita l shaker
(Innova 44R, New B runsw ick Scientific, USA) u n d e r flu o re s c e n t lig h t (40 p m o l m"2
s"1) a t 25 °C and 120 rpm . The air inside th e in c u b a to r was enriched w ith 2%
carbon d ioxide. In th e re a c to r e xp e rim e n ts th e c u ltu re m edia was e nriched w ith
10 m M N aH C 03.
A 3 L ja cke te d b io re a c to r (A p plikon B iotechnology, The N etherlands) was used to
p e rfo rm co n tin u o u s tu rb id o s ta t e xpe rim en ts. All sensors and reg ula tors o f th e
e xp e rim e n ta l set-up w e re co nn ecte d to an E z-controller e qu ip pe d w ith
Bioexpert® so ftw a re (A p plikon B iotechnology, The N etherlands). The c u ltu re was
illu m in a te d w ith tw o lig h t panels (20x20 cm ) w ith red (627 nm ) LED lights
(SL3500, Photon Systems In strum en ts, Czech Republic). The in c id e n t lig h t in te n s ity
was m easured w ith a PAR q u a n tu m sensor (m odel SA-190, LiCor Biosciences, USA)
b e fo re th e s ta rt o f each e xp e rim e n ta l run. The m ea surem e nts w e re d on e at
d iffe re n t heights and radial positions to d e te rm in e th e average in c id e n t p h o to n
flu x d e n sity (PFDavg). The average value fo r th e d iffe re n t e x p e rim e n ts a t h ig h -lig h t
in te n s ity was alw ays ~ 500 p m o l m"2 s'1, w h ile a t lo w lig h t in te n s ity th e average
in cid e n t p h o to n flu x d e n sity was ~200 p m o l m"2 s"1 The m easured and c o n tro lle d
process param eters w e re : pH, te m p e ra tu re , oxygen and carbon d io xide p artial
pressure in th e liqu id phase (P q2 and PCo2), liq u id level, s tirre r speed and o ptical
d e n sity (OD) (Sousa e t al., 2012).
40
oleoabundans
The cells w e re a dapted to th e tu rb id o s ta t co n d itio n s fo r a t least 3 days, b efo re

th e specific g ro w th rate (p) was d e te rm in e d fro m th e d ilu tio n rate. The o ptica l
d e n sity a t 750 (OD750) and 680 nm (O D 680) was m easured in a U V-visible
s p e c tro p h o to m e te r (UV-1650 PC, Schimadzu). The cell d ry w e ig h t c o n c e n tra tio n
and pig m e n ts c o n te n t w e re d e te rm in e d o ff line as w ell.
3.2.3. Dry w eig ht concentration

To d e te rm in e th e d ry w e ig h t co n c e n tra tio n , 5 mL sam ples in tr ip lo w e re w ashed
w ith 10 mL o f a m m o n iu m fo rm a te 0.5 M , filte re d th ro u g h a p re -w e ig he d glass
fib e r filte r (W h atm a n GF/F), and w ashed again w ith 40 mL o f a m m o n iu m fo rm a te
0.5 M . The filte rs w e re d rie d in an oven a t 95 °C, fo r 24 h, in a lu m in iu m trays,
cooled in a desiccator fo r a t least 2 hours, and th e n w eig he d on a 5 d ig it analytical
balance (ME235P-SD, Sartorius, G erm any).
3.2.4. Chlorophyll and carotenoids

C hlo ro ph yll and ca ro te n o id c o n te n t o f th e algae was d e te rm in e d in trip lic a te at
th e end o f each e xp e rim e n t. A 2 mL algal a liq u o t co lle cted fro m th e re a c to r was
c e n trifu g e d a t 3760 rpm and 4 °C d u rin g 10 m in (Allegra™ X-12 R C entrifuge). The
pellets w e re fro ze n a t - 80 °C, p rio r to fu rth e r analysis. C hlo ro ph yll was e xtra cte d
by th e adding 5 ml o f m e th a n o l (100 %) to th e biom ass p e lle t. The cells w e re
d isru p te d by u ltra so u n d (Sonorex Digitec, Bandelin) c o m b in e d w ith te m p e ra tu re
shock (in cu b a tio n a t 60 °C and 0 °C). The suspension was c e n trifu g e d a t 3760 rpm
and 4 °C d u rin g 10 m in. The su p e rn a ta n t was co lle cted and ch lo ro p h y ll and
ca ro te n o id c o n te n t w e re d e te rm in e d a t 470, 652 and 665 nm in a U V-visible
s p e c tro p h o to m e te r (UV-1650 PC, Schimadzu). M o d ifie d A rn o n 's e qu atio ns
(Lichten th aler, 1987) w e re used to calculate c h lo ro p h y ll and ca ro ten o id s
co n ce n tra tio n s in th e e xtra cts (Cuaresma e t al., 2011). C hlo ro ph yll and ca ro te n o id
c o n te n t w e re presented per gram o f biom ass w h ich was calculated based on th e
d ry w e ig h t co n c e n tra tio n s in th e sam ples used.
41
C hapter 3
3.3.1. Controlled cultivation o f algae at high and low light intensity

Figure 1 show s a typ ica l run a t 0.21 bar o f oxygen p artial pressure o f N eochloris
oleobundans c u ltiva te d a t high lig h t co n d itio n s. In th is e x p e rim e n t th e algae w e re
c u ltiv a te d using an average in cid e n t lig h t irra dian ce o f 500 p m o l m"2 s'1. The
accom panying average p h o to n flu x d e n sity experienced by th e algae inside th e
p h o to b io re a c to r was 230 p m o l m"2 s'1, using an e s tim a te d lig h t g ra d ie n t inside th e
p h o to b io re a c to r, as was described by Sousa e t al. (2012). The PI (p h oto syn the sis-
irra dian ce) curve fo r th is alga (Sousa e t al., 2012) show s th a t N eochloris
o le oabundans experiences n e a r-sa tu ra tio n co n d itio n s a t 230 p m o l m"2 s'1. W hen
g ro w in g th e algae a t an average lo w in cid e n t lig h t in te n s ity o f 200 p m o l m"2 s'1,
th e y experience 92 p m o l m"2 s"1 inside th e p h o to b io re a c to r w hich corresponds
w ith su b -sa tu ra tin g lig h t co n d itio n s according to th e PI curve.
2,0
OD (CU)
A Cx(g.Kg"1)
1,5
■ g (day 1)
1,0
0,5
0,0
0 2 4 6 8
Time (days)
Figure 1 - Graphical representation o f the partial oxygen pressure (Po2 ), optical density
(OD), dry weight concentration (Cx), pH and specific growth rate (p) o f Neochloris
oleoabundans in time at incident light intensity o f 5 0 0 pm ol m 2 s'1.
A fte r a batch p erio d o f 3 days th e algae c u ltu re reached an OD o f 1.4 CU and th e

tu rb id o s ta t o p e ra tio n was sta rted . D uring th e fir s t day o f tu rb id o s ta t c u ltiv a tio n
42
oleoabundans
th e o p tica l d en sity decreased to a co n sta n t set level o f 0.8 CU th a t corresponds to

a biom ass co n c e n tra tio n o f 0.42 ± 0.05 gD W L"1 (Fig. 1, Table 1) w h ile th e specific
g ro w th rate (p) rem ain ed co n sta n t a t 1.36 day"1.
3.3.2. Oxygen effects o f microalgal grow th at high and low light

intensity
The c u ltiv a tio n o f N eochloris oleoabundans was co nd ucte d a t 4 d iffe re n t oxygen
c o n ce n tra tio n s a t high lig h t co n d itio n s. Table 1 show s th e results o f th e
e xp e rim e n ts p e rfo rm e d . The results o b ta in e d a t lo w in c id e n t lig h t in te n s ity (200
p m o l m"2 s"1) w e re o b ta in e d fro m Sousa e t al. (2012) and added fo r com parison.
Table 1 - Specific growth rate (p), and biomass concentration (Cx) o f the microalgae
Neochloris oleoabundans at different partial oxygen and carbon dioxide pressures a t high
(500 pm ol m'2 s'1) incident light intensity. The results obtained a t low incident light
intensity (200 pm ol m'2 s'1) were obtained from Sousa et al. (2012)
N aH C 03 Po2 Pco2 p (d a y 1) Cx (g kg'1) p (d a y 1) Cx (g kg'1)

(m M ) (bar) (bar) ± Stdev ± Stdev ± Stdev ± Stdev
500 p m o l m"2 s"1 200 p m o l m"2 s"1
10 0.21 0.007 1.36 ± 0 .2 0 0.42 ± 0 .0 5 1.38 ± 0 .1 7 0.40 ± 0 .0 0 2
10 0.42 0.007 1.16 ± 0 .2 6 0.42 ± 0 .0 5 - -

10 0.63 0.007 0.93 ± 0 .1 4 0.42 ± 0 .0 2 1.36 ± 0 .1 8 0.41 ±0.04
10 0.84 0.007 0.68 ± 0 .2 8 0.43 ± 0.03 1.06 ± 0 .0 2 0.39 ± 0 .0 0 4
30 0.84 0.020 0.68 ± 0 .1 5 0.39 ± 0 .0 5 1.36 ± 0 .0 0 2 0.39 ± 0 .0 0 4
This ta b le show s th a t th e specific g ro w th rates decrease w ith an increase o f th e

p a rtia l oxygen pressure. The highest g ro w th rate value a t 500 p m o l m"2 s'1 (1.36
day"1) was o b ta in e d fo r N eochloris oleoabundans c u ltiv a te d a t 0.21 bar p artial
oxygen pressure, w h ile a t 0.84 bar p artial oxygen pressure th e g ro w th ra te was
reduced by a lm o st 50%. Previous w o rk a t lo w in c id e n t lig h t co n d itio n s o f 200
pm o l m"2 s"1 (Sousa e t al., 2012) had show n th a t th e in h ib itin g e ffe c t o f oxygen
could be o vercom e by a d d itio n o f e xtra C 0 2 in d ic a tin g th a t oxygen m ainly
in h ib ite d th e algae via re sp ira tio n . In th is e x p e rim e n t a t n e a r-sa tu ra tin g lig h t
co n d itio n s, th e b ica rb o n a te co n c e n tra tio n (N aH C 03) was increased fro m 10 to 30
m M as w ell. The hig he r b ica rb o n a te c o n ce n tra tio n , how eve r, did n o t re su lt in an
increase o f th e specific g ro w th rate. W h ile an increase o f th e b ica rb o n a te
43
C hapter 3
c o n c e n tra tio n p roved to be an e ffic ie n t m e th o d to d im inish in h ib itio n o f oxygen

via p h o to re s p ira tio n a t lo w lig h t co n d itio n s, it did n o t have any e ffe c t on g ro w th
a t th e high lig h t c o n d itio n s used in th is e xp e rim e n t.
The increase o f b ica rb o n a te and co n se q u e n tly th e increase o f C 0 2 to th e c u ltu re

m ed iu m did n o t help to reduce th e e ffe cts o f oxygen on th e specific g ro w th rate
a t high lig h t co n d itio n s. It is possible th a t th e e ffe c t o f a d d itio n a l C 0 2 in th e
m ed iu m c u ltu re did n o t c o n trib u te enough to th e o vercom e th e in h ib ito ry e ffe c t
by p h o to re s p ira tio n . A t high lig h t inten sitie s, th e local c o n c e n tra tio n o f oxygen at
th e Rubisco site is hig he r and th e C 0 2 is lo w e r th a n in th e m e d iu m c u ltu re . The
a d d itio n o f C 0 2 in th e m ed iu m a t th ese co n d itio n s m ig h t n o t be e ffe c tiv e enough
to change th e local ra tio C 0 2/ 0 2 in th e v ic in ity o f th e Rubisco and th e re is hardly
any e ffe c t by th is a d d itio n . On to p o f th e in h ib ito ry e ffe cts o f p h o to re s p ira tio n
also p h o to in h ib itio n is expected to o ccur a t high lig h t co nd itio ns.
W hen c u ltiv a tin g o th e r m icroalgal species like Phaeodactylum tric o rn u tu m

(M o lin a e t al., 2001); C hlorella so ro kin ia n a (Ugwu e t al., 2007), S piru lin a p latensis
a t high lig h t in te n sity, a sim ila r decrease in specific g ro w th rate was observed at
e levated oxygen co n ce n tra tio n s. The described e xpe rim en ts, h ow eve r, w e re n o t
p e rfo rm e d a t c o n tro lle d co n d itio n s o f oxygen. It is th e re fo re n o t possible to
distin gu ish b etw e e n th e d ire c t e ffe cts o f lig h t and o f oxygen on th e g ro w th o f th e
algae. In th e p re sen t study, th e e x p e rim e n t was p e rfo rm e d a t c o n tro lle d p artial
oxygen pressures and irradiance. These studies claim th a t th e cells phase
p h o to in h ib itio n effects, b u t it is n o t clear, if e levated oxygen levels in th e m edium
s tim u la te p h o to -o x id a tio n and th e re b y c o n trib u te to a d d itio n a l p h o to -in h ib ito ry
e ffe cts on g ro w th .
3.3.3. Oxygen effects of pigment content at high and low light

intensity
Figure 2 presents th e c h lo ro p h yll c o n te n t o f N eochloris oleoabundans m easured
a t d iffe re n t p artial oxygen pressures a t tw o d iffe re n t lig h t co n d itio n s. The cells
show p h o to -a c c lim a tio n ; since th e average ch lo ro p h y ll c o n te n t of N.
o le oabundans g ro w n a t lo w lig h t in te n s ity (200 p m o l m"2 s"1) is a b o u t 1.9 tim e s
h ig he r th a n w h e n cu ltiv a te d a t high lig h t in te n s ity (500 p m o l m"2 s"1). P h o to
a cclim a tio n is a process in w h ich th e p h o to s y n th e tic p ig m e n t c o n te n t is reduced
44
Effect o f oxygen a t lo w a n d high lig h t in te n s ity on the g ro w th o f N eochloris
oleoabundans
as a p ro te c tio n m echanism o f th e p h o to s y n th e tic apparatus against increased

irradiance (A n em ae t e t al., 2010). Such a clear d ep en de ncy o f c h lo ro p h y ll c o n te n t
per cell in response to d iffe re n t lig h t in te n sitie s is a co m m on m echanism am ong
m icroorganism s th a t p e rfo rm p hotosynthesis. C hlo ro ph yll is a lig h t-h a rve stin g
p ig m e n t th a t, u nd e r lo w light, increases u n til th e cells becom e o p tic a lly dark; and
u nd er high light, it decreases, resu ltin g in cells ra th e r tra n s p a re n t.
A t b oth lig h t in te n sitie s no e ffe c t o f p artial oxygen pressures on c h lo ro p h y ll

c o n te n t was fo u n d . A t 200 p m ol m 2 s'1 th e a m o u n t o f c h lo ro p h y ll ranged fro m
23.8 at Pq2 = 0.63 bar to 28.2 mgChl g D W '1 at Po2 = 0.84 bar, w h ile at an in c id e n t
lig h t in te n s ity o f 500 p m ol m '2 s'1, ch lo ro p h yll c o n te n t ranged fro m 11.3 m easured
at P0_= 0.42 bar till 16.2 mgChl g D W '1 a t P0, = 0.63 bar.
200 nmol.m .
500 nmol .m' .s
Q 20
0,21 0,42 0,63 0,84 0,84*
P02 (bar)
Figure 2 - Chlorophyll content in Neochloris oleoabundans cultivated at sub-saturating

light intensity (200 pm ol m 2 s'1} and near saturating light intensity (500 pm ol m 2 s'1).
A t Po2=0.84 the bicarbonate concentration in the culture media was 10 m M and at
Po2=0.84* the bicarbonate concentration was increased to 30 mM.
Figure 2 shows th a t no sig n ifica n t effects o f p artial oxygen pressure and partial
carbon d io xide pressure on th e ch lo ro p h y ll c o n te n t was fo u n d a t b oth lig h t
inten sitie s; no loss o r dam age o f ch lo ro p h y ll seem to occur due to oxygen
a ccum ula tion o r increase o f carbon dioxide. In general, th e same increasing tre n d
45
C hapter 3
o f c h lo ro p h yll c o n te n t upon a decrease o f Irradiance is re p o rte d fo r Thallasiosira

pseudonana (Valenzuela-Espinoza e t al., 2007) and S pirulina p la te nsis (A n em ae t e t
al., 2010). C hlo ro ph yll co n ce n tra tio n s in Thallasiosira pseudonana w e re 1.74 m g/L
a t low lig h t in te n s ity (50 p m o l m"2 s"1) and 0.47 m g/L a t high lig h t irra dian ce (750
pm o l m"2 s"1) (Valenzuela-Espinoza e t al., 2007). D uring th e g ro w th o f S pirulina
platensis, th e highest ch lo ro p h yll c o n te n t (14.6 mg g"1) was d e te c te d a t in c id e n t
lig h t in te n s ity o f ~40 p m o l m"2 s"1 and sm allest c h lo ro p h y ll c o n te n ts (6.2 mg g"1)
w e re fo u n d w h e n c u ltiv a tin g S piru lin a u nd er lig h t in te n s ity o f 100 p m o l m"2 s"1 in

th is stu d y (A nem aet e t al., 2010).
The a ccu m u la tio n o f oxygen in th e p h o to b io re a c to r was expected to induce extra

fo rm a tio n o f oxygen radicals and singlet oxygen. Singlet oxygen dam ages p ro te in
in ch lo ro p la sts and ina ctivates e le ctro n tra n s p o rt m echanism (Ledford & Niyogi,
2005; M u ra ta e t al., 2007). The life tim e o f singlet oxygen is ra th e r sh ort. Singlet
oxygen th u s reacts w ith th e m olecules in its v ic in ity such as pigm ents w h ich are
invo lved in th e e le ctro n tra n s p o rt m echanism and p ro te in s pre sen t in th e
ch lo ro p la st. Figure 2 shows, h o w e ve r th a t th e c h lo ro p h y ll was n o t dam aged by
th e oxygen p re sen t in th e m ed iu m , in d ica tin g th a t no a d d itio n a l sing let oxygen
was fo rm e d a t hig he r oxygen co n ce n tra tio n s a t th e lig h t c o n d itio n s used.
C arotenoids are know n to p ro te c t th e cells against p h o to -in h ib itio n a t high lig h t

in te n s ity and increased in h ib itio n by p h o to -o x id a tio n is accom panied by increased
p ig m e n t c o n te n t (Falkow ski & Raven, 2007). C arotenoids play an im p o rta n t role in
p ho tosyn th esis; located in th e c h lo ro p la st th e y c o n trib u te to lig h t harvesting,
dissip atio n o f lig h t energy, scavenging o f tr ip le t c h lo ro p h y ll and singlet oxygen,
and m a in ta in in g th e p ho tosystem s tru ctu re . They are claim ed to be a m a jo r
a n tio x id a n t defense (D em m ig-A dam s & Adam s, 2002). Figure 3 show s th a t th e
ca ro te n o id c o n te n t o f N eochloris oleoabundans g ro w n a t lo w lig h t in te n s ity (200
p m o l m"2 s"1) is indeed lo w e r th a n th e c a ro te n o id c o n te n t o f th e cells c u ltiv a te d at
high lig h t in te n s ity (500 p m o l m"2 s"1).
46
E ffect o f oxygen a t lo w a n d high lig h t in te n s ity on the g ro w th o f N eochloris
oleoabundans
200 |amol.m"2 s " 1
500 )amol.m"2 .s "1
^ 3
Q
U)
d)
E
0,21 0,42 0,63 0,84 0,84*
P0 2 (bar)
Figure 3 - Carotenoids content in Neochloris oleoabundans cultivated at sub-saturating

light intensity ( 200 ¡umol m'2 s'1) and near saturating light intensity ( 500 iim oI m'2 s'1).
A t Po2=0.84 the bicarbonate concentration in the culture media was lO m M and at
P02=0.84* the bicarbonate concentration was increased to 30mM.
A t elevated oxygen c o n ce n tra tio n s in th e p h o to b io re a c to r, extra fo rm a tio n o f

oxygen radicals and sing let oxygen was expected and th ose w o u ld induce extra
p ig m e n t p ro d u c tio n to p ro te c t th e cells by q uenching th e e le ctron s and act as
a n ti-o x id a n t against th e ROS fo rm e d . The ca ro te n o id c o n te n t in cells o f N eochloris
o leoabundans c u ltiva te d at elevated p artial oxygen pressures, h ow eve r, is hardly
a ffe cted . A t an in cid e n t lig h t in te n s ity o f 200 pm o l m '2 s'1a m axim um o f 2.7 mgCar
g D W 1 was m easured at a p artial oxygen pressure o f 0.84 bar and a m in im u m o f
2.3 mgCar g D W '1 at P02 = 0.63 bar. A t high lig h t in te n s ity (500 p m o l m '2 s'1) th e
highest value is 4.9 mgCar g D W '1and th e lo w e st 3.1 mgCar g D W '1.
Vonshak e t al. (1996) refers to p h o to in h ib itio n as a tim e and lig h t d e p e n d e n t

decline in p ho tosyn th esis th a t happens in a firs t stage o f th e exposure o f th e algae
to high lig h t and oxygen and to p h o to -o x id a tio n in a secondary stage leading to
dam age a n d /o r death o f th e cells. O ur results ind icate th a t at th is lig h t in te n s ity
th e cells w e re indeed exposed to p h o to in h ib itio n b u t no re la tio n b etw e en
c a ro te n o id c o n te n t and oxygen co n ce n tra tio n (figu re 3) was fo u n d , ind ica tin g th a t
47
C hapter 3
e levated oxygen co n ce n tra tio n s in th e m ed iu m do n o t cause a d d itio n a l p h o to -

o xid a tive dam age a t su b-satu ratin g and n e a r-sa tu ra tin g lig h t co nd itio ns.
3.4. Conclusions
High oxygen co n ce n tra tio n s n eg ative ly a ffe cte d th e g ro w th ra te o f N eochloris
o le oabundans a t high lig h t co n d itio n s. A t such co n d itio n s, p h o to re s p ira tio n
co m b in e d w ith p h o to in h ib itio n o f th e c u ltu re is bound to occur, resu ltin g in th e
d e te rio ra tio n of m icroalgae cell v ia b ility and th e decrease in N eochloris
o le oabundans g ro w th rate. A 3 tim e s increase in b ica rb o n a te a d d itio n did n o t
sh ow any p ositive e ffe c t o f th e overall g ro w th o f th e algae a t n e a r-sa tu ra tin g ligh t
c o n tra ry to w h a t happens a t su b -sa tu ra tin g lig h t inten sitie s. This indicates th a t
a d d itio n o f extra ca rb on ate to th e m ed iu m to o vercom e th e p h o to re s p ira tio n
effects, is in s u ffic ie n t to co m pensate fo r th e loss o f biom ass due to th e co m b in e d
p h o to re s p ira tio n and p h o to in h ib itio n a t n e a r-sa tu ra tin g lig h t co n d itio n s. The
e levated oxygen co n c e n tra tio n in th e g ro w th m ed iu m did n o t a ffe c t ch lo ro p h y ll
and ca ro te n o id c o n te n t a t sub- and n e a r-sa tu ra tin g lig h t co n d itio n s, ind icatin g
th a t th e e levated oxygen co n c e n tra tio n in th e m ed iu m did n o t c o n trib u te to th e
p h o to in h ib itio n e ffe cts experienced a t th e hig he r lig h t inten sitie s. These results
in d ica te th a t th e p h o to in h ib itio n e ffe cts are o nly due to th e increased irra dian ce
used and n o t due to a ccum ula tion o f th e oxygen in th e m ed iu m as such. The
p h o to in h ib itio n e ffe cts can th u s o nly be p re ve n te d by w o rk in g a t su b-satu ratin g
lig h t co n d itio n s ra th e r th a n a t n ea r-sa tu ra tin g lig h t c o n d itio n s. For large-scale
o u td o o r c u ltiv a tio n o f m icro-algae o u r results ind icate th a t re a c to r co n fig u ra tio n s
th a t a llo w spatial d ilu tio n o f lig h t should be used, in c o m b in a tio n w ith a d d itio n o f
ca rb on ate. In th ese typ es o f p h o to b io re a c to rs th e algae g ro w a t su b-satu ratin g
lig h t co n d itio n s and w ith th e a d d itio n o f carbon d io xide th e p h o to re s p ira tio n
e ffe cts w ill be m in im ize d. This reduces th e need fo r degassing to rem ove th e
surplus o f oxygen fro m th e m ed iu m . In th is w ay, th e to ta l energy and costs
re q u ire d fo r degassing can be decreased.
48
oleoabundans

EZ/Kompas p ro gram o f th e "S am en w erking sverba n d N o o rd -N e d e rla n d ". The
discussions and th e ir fin a n cia l su pp ort.
3.6. References
A n em a et, I.D., Bekker, M ., H elling w e rf, K.J. 2010. Algal p h o tosyn th esis as th e
p rim a ry d riv e r fo r a sustainable d e ve lo p m e n t in energy, fe ed , and fo o d
p ro d u c tio n . M a rin e B iotechnology, DOI 1 0 .1 0 0 7 /s l0 1 2 6 -0 1 0 -9 3 1 1 -l.
Bader, M.R., von C aem m erer, S., Ruuska, S., Nakano, H. 2000. Electron flo w to
oxygen in h ig he r plants and algae: rates and c o n tro l o f d ire c t p h o to re d u c tio n
(M e h le r rea ction ) and rubisco oxygenase. Philosophical Transactions o f th e Royal
Society o f London Series B-Biological Sciences, 355(1402), 1433-1445.
Chisti, Y. 2007. Biodiesel fro m m icroalgae. B io tech n olog y Advances, 25, 294-306.
Cuaresma, M ., Janssen, M ., Vilchez, C., W ijffe ls , R.H. 2011. H orizontal o r vertica l

p h o to b io re a cto rs? H ow to im p ro ve m icroalgae p h o to s y n th e tic e fficiency.
D em m ig-A dam s, B., Adam s, W .W . 2002. A n tio x id a n ts in P hotosynthesis and

H um an N u tritio n . Science, 298 (5601 ), 2149- 2153
Falkowski, P.G., Raven, J.A. 2007. A q ua tic p ho tosyn th esis. P rinceton U nive rsity
Press, P rinceton, NJ.
G ouveia, L., M arques, A .E., da Silva, T.L., Reis, A. 2009. N eochloris oleabundans
UTEX #1185: a su ita b le ren e w a b le lip id source fo r b io fu e l p ro d u c tio n . Journal o f
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fo r M icro b io lo g y, 8, 229-239.
Hakala, M., Tuo m in en , I., Keranen, M., T yystjarvi, T., T yystjarvi, E. 2005. Evidence
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P hotosystem II. Biochim ica Et Biophysica A cta-B ioenergetics, 1706(1-2), 68-80.
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p ho tosystem II and related p ro te c tio n m echanism . Photosynthesis Research,
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m an ag em en t in plants and algae. Plant Cell and E n viro nm e nt, 28(8), 1037-1045.
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on cell g ro w th and lipid a ccu m u la tio n o f green alga N eochloris oleoabundans.
A pplie d M ic ro b io lo g y and B iotechnology, 81(4), 629-636.
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Norsker, N.H., Barbosa, M.J., V erm uë, M .H ., W ijffe ls , R.H. 2011. M icroalgal
p ro d u c tio n - A close loo k a t th e econom ics. B io tech n olog y Advances, 29(1), 24-27.
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Santos, A .M ., Janssen, M., Lamers, P.P., Evers, W .A.C., W ijffe ls , R.H. 2012. G ro w th
o f oil a ccum ula ting m icroalga N eochloris o le oabundans u n d e r alkaline-saline
co n d itio n s. B ioresource Technology, 104, 593-599.
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C., Kruse, O., H ankam er, B. 2008. Second G e ne ra tio n Biofuels: H igh-E fficiency
M icroalgae fo r Biodiesel P roduction. B ioenergy Research, 1, 20-43
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o f th e m icroalgae N eochloris oleoabundans a t high p a rtia l oxygen pressures and
su b-satu ratin g lig h t in te n sity. B ioresource Technology, 104, 565-570.
Tornabene, T.G., Holzer, G., Lien, S., Burris, N. 1983. Lipid c o m p o s itio n o f th e
n itro g e n starved green alga N eochloris oleoabundans. Enzyme and M icro b ia l
Technology, 5, 435-440.
T orzillo, G., B ernardini, P., M asojidek, J. 1998. O n-line m o n ito rin g o f c h lo ro p h yll
high oxygen co n c e n tra tio n and lo w te m p e ra tu re and its e ffe c t on th e p ro d u c tiv ity
o f o u td o o r cu ltu re s o f S pirulina p la te nsis (cyanobacteria). Journal o f Phycology, 34
5 0 4 -5 1 0
148(2), 960-968.
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N unez-C ebrero, F. 2007. G ro w th and accessory p ig m e n t to c h lo ro p h y ll a ratio s o f
Thalassiosira pseudonana (B acillariophyceae) c u ltu re d u n d e r d iffe re n t irradiances.
H idrob iolog ica, 17(3), 249-255.
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Vonshak, A., T orzillo, G., Accolla, P., Tom aselli, L. 1996. Light and oxygen stress in
S piru lin a p la te nsis (cyanobacteria) g ro w n o u td o o rs in tu b u la r reactors. Physiologia
P lantarum , 97(1), 175-179.

295.
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oleoabundans
53
C hapter 4
54
E ffect o f d yna m ic oxygen co n ce n tra tio n on th e g ro w th o f N eochloris oleoabundans
a t s u b -s a tu ra tin g lig h t co nd itio ns
Chapter 4 . E ffe c t o f D y n a m ic O xyg en C o n c e n tr a tio n s o n th e

GROWTH OF NEOCHOLORIS OLEOABUNDANS AT SUB-SATURATING
LIGHT CONDITIONS
Claudia Sousa1' 2, D im ita r V a lev1,2, M arian H. V e rm u ë 2and Rene H. W ijffe ls 2
the Netherlands
Abstract - T u b u la r p h o to b io re a c to rs fo r m icro-algae p ro d u c tio n are considered

econ om ically-fe a sib le, but o n ly if th e energy needed to rem ove th e
p h o to s y n th e tic a lly produced oxygen can be reduced considerably. In th is study,
th e e ffe cts o f th e increase o f th e oxygen co n c e n tra tio n fo llo w e d by a decrease o f
th e oxygen in th e degasser w e re sim u la te d in a CSTR a t fu lly c o n tro lle d co n d itio n s
a t su b-satu ratin g lig h t in te n s ity and th e e ffe c t o f a 10 tim e s e lo n g a tio n o f th e
residence tim e a t in th e solar rece ive r was inve stiga te d. T h e re fo re 3 d iffe re n t ligh t
regim es w e re used: co n tin u o u s lig h t; 30 m in utes lig h t on fo llo w e d by 6 m in utes
lig h t o ff and 300 m in u te s lig h t on fo llo w e d by 6 m in u te s lig h t o ff. The specific
g ro w th ra te m easured a t co n sta n t lo w oxygen c o n c e n tra tio n Pq2 = 0.21 bar d u rin g
th ese th re e lig h t regim es w e re 1.14 ± 0.06, 0.80 ± 0.16 and 1.09 ± 0.05 day"1
respectively. The e ffe c t o f d yna m ica lly changing oxygen co n c e n tra tio n s fro m Po2 =
0.21 bar to Po2 = 0.63 bar fo llo w e d by su bse qu en t degassing to Po2 = 0.21 bar
d u rin g th e d ark p e rio d resu lted in sim ila r specific g ro w th rates. The decrease o f
th e algae specific g ro w th observed w h e n a pplying d iffe re n t lig h t regim es, shows
th a t th e exposure o f th e algae cells to d ark periods in th e degasser has bigger
n egative im p a ct th a n th e te m p o ra ry exposure to a ccum ula ting oxygen
c o n ce n tra tio n s in th e solar receiver. Based on th e observed algae physiology
u n d e r dyna m ic oxygen co n c e n tra tio n , reducing th e n u m b e r o f degassing u nits and
increasing th e ir degassing capacity w ill re su lt in su bsta ntia l savings in capital and
energy costs.
Key words: tu b u la r p h o to b io re a c to rs , dyna m ic oxygen, lig h t regim e, N eochloris

oleoabundans
Sousa, C., Valev, D., Vermuë, M.H., Wijffels, R.H. 2013. Effect o f dynamic oxygen
concentration on the growth o f Neochloris oleabundans at sub-saturating light conditions.
Bioresource Technology, Accepted fo r publication. ¡-¡-
C hapter 4
4.1. Introduction
Lipid-rich m icroalgae such as N eochloris oleoabundans are considered to be a
ren ew a ble resource fo r b io fu e l p ro d u c tio n (W ijffe ls e t al., 2010). A lth o u g h these
m icroalgae show high g ro w th rates and high lip id c o n te n t, large-scale o u td o o r
p ro d u c tio n o f m icroalgae is still n o t e con om ically feasible. The fe a s ib ility s tu d y o f
N orsker e t al. (2011) show s th a t a tu b u la r p h o to b io re a c to r (PBR) can be used as
an e con om ically-fe a sib le system fo r p ro d u c tio n o f algae, b u t o n ly if th e energy
c o n su m p tio n is conside rab ly reduced. The c u rre n t m ain b o ttle n e c k in th is
p ro d u c tio n system is th e energy needed fo r c irc u la tio n o f th e liqu id th ro u g h th e
tu b e s o f th e p h o to b io re a c to r and fo r exchange o f gases in th e degasser (N orsker
e t al., 2011). This c ircu la tio n and gas exchange is needed to supply th e algae w ith
ligh t, C 0 2 and to rem ove th e oxygen produced.
The oxygen needs to be rem ove d as it causes p h o to re s p ira tio n . In

p h o to re s p ira tio n 0 2 com petes w ith C 0 2 fo r th e key enzym e in th e Calvin cycle,
RuBiSco, w h ich is responsible fo r in c o rp o ra tin g ino rg a nic carbon in organic
m olecules, resu ltin g in less g ro w th o f th e algae and lo w e r biom ass yield (Foyer e t
al., 2009; M iro n e t al., 1999). A n o th e r process w hich lim its m icroalgae g ro w th is
p h o to in h ib itio n . This process, how eve r, m ainly occurs at near- and o v e r
s a tu ra tin g lig h t in te n sitie s and is h ardly re le va n t a t su b -sa tu ra tin g lig h t in te n sitie s
(Kliphuis e t al., 2011; Raso e t al., 2011; Sousa e t al., 2012).
Previous w o rk on th e e ffe c t o f oxygen a t su b-satu ratin g lig h t on m icroalgae

g ro w th show ed th e expected decrease in specific g ro w th rate w ith increasing
oxygen co n c e n tra tio n (Kliphuis e t al., 2011; Raso e t al., 2011; Sousa e t al., 2012).
Ali studies m e n tio n e d w e re p e rfo rm e d a t c o n s ta n t oxygen co n c e n tra tio n s . In
practice, h ow eve r, th e algae c u ltu re d in closed tu b u la r p h o to b io re a c to r systems
experience an increasing oxygen co n ce n tra tio n g ra d ie n t along th e length o f th e
tubes, and th is m ay induce a decreasing g ro w th rate along th e tu b e s (Ugwu e t al.,
2008). In a d d itio n , a n o n -illu m in a te d degassing u n it is placed a t th e end o f th e
tu b e to rem ove th e accum ula ted oxygen. In th is stu dy th e e ffe c t o f dynam ic
changing oxygen co n ce n tra tio n s on th e algal g ro w th in p h o to b io re a c to rs w ill be
e valuated, also ta kin g th e e ffe c t o f th e residence tim e o f th e algae in th e dark
degasser in to account. In a d d itio n , th e e ffe c t o f exten sion o f th e exposure tim e to
high oxygen co n ce n tra tio n s w ill be inve stiga te d. A possible extension o f th is
56
residence tim e w o u ld m ean th a t less degassing u nits are needed and th is w o u ld

re su lt in a lo w e r o verall p o w e r co n su m p tio n fo r degassing co m b in e d w ith higher
o verall p ro d u c tiv ity in th e system , as th e algae spend re la tiv e ly little tim e in th e
dark.
To m im ic th e dynam ic changes in oxygen c o n c e n tra tio n o ccurrin g in tu b u la r

system s, a fu lly c o n tro lle d C ontinuous S tirred Tank Reactor (CSTR) o p e ra te d in
tu rb id o s ta t m ode was used to c u ltu re N eochloris o le oabundans a t su b-satu ratin g
lig h t in te n sity. D uring th e tu rb id o s ta t run, oxygen was a llo w e d to build up fro m
Po2 = 0.21 to 0.63 bar and th e n was ra p id ly decreased d urin g a s h o rt tim e o f
darkness to th e s ta rtin g level w h ile th e specific g ro w th ra te was m o n ito re d . The

e xp e rim e n ts w e re rep ea te d w h ile e xte n d in g th e tim e a t w h ich th e algae w ere
exposed to high p a rtia l oxygen pressures in o rd e r to d e te rm in e th e e ffe c t on th e
specific g ro w th ra te o f th e algae.
4.2. Materials and methods
4.2.1. Cultures and medium

N eochloris oleoabundans (UTEX 1185) was c u ltu re d and m a in ta in e d in 250 ml
E rlenm eyer flasks in 100m l o f a dapted f/2 m ed iu m (G uillard & R yther, 1962). The
flasks w e re closed w ith porous stoppers (Bio-silico, H irschm ann Laborgeräte
GmbH & Co.KG, G erm any) and placed in an in c u b a to r w ith an o rb ita l shaker
(Innova 44R, New B runsw ick S cientific, USA) u n d e r flu o re s c e n t lig h t (40 p m o l m"2
s"1) a t 25 °C and 120 rpm . The air inside th e in c u b a to r was enriched w ith 2%
carbon d ioxide. The m ed iu m used fo r th e re a c to r runs was enrich e d w ith 10 m M
N aH C 03. Both m edia w e re filte r-s te riliz e d using 0.22 pm filte rs and k e p t a t pH o f
7.8.
A 3L ja cke te d b io re a c to r (A pplikon B iotechnology, The N etherlands), equ ip pe d
w ith m arine im p e lle r was o p e ra te d in tu rb id o s ta t m ode. The illu m in a te d surface
o f th e re a c to r was 0.061 m 2. An E z-controller o p e ra tin g w ith Bioexpert® s o ftw a re
(A p plikon B iotechnology, The N etherlands) was used fo r m o n ito rin g and c o n tro l.
The o n -lin e m easured process p a ram eters w e re pH, te m p e ra tu re , p a rtia l oxygen
57
C hapter 4
pressure in th e liq u id phase (Po2), liq u id level, s tirre r speed and o p tica l d en sity
(OD). D uring th e run th e pH was c o n tro lle d a t pH 7.8 by a u to m a tic a d d itio n o f

gaseous carbon d io xide (C 0 2), th e te m p e ra tu re was m a in ta in e d a t 25 °C and th e
o p tica l d e n sity (OD) was c o n tro lle d by a tu rb id ity sensor (ASD19-N, O ptek,
G erm any) co nnected to a p e ris ta ltic m ed iu m p um p to keep a co n sta n t o ptica l
density. The liq u id level in th e re a c to r was c o n tro lle d a t ~ 2L by a level sensor
co nn ecte d to a p e ris ta ltic p um p fo r rem ovin g th e excess o f c u ltu re , w h ich was
a ctivate d w hen necessary. D uring th e e xp e rim e n ta l run, dyna m ic oxygen
c o n ce n tra tio n s w e re app lie d: th e oxygen was a llo w e d to b uild up to Pq2 = 0.63 bar
and th e n degassed to Po2 = 0.21 bar. The p a rtia l oxygen pressure was decreased
to th e desired levels by th e a u to m a tic a d d itio n o f gaseous d in itro g e n (N 2). The
p a rtia l oxygen pressure was m o n ito re d by a Clark ty p e Pq2 sensor (L o w D rift
sensor, Applisens, The N etherlands). C alibra tion o f th e sensor was p e rfo rm e d

inside th e re a c to r w ith filte re d a da pte d f /2 m ed iu m , b e fo re in o c u la tio n , using
p ure 0 2 giving a p a rtia l oxygen pressure (Po2) o f 1 bar. The cells w e re a llo w e d to
a da pt to th e tu rb id o s ta t co n d itio n s, b e fo re th e specific g ro w th rate (p) was
d e te rm in e d fro m th e d ilu tio n rate.
4.2.3. Light regim e

Light was p ro vid e d by tw o LED lig h t panels (20x20 cm ) (SL3500, Photon Systems
In strum en ts, Czech Republic). The lig h t sources w e re p o sitio n e d a t b o th sides o f
th e re a cto r and a p late o f opal glass was placed in fr o n t o f each o f th e lig h t panels,
to ensure hom ogeneous lig h t d is trib u tio n . In a d d itio n , re fle c tiv e m a te ria l was
placed a ro u n d th e rea ctor. A PAR q u a n tu m sensor (m odel SA-190, LiCor
Biosciences, USA) was used to m easure th e average in c id e n t p h o to n flu x den sity
(PFDavg) on th e re a c to r surface. The average value in c id e n t p h o to n flu x d en sity
was 198 p m ol m"2 s"1. The PI (P hoto synthe sis-irra dia nce ) curve fo r th is algae show s
th a t N eochloris oleoabudans indeed experiences su b -sa tu ra tin g lig h t co n d itio n s at
th e m easured PFDavg (Sousa e t al., 2012).
The lig h t sources w e re co nnected to a Siemens PLC Relay (LOGO!) lig h t c o n tro lle r
to sim u late th e dark p erio d in th e degasser o f a tu b u la r PBR system . T w o d iffe re n t
tim e regim es fo r th e lig h t w e re used, related w ith th e oxygen co n d itio n s applied
d u rin g th e e xpe rim en ts. The firs t one was 30 m in u te s lig h t "O n " and 6 m in u te s
58
lig h t "O ff" (3 0 /6 regim e). This regim e was a pplied d u rin g dyna m ic oxygen
co n d itio n s, w he n th e algae w e re a llo w e d to p ro du ce and a ccum ula te oxygen fro m
Po2 = 0.21 bar up to Pq2 = 0.63 bar. A fte r th is phase a degassing phase was
in itia te d . D uring th e degassing phase th e lig h t c o n tro lle r sw itches to Lights "O ff"
and rapid degassing o f th e liq u id v o lu m e is in itia te d to b ring th e oxygen
c o n ce n tra tio n s back to s ta rtin g levels o f Pq2 = 0.21 bar in 6 m in utes. The second
tim e regim e was o p e ra te d a t 300 m in utes lig h t "O n " and 6 m in utes lig h t "O ff"
(3 0 0 /6 regim e). This regim e was a pplied d u rin g c u ltiv a tio n o f th e algae a t high
oxygen levels (P q2 = 0.63 bar) fo r 300 m in utes. The degassing phase was
p e rfo rm e d a t six m in u te s lights "O ff".
4.2.4. Dry w eig ht concentration

T rip lica te sam ples o f 5 ml w e re co lle cted on a d aily base fo r d ry w e ig h t
d e te rm in a tio n . The sam ples w e re d ilu te d w ith 10 m l a m m o n iu m fo rm a te (0.5M )
and filte re d o ve r p re -w e ig he d glass fib re -filte rs (W h atm a n GF/F). An a d d itio n a l 40
m l o f a m m o n iu m fo rm a te (0.5M ) was used fo r w ashing. Filters w ith biom ass w e re
d rie d a t 95 ° C fo r 24 hours in a lu m in iu m trays, cooled in desiccator fo r 2 hours and
w e ig h te d on a 5 d ig it a nalytical balance (ME235P-SD, Sartorius, G erm any).
4.2.5. Chlorophyll and Carotenoid determ ination

The d yn a m ica lly changing oxygen co n ce n tra tio n s b u t especially th e d iffe re n t lig h t
darkness regim es a pplied could cause a d d itio n a l p h o to -a c c lim a tio n and p h o to
in h ib itio n effects. C hlo ro ph yll and ca ro te n o id s are b o th pig m e n ts fo r ligh t
h arvesting and th e la tte r serve as p h o to p ro te c tiv e pig m e n ts to p ro te c t th e
p h o to s y n th e tic m ach in e ry fro m excess o f lig h t by scavenging rea ctive oxygen
species and sing let oxygen (D em m ig-A dam s & Adam s, 2002; Falkowski & Raven,
2007; T ria n ta p h ylid è s & Havaux, 2009; Vilchez e t al., 2011). A lth o u g h th e e ffe c t o f
lig h t on th e c a ro te n o id c o n te n t is described in d e ta il (B ritto n e t al., 1999; Lamers
e t al., 2008) it is n o t kn ow n if hig he r oxygen co n c e n tra tio n s in th e m ed iu m a ffe c t
th e c h lo ro p h yll and c a ro te n o id c o n te n t as w e ll. To v e rify if th ese p h o to -p ro te c tiv e
m echanism s w e re a ctivate d w he n th e cells are subjected to dyna m ic oxygen
co n ce n tra tio n s, th e ch lo ro p h yll and to ta l ca ro te n o id s w e re d e te rm in e d . T rip lic a te
sam ples w e re co lle cted on a d aily base and c e n trifu g e d a t 3750 rpm fo r 10 m in. at
59
C hapter 4
4 °C and su bse qu en tly th e pellets froze n a t -80 °C. The e x tra c tio n was done by
a d d itio n o f 5 mL o f 100% m e th a n o l to each tu b e . The tu b e s w e re th e n placed fo r
5 m in. in u ltra so u n d bath (Sonorex Digitec, Bandelin). A fte rw a rd s th e samples
w e re incu ba te d fo r 40 m in a t 60 °C and th e n fo r 15 m in a t 0 °C. The suspension
was ce n trifu g e d once m o re a t th e same co n d itio n s (3750 rpm , 10 m in. and 4 °C).
The su p e rn a ta n t co lle cted and c h lo ro p h yll and ca ro te n o id d e te rm in e d a t 470 nm,
652 nm and 665 nm in a U V-visible s p e c tro p h o to m e te r (UV_1650 PC, Schimadzu).
The e qu atio ns used to d e te rm in e th e ch lo ro p h y ll and c a ro te n o id c o n te n t w e re
m o d ifie d A rn o n 's e qu atio ns (Lichten th aler, 1987). C hlo ro ph yll and ca ro te n o id
c o n te n t w e re expressed per gram o f biomass, calculated based on th e d ry w e ig h t
c o n c e n tra tio n o f th e sam ples used (Cuaresma Franco, 2011).
4.3.1. Effect o f th e applied light-regim e and the dynam ically

changing 0 2 on algal grow th
The specific g ro w th rate o f N eochloris oleoabundans u n d e r dyna m ic oxygen
c o n c e n tra tio n and su b-satu rate d lig h t co n d itio n s was d e te rm in e d . The
e xp e rim e n ta l run can be divid ed in six d iffe re n t phases. In itially, th e
p h o to b io re a c to r was ino cula ted w ith N eochloris oleoabundans and th e cells w e re
g ro w n batch w ise (Figure 1 - Phase 1) u n til th e o p tica l d en sity was above 0.8 CU,
reaching a biom ass co n ce n tra tio n o f 1.05 ± 0.02 g L"1. In Phase 2 th e algal c u ltu re
was d ilu te d to th e desired o ptica l d e n sity w ith th e a d d itio n o f fresh c u ltu rin g
m ed iu m and a llo w e d to a d ju st to co n tin u o u s su b-satu ratin g lig h t co n d itio n s and
c o n sta n t p a rtia l oxygen pressure o f 0.21 bar fo r 2 days. A fte r th is in itia l
a cclim atio n phase, th e o ptica l d e n sity was k e p t c o n s ta n t co rresp on din g to a
biom ass co n ce n tra tio n o f 0.55 ± 0.02 g L"1 and show ed a specific g ro w th ra te o f
1.14 ± 0 .0 6 d a y 1 (Table 1).
60
2.0
-------p o ; (bar)
------- OD (CU)
■ n (c )ay’ 1)
1.5 - A c x (gDW.L’ 1)
a P2
P1 P3 P4 P5 P6
X
O . . . .
S 10
CL ■
a
o
0.5
j- ^ tfMMÉTfár
0.0
0 5 10 15 20
Time ( days)
Figure 1 - Graphical representation o f partial oxygen pressure (P02) in bar;

optical density (OD) in CU; specific growth rate (p) in d a y1;
biomass concentration (Cx) in g L 1 versus time in days. P#= Phase
D uring th is e x p e rim e n t a typ ica l circadian (daily) rh y th m in 0 2 p ro d u c tio n can be

observed (Figure 1). Circadian rh yth m s are endogenous biological program s th a t
tim e d iffe re n t physiological processes to occur a t o p tim a l phases d u rin g th e daily
cycle, such as cell division, gene expression, etc. (Suzuki & Johnson, 2001). This
circadian rh y th m influences th e d e te rm in a tio n o f th e specific g ro w th rate and cell
c o n c e n tra tio n , and th e re fo re sam ples w e re ta ke n a t d aily intervals.
In Phase 3 th e oxygen co n c e n tra tio n was c o n tro lle d a t Po2 = 0.21 bar, b u t n ow th e
lig h t was tu rn e d on fo r 30 m in u te s and tu rn e d o ff d u rin g 6 m in utes (lig h t regim e
3 0 /6 ) to m im ic th e darkness d urin g degassing. The biom ass c o n c e n tra tio n was
c o n sta n t a t 0.55 ± 0.01 g L"1 and a fte r 4 days th e specific g ro w th ra te was
d e te rm in e d to be 0.8 ± 0.16 day"1, show ing th a t th e algal g ro w th rate decreased
61
C hapter 4
due to th e lig h t regim e a pplied in th e e xpe rim en ts. This m easured specific g ro w th
ra te was used as re fe ren ce value fo r th e specific g ro w th ra te m easured d u rin g th e
phase in w h ich th e e ffe c t o f d yna m ica lly changing oxygen c o n c e n tra tio n is a pplied
a t th e same lig h t regim e o f 3 0/6 .
In Phase 4 th e oxygen was co n tro lle d a t a c o n s ta n t p a rtia l pressure o f 0.21 bar

and a lig h t regim e co rre sp o n d in g to 300 m in u te s lights on and 6 m in u te s lights o ff
(3 0 0 /6 ) was applied. The biom ass co n ce n tra tio n was again 0.55 ± 0.01 g L"1 and
th e specific g ro w th rate o b ta in e d a fte r 3 days was 1.09 ± 0.05 day"1 and th u s o nly
4% lo w e r th a n th e g ro w th rate a t co n tin u o u s lig h t and a b o u t 1.4 tim e s higher
th a n a t a lig h t regim e o f 3 0 /6 . This specific g ro w th rate served as re fe ren ce fo r th e
fin a l phase o f th e e x p e rim e n t w h e n th e algae g ro w th s w e re inve stiga te d w he n
exposed to high oxygen levels fo r 300 m inutes.
Table 1 - Average biomass concentration, specific growth rate and biomass yield on
photons o f Neochloris oleoabundans during each phase o f the experimental run
Cx ± Stdev p ± Stdev Yx,ph ± Stdev

(g L 1) (d a y 1) (g m o l-p h '1)
P I - Batch 1.05 ± 0 .0 2 N /A N /A
P2 - C ontinuous lig h t -
0.55 ± 0 .0 2 1.14 ± 0 .0 6 1.15 ± 0 .0 4
Po2=0.21 bar
P3 - Light regim e 3 0 /6 -
0.55 ± 0 .0 1 0.80 ± 0 .1 6 0.97 ± 0 .0 3
Po2=0.21 bar
P4 - Light regim e 3 0 0 /6 -
0.55 ± 0 .0 1 1.09 ± 0 .0 5 1.12±0.02
Po2=0.21 bar
P5 - Light regim e 3 0 /6 -
0.49 ± 0 .0 1 0.82 ± 0 .0 4 0.88 ± 0 .0 2
D ynam ic PO2= 0 .2 1/0 .6 3 bar
P6 - Light regim e 3 0 0 /6 -
0.51 ± 0 .0 3 1.07 ± 0 .1 2 1.01 ± 0 .0 7
D ynam ic PO2= 0 .2 1/0 .6 3 bar
D uring Phase 5 dyna m ic oxygen c o n d itio n s w e re a pplied and th e re a c to r was

o p e ra te d a t a lig h t regim e o f 30 m in u te s lights "O n " and 6 m in u te s lights "O ff".
The oxygen was a llo w e d to build up fro m Po2 = 0.21 bar to Po2 = 0.63 bar fo r 30
m in u te s and th e n was fo rce d to decrease to s ta rtin g level d u rin g 6 m in u te s o f
degassing. A t th o se c o n d itio n s a specific g ro w th rate o f 0.82 ± 0.04 d a y '1 (Table 1)
was m easured w h ich does n o t d iffe r fro m th e specific g ro w th rate o f 0.80 ± 0.16
62
day"1 m easured in th e refe ren ce e xp e rim e n t (Phase 3).This show s th a t th e

dyna m ic change in oxygen co n ce n tra tio n s as such does n o t c o n trib u te to th e
decrease o f th e g ro w th ra te a t th e su b-satu ratin g lig h t co n d itio n s used.
The fin a l phase (Phase 6) o f th is e xp e rim e n t was p e rfo rm e d using an exposure

tim e a t high oxygen co n c e n tra tio n o f Po2 = 0.63 bar fo r 300 m in, fo llo w e d by 6
m in u te s fo r degassing and th e lig h t supply c o n tro lle d using th e 3 0 0 /6 lig h t regim e.
The specific g ro w th o f th e algae m easured was 1.07 ± 0.12 d a y '1. W ith o n ly a
m in o r decrease in g ro w th rate co m p ared w ith th e re fe ren ce e x p e rim e n t (Phase
4), th is re su lt co n firm s once again th a t th e decrease in th e specific g ro w th ra te o f
N eochloris oleoabundans is not caused by th e b u ild -u p of th e oxygen
c o n c e n tra tio n b u t th a t th e lig h t regim e a pplied is fa r m ore im p o rta n t fo r th e
g ro w th o f th e algae.
W h e n co m p aring th e specific g ro w th ra te a t c o n s ta n t oxygen c o n c e n tra tio n

(P o2=0.21 bar) and a pplying a lig h t regim e 3 0 /6 (P3), w ith th e one o b ta in e d at
co n tin u o u s lig h t and co n sta n t P02= 0.21 bar (P2), th e d ark perio d th a t algae w e re
expe rie ncing resu lted in a decrease o f th e specific g ro w th rate fro m 1.14 ± 0.06
day"1 d o w n to 0.80 ± 0.16 day"1. A t co n sta n t oxygen c o n c e n tra tio n (Po2= 0.21),
lig h t regim e 3 0 0 /6 (P4) and co n sta n t oxygen c o n c e n tra tio n (Po2= 0.21 bar),
co n tin u o u s lig h t (P2) th e decrease in g ro w th rate was less p ro fo u n d . A t a lig h t
regim e 3 0 0 /6 th e algae w e re exposed to dark periods less o fte n (com pared w ith
lig h t regim e 3 0 /6 ) and th e decrease in th e specific g ro w th ra te was low e r. The
dyna m ic oxygen c o n d itio n s fo r th e b o th lig h t regim es (3 0 /6 and 3 0 0 /6 ) (P5 and
P6) did n o t give rem arkab le changes in th e specific g ro w th ra te co m p ared w ith
th e co rresp on din g re fe ren ce (P3 and P4) g ro w th rates o b ta in e d a t co n sta n t
oxygen co n ce n tra tio n (Po2= 0.21 bar).
4.3.2. Effect on biomass yield on photons

In Table 1 th e biom ass yield on p ho ton s, calculated based on th e biom ass
p ro d u c tio n rate and lig h t supply rate, is expressed as th e a m o u n t o f lig h t energy
th a t is co n ve rte d in to biom ass per m ol o f p h o to n s supplied in th e PAR range (g
m ol-ph "1). U nder dyna m ic oxygen co n ce n tra tio n s a biom ass yield on p h o to n s o f
0.88 ± 0.02 g m ol-ph "1 (P5) and 1.01 ± 0.07 g m ol-ph "1 (P6) was calculated. The
63
C hapter 4
m axim um biom ass yield on p h o to n s calculated in th is e x p e rim e n t was 1.15 ± 0.04

g m ol-ph "1 (P2) u n d e r co n tin u o u s lig h t and c o n s ta n t oxygen c o n c e n tra tio n
(Po2=0.21 bar). This value is hig he r th a n th e value fo r N eochloris oleoabundans
o b ta in e d by Pruvost et al. (2009). They re p o rte d a v o lu m e tric biomass

p ro d u c tiv ity fo r N eochloris o le oabundans o f 0.55 kg m"3 day"1 and w he n th is value
is co m b in e d w ith th e p ro vid e d data o f in c id e n t lig h t flu x and th e illu m in a te d
surface to vo lu m e ra tio o f th e re a c to r used a t co n tin u o u s lig h t co n d itio n s (lig h t-
lim ite d g ro w th ), a biom ass yield on p h o to n s o f 0.71 g m ol-ph "1 is calculated.
N eochloris o le oabundans exposed to d ynam ic oxygen co n c e n tra tio n s and sub-
s a tu ra tin g lig h t co n d itio n s still e xhibits sim ila r high biom ass yield on p ho ton s. In
previous w o rk on th e e ffe c t o f co n tin u o u s oxygen c o n c e n tra tio n s (Po2=0.63 bar)
on th e algal g ro w th o f N eochoris o le oabundans a t s u b-satu ratin g lig h t in te n sitie s a

biom ass yield on p h o to n s o f 1.07 ± 0.10 g m ol-ph "1 was o b ta in e d (Sousa e t al.,
2012). These results clearly show th e e ffe c t o f te m p o ra ry exposure o f th e algae to
d ark regim es. Evaluating th e results fro m P3 and P4 it is fa ir to say, th a t once th e
lig h t exposure was increased by a fa c to r 10 w e w o u ld e xpect a larger increase in
specific g ro w th ra te th a n th e 1.4 tim e s fo u n d . In a d d itio n , a s im ila r biom ass yield
in p h o to n s w o u ld be expected d u rin g th e w h o le e x p e rim e n t. But w e have to ta ke
in co n sid e ra tio n , th a t n o t all th e lig h t th a t fa lls on th e p h o to s y n th e tic a ntenna
com plexes is absorbed. A p a rt o f th e lig h t is absorbed and used fo r pho tosyn th esis
b u t a n o th e r p a rt ju s t passes th ro u g h th e cells. A n o th e r fa c to r to consider is th e
energy re q u ire d fo r m aintenance. All th e processes needed fo r th e algae to
survive except g ro w th , also re q u ire energy and d u rin g th e darkness periods,
energy is needed fo r re sp ira tio n and storage o f co m p ou nd s (Vejrazka e t al., 2011).
4.3.3. Chlorophyll and carotenoid content

To v e rify if a p p lica tio n o f d iffe re n t lig h t regim es and d yna m ica lly changing oxygen
c o n c e n tra tio n lead to a d d itio n a l p h o to -a c c lim a tio n and p h o to -in h ib itio n e ffects
on th e g ro w th rate, th e ch lo ro p h yll and ca ro te n o id c o n te n t o f th e algae was
m easured d u rin g th e su bse qu en t phases o f th e e x p e rim e n t (Figure 2).
64
E ffect o f d yna m ic oxygen co n ce n tra tio n on the g ro w th o f N eochloris oleoabundans
Chla+Chlb (mg g'1)

P1 P2 P3 P4
I 1 Car ( mg g'1)
30 -
P5 P6
es
O
20 -
O
+
CS
10 -
10 15 20
Time (days)
Figure 2 - Total chlorophyll content and carotenoids in Neochloris oleoabundans

a t different phases in the experimental run. P= Phase
(see Table 1 fo r the conditions a t which the samples were taken)
The ch lo ro p h yll c o n te n t o f th e algae was fo llo w e d d urin g th e w h o le e x p e rim e n ta l

run and th e cells show typ ical p h o to -a cclim a tio n effects. C hlo ro ph yll c o n te n t
decreased w he n m ore lig h t was available per cell and increased in case less ligh t
was pro vid e d The highest a m o u n t o f c h lo ro p h y ll per d ry w e ig h t was m easured in
P I (29 mg g"1) at th e end o f th e batch, w he n th e biom ass d e n sity inside th e
p h o to b io re a c to r was re la tive ly high and less lig h t was available per cell. D uring
tu rb id o s ta t o p e ra tio n , th e c h lo ro p h yll c o n te n t was in general low e r, as th e
biom ass d en sity was ke pt co n sta n t at relative lo w biom ass co n c e n tra tio n (Figure
l).T h e d ifferen ce s in c h lo ro p h yll c o n te n t observed d u rin g th e d iffe re n t tu rb id o s ta t
phases was n o t caused by th e h igher oxygen co n c e n tra tio n s applied b u t due to
th e d iffe re n t exposure tim e s o f th e algae to th e light. The s h o rte s t (P3 and P5)
periods o f exposure resulted in h igher c h lo ro p h y ll c o n c e n tra tio n s and th e longest
exposures o f tim e (P4 and P6) in lo w e r co n ce n tra tio n s. No d ifferen ce s can be
observed w hen co m p aring th e average c h lo ro p h y ll c o n te n t o f th e cells exposed to
65
C hapter 4
th e same lig h t regim e, b u t a t lo w oxygen c o n c e n tra tio n (P3 and P4) and th e
ch lo ro p h yll c o n te n t o f th e cells subjected to d yna m ica lly changing oxygen
co n ce n tra tio n s (P5 and P6).
C arotenoids are kn ow n as p h o to p ro te c tiv e pigm ents. T h e ir c o n te n t n o rm a lly

increases w he n th e algae cells are exposed to high lig h t in te n sitie s (D ubinsky &
S tam bler, 2009). The reason fo r th a t is th e ir fu n c tio n to dissipate excess energy o f
e xited ch lo ro p h yll and also e lim in a tin g ROS (Law lor, 2001). The a m o u n t o f
c a ro te n o id s rem ain ed co n sta n t o ve r th e w h o le e x p e rim e n t. N e ith e r th e oxygen
levels in th e m ed iu m , n o r th e lig h t regim es a pp lie d a ffe cte d th e ca ro te n o id
c o n te n t, w h ich indicates th a t a d d itio n a l p h o to -in h ib itio n e ffe cts did n o t in te rfe re
a t th e lig h t co n d itio n s used. In a d d itio n , th e a pplied oxygen c o n c e n tra tio n did n o t
lead to e xtra fo rm a tio n o f th is p h o to p ro te c tiv e p ig m e n t, d e m o n s tra tin g th a t
oxygen as such does n o t induce p h o to in h ib itio n .
4.3.4. Final remarks

In closed p h o to b io re a c to r systems, oxygen a ccu m u la tio n leads to an increase o f
th e O 2 /C O 2 ra tio in th e m ed iu m , p ro m o tin g th e oxygenase a c tiv ity o f th e enzym e
and a c tiva tin g th e p h o to re s p ira to ry p ath w ay. Dissolved oxygen c o n c e n tra tio n s in
p h o to b io re a c to rs can easily increase up to 4 tim e s air s a tu ra tio n (Carvalho e t al.,
2006; W eissm an e t al., 1988). The oxygen b u ild -u p and a ccu m u la tio n is a
p a rtic u la rly serious p ro ble m w hich u ltim a te ly results in decreases in specific
g ro w th rates and biom ass p ro d u c tiv itie s . Oxygen c o n c e n tra tio n s above 1.5 to 2
tim e s air sa tu ra tio n in h ib it algal g ro w th (Ota e t al., 2011; Raso e t al., 2011; Ugwu
e t al., 2007). For w o rk p e rfo rm e d a t o u td o o r c o n d itio n s, th e oxygen p ro b le m is
ta ke n in to co n sid e ra tio n by w o rk in g a t oxygen co n c e n tra tio n s b e lo w in h ib itin g
levels fo r th e m icroalgal cu ltures. The m o st c o m m o n ly used stra te g y to m a in ta in
th e oxygen level b e lo w in h ib itin g levels is th e im p le m e n ta tio n o f degassers
(Fernandez e t al., 2001; Fuentes e t al., 1999; Pirt e t al., 1983; Richm ond e t al.,
1993; Travieso e t al., 2001). In a re vie w o f enclosed system designs and
p erform an ces Carvalho e t al. (2006) state th a t a universa lly o p tim u m gas tra n s fe r
device does n o t exist. In all th e cases, an overall decrease in specific g ro w th rates
u n d e r oxygen a ccu m u la tio n was observed a n d /o r expected. Based on th a t, a
lo w e r g ro w th rate was also expected w hen d yna m ica lly changing oxygen
66
c o n ce n tra tio n s w e re a pplied, once th e m icroalgae cu ltu re s w e re a n yh o w exposed

to high oxygen co n ce n tra tio n s fo r a perio d o f tim e . The results o b ta in e d in th is
s tu d y h ow eve r, show ed no s ig n ifica n t change in th e specific g ro w th rate caused
by th e high oxygen c o n ce n tra tio n . In a d d itio n , high oxygen co n c e n tra tio n s did n o t
lead to changes in p ig m e n ta tio n , w h ich indicates th a t th e cells did n o t a ctiva te
p ro te c tiv e m echanism s against p h o to o x id a tiv e dam age. The o b se rva tio n th a t th e
g ro w th ra te and th e biom ass yield on p ho ton s is n o t a ffe cte d by th e d yna m ica lly
changing oxygen co n ce n tra tio n could be explained by th e presence o f a carbon
c o n ce n tra tin g m echanism (CCM) in N eochloris o le oabundans th a t is a ctivate d a t
e levated oxygen co n ce n tra tio n s in th e m ed iu m . CCM's are considered responsible
fo r m in im izin g p h o to re s p ira tio n by increasing th e dissolved ino rg a nic carbon
c o n c e n tra tio n in th e cell via active tra n s p o rt o f C 0 2 and H C 03" (Kaplan &
R einhold, 1999). This increase in C 0 2 co n c e n tra tio n helps th e ca rb o xyla tio n
rea ction and in h ib its th e oxygenase a c tiv ity o f th e Rubisco. C onsequently
increases p h o tosyn th esis and reduces p h o to re s p ira tio n (H uertas e t al., 2000).
4.4. Conclusions
The e ffe c t o f dyna m ic oxygen co n ce n tra tio n s and lig h t/d a rk regim e o f lig h t on
g ro w th o f N eochloris oleoabundans was e valuated. Gradual increase o f th e
oxygen co n ce n tra tio n fro m Po2 = 0.21 bar up to 0.63 bar, fo llo w e d by p e rio d o f
rapid degassing did n o t bring sig n ifica n t decrease in th e specific g ro w th rate.
F u rth e rm o re even w h e n th e algae w e re exposed fo r 300 m in u te s a t high oxygen
c o n c e n tra tio n , th e re was no sig n ifica n t change in th e specific g ro w th rate o f th e
algae N eochloris oleoabundans. O b ta in ed results sh ow th a t th e residence tim e o f
th e algae on th e solar rece ive r could be increased up to 10 x w ith o u t degassing. A
s ig n ifica n t decrease o f th e algae specific g ro w th was observed w he n a pplying lig h t
regim e o f 30 m in u te s lig h t "O n " and 6 m in u te s lights "O ff", p ro vin g th a t th e
exposure o f th e algae cells to d ark periods in th e degasser has a bigger negative
im p a ct th a n th e exposure to high oxygen c o n c e n tra tio n as such. The results o f th is
stu dy clearly show th a t o p tim iz a tio n o f closed p h o to b io re a c to rs based on th e
observed algae physiology u n d e r dyna m ic oxygen c o n c e n tra tio n w ill re su lt n o t
o n ly in a re d u ctio n o f th e n u m b e r o f th e degassing units, b u t also by doing so it
w ill c o n trib u te fo r keeping hig he r g ro w th ra te and h ig he r biom ass p ro d u c tio n
67
C hapter 4
respectively. Reducing th e a m o u n t o f degassing units and increasing th e ir

degassing capacity, to m in im ize th e tim e o f darkness th e algae are exposed to , w ill
re su lt in su bsta ntia l savings in design and o p e ra tio n o f plants fo r m icroalgae
p ro d u ctio n .
discussions and th e ir fin a n cia l su p p o rt.
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Fernandez, F.G.A., Sevilla, J.M.F., Perez, J.A.S., G rim a, E.M., Chisti, M.Y. 2001.
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and e x p lo itin g c a ro te n o id a ccu m u la tio n in D un aliella sa lin a fo r ce ll-fa c to ry
a pp lica tion s. Trends in B iotechnology, 26(11), 631-638.
Law lor, D.W. 2001. P hotosynthesis. Bios S cientific Publishers, O xford.
M iro n , A.S., Gomez, A.C., Camacho, F.G., G rim a, E.M., Chisti, Y. 1999. C om parative
e va lu a tio n of co m p a ct p h o to b io re a c to rs fo r large-scale m o n o c u ltu re of
m icroalgae. Journal o f B iotechnology, 70(1-3), 249-270.
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Norsker, N.-H., Barbosa, M.J., V erm uë, M .H., W ijffe ls , R.H. 2011. M icroalgal
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Ota, M ., Kato, Y., W a ta na be , M., Sato, Y., Sm ith Jr, R.L., Rosello-Sastre, R., Posten,
C., Inornata, H. 2011. Effects o f n itra te and oxygen on p h o to a u to tro p h ic lipid
p ro d u c tio n fro m Chlorococcum litto ra le . B ioresource Technology, 102(3), 3286-
3292.
Pirt, S.J., Lee, Y.K., W alach, M.R., W a tts Pirt, M., Balyuzi, H .H.M ., Bazin, M.J. 1983.
A tu b u la r b io re a c to r fo r p h o to s y n th e tic p ro d u c tio n o f biom ass fro m carbon
d io xide : Design and p e rfo rm a n ce . Journal of Chemical T echnology and
B iotechnology, 33B, 35-58.
c o n c e n tra tio n on th e g ro w th o f N annochloropsis sp. a t lo w lig h t in te n s ity . Journal
R ichm ond, A., Boussiba, S., Vonshak, A., Kopel, R. 1993. A new tu b u la r re a c to r fo r
mass p ro d u c tio n o f m icroalgae o u td o o rs. Journal o f A pplie d Phycology, 5(3), 327-
332.
Sousa, C., de W in te r, L., Janssen, M ., V erm uë, M .H ., W ijffe ls , R.H. 2012. G ro w th o f

th e m icroalgae N eochloris o le oabundans a t high p a rtia l oxygen pressures and sub-
s a tu ra tin g lig h t in te n sity. B ioresource Technology, 104(0), 565-570.
Suzuki, L., Johnson, C.H. 2001. Algae kn o w th e tim e o f day: Circadian and
p h o to p e rio d ic program s. Journal o f Phycology, 37, 933-942.
Travieso, L., Haii, D.O., Rao, K.K., Benitez, F., Sanchez, E., Borja, R. 2001. A helical
tu b u la r p h o to b io re a c to r p ro du cing Spirulina in a s e m ico n tin u o u s m ode.
In te rn a tio n a l B io d e te rio ra tio n & B iodégradation, 47(3), 151-155.
T ria ntap hylidè s, C., Havaux, M . 2009. Singlet oxygen in plants: p ro d u c tio n ,
d e to x ific a tio n and signaling. Trends in Plant Science, 14(4), 219-228.
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Ugwu, C.U., Aoyagi, H., U chiyam a, H. 2007. In flue nce o f Irradiance, dissolved
Ugwu, C.U., Aoyagi, H., Uchiyam a, H. 2008. P h o to b io re a cto rs fo r mass c u ltiv a tio n
o f algae. Bioresource Technology, 99(10), 4021-4028.
Vej'razka, C., Janssen, M ., S treefland, M ., W ijffe ls , R.H. 2011. P h o to syn th e tic

Efficiency o f C hlam ydom onas re in h a rd tii in Flashing Light. B io tech n olog y and
B ioengineering, 108(12), 2905-2913.
Vilchez, C., Forjan, E., Cuaresma, M ., Bedm ar, F., Garbayo, I., Vega, J.M. 2011.
M a rin e C arotenoids: Biological Functions and C om m ercial A p plica tion s. M a rin e
Drugs, 9(3), 319-333.
W eissm an, J.C., G oebel, R.P., Benem ann, J.R. 1988. P h o to b io re a c to r design:
M ixing, carbon u tiliz a tio n , and oxygen a ccum ula tion . B io tech n olog y and

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295.
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72
a t hig h lig h t co nd itio ns
Chapter 5. E ffe c t o f D y n a m ic O xyg en C o n c e n tr a tio n s o n th e

g ro w th of N e o c h l o r is oleoabundans at hig h l ig h t
CONDITIONS
Claudia Sousa1' 2, M arian H. V e rm u ë 2and Rene H. W ijffe ls 2
1 Wetsus, P.O. Box 1113, 8900 CC Leeuwarden, the Netherlands
the Netherlands
A b s tra c t - D ynam ically changing oxygen c o n c e n tra tio n s experienced in closed

p h o to -b io re a c to r system w e re sim u la te d in CSTR a t high lig h t in te n sity. The e ffe c t
of 10 tim e s e lo n g a tio n o f th e residence tim e in th e solar receiver was
inve stiga te d. W h e n th e algae w e re exposed to c o n s ta n t oxygen c o n c e n tra tio n and
c o n sta n t high lig h t th e specific g ro w th rate was 1.29 ± 0.08 day"1. Using a ligh t
regim e o f 30 m in u te s lig h t ON fo llo w e d by 6 m in utes lights OFF and degassing
resu lted in a specific g ro w th rate o f 0.84 ± 0.09 day"1, e lo n g a tio n o f th e tim e
(lights ON) to 300 m in u te s resu lted in 1.18 ± 0.05 day"1. W hen d yna m ica lly
changing oxygen co n ce n tra tio n s w e re applied, s im ila r specific g ro w th rates w e re
o b ta in e d . These results in d ica te th a t algae do n o t experience th e expected p h o to -
o xid a tive in h ib itio n caused by high oxygen co n c e n tra tio n in c o m b in a tio n w ith high
ligh t, as long as th e oxygen is rem ove d via reg ula r degassing. The te m p o ra ry
exposure o f th e algae to th e darkness in th e degasser has m ore im p a ct on th e
p ro d u c tiv ity .
Key w o rd s : tu b u la r p h o to b io re a c to rs , dyna m ic oxygen, N eochloris oleoabundans,

high lig h t inte n sitie s
Submitted fo r publication
73
C hapter 5
5 .1 . In tro d u c tio n
High g ro w th rates and high lipid c o n te n t co m b in e d w ith th e n o n -co m p e titive n e ss

w ith fo o d , m ake m icroalgae one o f th e m ost a ttra c tiv e resource fo r biodiesel
p ro d u c tio n (Gong & Jiang, 2011; W ijffe ls e t al., 2010). To m ake large-scale
o u td o o r p ro d u c tio n o f m icroalgae fo r biodiesel e con om ically feasible, design and
d e v e lo p m e n t o f p h o to b io re a c to rs fo r m icroalgal c u ltiv a tio n is su bject o f m any
studies th a t focus on m axim iza tion o f th e m icroalgae p ro d u c tio n (N orsker e t al.,
2011; Singh & Sharma, 2012; W ijffe ls & Barbosa, 2010). T u b u la r p h o to b io re a c to rs
are p resented as a cost e ffic ie n t system w ith ro o m fo r im p ro v e m e n t (N orsker et
al., 2011). These types o f closed p h o to b io re a c to rs are a lready used as o u td o o r
c u ltiv a tio n system s fo r m icroalgae (M o lin a e t al., 2001; Pirt e t al., 1983; T orzillo et
al., 1993; T redici & Z itte lli, 1998) b u t a lo t o f energy is used fo r th e C 0 2 supply and
to p re ve n t 0 2 a ccu m u la tio n (N orsker e t al., 2011). Studies on th e e ffe c t o f oxygen
on m icroalgae g ro w th a t co n sta n t oxygen co n c e n tra tio n s and c o n s ta n t lig h t
in te n sitie s revealed th a t 0 2 a ccu m u la tio n leads to a decrease in specific g ro w th
rate (Kliphuis e t al., 2011; Raso e t al., 2011; Sousa e t al., 2012). In practice,
how eve r, algae cu ltu re d in tu b u la r p h o to b io re a c to r system s are n o t su bjected to
c o n sta n t oxygen co n ce n tra tio n s, b u t th e y experience an increasing oxygen
g ra d ie n t along th e tu b e length and a t th e end o f th e tu b e th e high oxygen level
could lead to decreasing g ro w th rate (Ugwu e t al., 2008). T h e re fo re a t th e end o f
th e tu b e a degassing u n it is placed, w h e re th e a ccum ula ted oxygen is rem ove d to .
To te s t if indeed, th e a ccu m u la tio n o f oxygen caused a decrease o f th e g ro w th
rate, Sousa e t al (2013b) m easured th e in-vivo g ro w th rate o f N eochloris
o le oabundans a t d yn a m ica lly changing oxygen co n c e n tra tio n s (PO2= 0 .2 1/0 .6 3 bar)
and lo w lig h t in te n s ity (200 p m o l m"2 s"1) fo llo w e d by a s h o rt d ark p e rio d o f

degassing to sim u la te th e d yn a m ica lly changing oxygen and lig h t c o n d itio n s
experienced by algae in a tu b u la r p h o to b io re a c to r system . Surprisingly, no
s ig n ifica n t decrease in specific g ro w th rate was fo u n d . A t th ese lo w lig h t
inten sitie s, th e g ro w rate decreased o n ly due to th e e ffe c t o f less lig h t a v a ila b ility
d u rin g th e sim u la te d d ark periods in th e degasser. This ind icate d th a t 0 2
a ccu m u la tio n did n o t lead to a d d itio n a l p h o to re s p ira tio n e ffe cts as long as th e 0 2
was rem ove d fre q u e n tly (Sousa e t al., 2013b).
74
In o u td o o r co n d itio n s lig h t co n d itio n s va ry o ve r th e day and o ve r th e seasons.

M o re o ve r, especially d u rin g th e sta rt-u p o f th e cu ltu re s w h e n th e biom ass
c o n c e n tra tio n is still low , th e algae m ay also experience hig he r lig h t inten sitie s. A t
high lig h t inten sitie s, th e c o m b in a tio n o f high 0 2 and high lig h t a d d itio n a l
p h o to in h ib itio n e ffe cts can be expected (Kliphuis e t al., 2011; Raso e t al., 2011;
Sousa e t al., 2012) due to th e fo rm a tio n o f oxygen radicals and o th e r reactive
oxygen species (ROS) (M u ra ta e t al., 2007) and fo rm a tio n o f th e h ig hly reactive
singlet oxygen via p h o to a c tiv a tio n (T rian ta ph ylid es e t al., 2008). It is n o t clear
how eve r, h ow th e algae w ill respond on th e d yn a m ica lly changing oxygen
co n ce n tra tio n s in a tu b u la r p h o to b io re a c to r system w h ile th e algae are exposed
to high lig h t in te n sitie s fo llo w e d by sh o rt periods o f darkness in th e degasser. To
s im u la te th e se c u ltiv a tio n co n d itio n s, a fu lly c o n tro lle d C ontinuous S tirred Tank
Reactor (CSTR) o p e ra te d in tu rb id o s ta t m ode was used to c u ltu re N eochloris
o le oabundans (UTEX1185) a t high lig h t in te n s ity (500 p m o l m"2 s"1) and th e specific
g ro w th rate was m easured. In a d d itio n , th e tim e a t w hich th e algae w e re exposed
to high p a rtia l oxygen pressures was e xten de d to d e te rm in e th e e ffe c t o f a te n
tim e s lon ge r residence tim e a t high lig h t co n d itio n s on th e specific g ro w th ra te o f
th e algae. This e x p e rim e n t was included because in previous w o rk a t lo w ligh t
co n d itio n s th e e lo n g a tio n o f th e tim e sp en t in th e tubes, did n o t a ffe c t th e specific
g ro w th rate. If extension o f th e tim e spend a t high oxygen c o n c e n tra tio n and high
lig h t irra dian ce does n o t a ffe c t th e g ro w th rate, th is w o u ld in d ica te th a t th e
p e rio d th a t cells spend in th e dark degasser can be decreased considerably,
leading to hig he r p ro d u c tiv itie s .
5.2. Material and method
5.2.1. Culture and photobioreactor system

N eochloris o le oabundans (UTEX 1185) was p re -c u ltu re d in a dapted f /2 m edium
(G uillard & Ryther, 1962) and used as ino culu m s in 3L b io re a c to r (A pplikon
B iotechnology, The N etherlands), o p e ra te d in tu rb id o s ta t m ode. The re a c to r
system and c o n tro llin g apparatus is described by Sousa e t al. (2012).
75
C hapter 5
5.2.2. Light regim e

Light was p ro vid e d by tw o LED lig h t panels (20x20 cm ) (SL3500, Photon Systems
In strum en ts, Czech Republic), w h ich w e re set to supply an average in c id e n t lig h t
o f ~500 p m o l m"2 s"1 on th e surface o f th e re a c to r w alls. The lig h t sources w ere
co nn ecte d to a Siemens PLC Relay (LOGO!) lig h t c o n tro lle r to sim u la te th e
darkness p e rio d in th e degasser o f a tu b u la r PBR system . T w o d iffe re n t tim e
regim es fo r th e lig h t w e re used, re la te d w ith th e oxygen c o n d itio n s applied d u rin g
th e e xpe rim en ts. The fir s t one was 30 m in utes lig h t "O n " and 6 m in u te s lig h t "O ff"
(3 0 /6 regim e). This regim e was a pplied d u rin g dyna m ic oxygen co n d itio n s, w he n
th e algae w e re a llo w e d to p ro du ce and accum ula te oxygen fro m PO2=0.21 bar up
to Pq2=0.63 bar. A fte r th is phase a degassing phase was in itia te d . D uring th e
degassing phase th e lig h t c o n tro lle r sw itch to Lights "O ff" regim e a llo w in g rapid
degassing o f th e liq u id v o lu m e back to s ta rtin g oxygen levels o f Po2=0.21 bar in 6
m in utes.
The second tim e regim e th a t was used, o p e ra te d a t 300 m in utes lig h t "O n " and 6
m in u te s lig h t "O ff" (3 0 0 /6 regim e). This regim e was applied d u rin g c u ltu rin g th e
algae a t high oxygen levels (P q2=0.63 bar) fo r 300 m in utes. The degassing phase
was p e rfo rm e d a t six m in u te s lights "O ff".
5.2.3. O ff-line analysis o f samples

T rip lica te sam ples o f 5 ml w e re co lle cted on a d aily base fo r d ry w e ig h t
d e te rm in a tio n . The sam ples w e re d ilu te d w ith 10 m l a m m o n iu m fo rm a te (0.5M )
and filte re d using p re -w e ig he d glass fib e r-filte rs (W h atm a n GF/F). An a d d itio n a l
40 m l o f a m m o n iu m fo rm a te (0.5M ) was used fo r w ashing th e biom ass on th e
filte rs . Filters w ith biom ass w e re d rie d a t 95 °C fo r 24 hours in a lu m in iu m trays,
cooled in d esiccator fo r 2 hours and w eighed on a 5 d ig it a nalytical balance
(ME235P-SD, Sartorius, G erm any).
For ch lo ro p h yll and to ta l ca ro te n o id d e te rm in a tio n sam ples w e re co lle cted on a

d aily base and c e n trifu g e d a t 3750 rpm fo r 10 m in. a t 4 °C. The pellets w e re froze n
a t -80°C. The p ig m e n t e x tra ctio n was done by a d d itio n o f 5m L o f 100% m eth an o l
to each tu b e . The tu b e s w e re th e n placed fo r 5 m in. in u ltra so n ic bath (Sonorex
Digitec, Bandelin). A fte rw a rd s th e sam ples w e re incu ba te d fo r 40 m in a t 60 °C and
76
fo r a n o th e r 15 m in a t 0 °C and c e n trifu g e d once m o re a t th e same co n d itio n s. The

s u p e rn a ta n t was collected and th e ch lo ro p h y ll and ca ro te n o id was d e te rm in e d by
m easuring th e o ptica l d e n sity a t 470nm , 652 nm and 665 nm in a U V-visible
s p e c tro p h o to m e te r (UV_1650 PC, Schimadzu). M o d ifie d A rn o n 's e qu atio ns w e re
used to d e te rm in e th e ch lo ro p h yll and ca ro te n o id c o n te n t (Lichten th aler, 1987).
C hlo ro ph yll and ca ro te n o id c o n te n t w e re expressed per gram o f biomass,
calculated based on th e d ry w e ig h t co n ce n tra tio n o f th e sam ples used (Cuaresma
e t al., 2011).
5.3.1. Effect o f dynamic 0 2 and light regim e on algal grow th

Figure 1 show s tw o typ ical b io re a c to r runs to d e te rm in e th e e ffe cts o f co n sta n t
and d yn a m ica lly changing oxygen co n ce n tra tio n w h ile using d iffe re n t tim e
regim es fo r oxygen b u ild -u p a t high lig h t co n d itio n s fo llo w e d by a perio d o f
darkness d u rin g w hich th e oxygen is rem oved.
2.0
P02 (bar)
OD (CU)
P3
* Cx(gDW.L*1)
1.5 ■ n (day-1)
i
X
U
s 10
CL
a
o
0.5
0.0
0 2 4 6 8 10 12
Tim e (days)
Figure 1 - Graphical representation o f partial oxygen pressure (Po2) in bar; optical density
(OD) in CU; specific growth rate (p) in d a y 1; biomass concentration (CJ in g L'1 versus time
in days on a typical experimental run.
(P2) Continuous Light - Constant PO2=0.21 bar; (P3) Light Regime 30/6 - Constant PO2=0.21
bar; (P4) Light Regime 300/6 - Constant PO2=0.21 bar; (P5) Light Regime 30/6 - Dynamic
Po2=0.21/0.63 bar; (P6) Light Regime 300/6 - Dynamic PO2=0.21/0.63 bar.
77
C hapter 5
The e xp e rim e n ta l run re p re sen te d in Figure 1 a) can be divid ed in 4 d iffe re n t

phases. In itia lly, th e p h o to b io re a c to r was in o cu la te d w ith N eochloris
o le oabundans and th e cells w e re g ro w n batch w ise (Figure la - Phase P I) u n til an
o ptica l d e n sity o f 0.8 CU was reached, co rre sp o n d in g to a biom ass c o n c e n tra tio n
o f 0.50 ± 0.02 g L 1. In Phase P2 th e algal c u ltu re was a llo w e d to a d ju st to
co n tin u o u s high lig h t co n d itio n s and a co n s ta n t p a rtia l oxygen pressure o f 0.21
bar. A fte r an in itia l a cclim atio n p erio d o f 2 days a t a c o n tro lle d o ptica l d e n sity o f
0.8 CU th e specific g ro w th rate was d e te rm in e d fro m th e m ed iu m o u tflo w o f th e
p h o to b io re a c to r and a t th ese co n d itio n s th e specific g ro w th rate was fo u n d to be
1.29 ± 0.08 day"1 (Figure la , Table 1). Phase P3 corresponds to a lig h t regim e 30
m in u te s lig h t ON and 6 m in u te s lig h t OFF a t c o n s ta n t Po2 o f 0.21 bar and Phase P4
300 m in utes lig h t ON and 6 m in utes lig h t OFF m easured a t th e same c o n s ta n t Pq2
o f 0.21 bar.
In Figure 1 b), th e m easured data in th e e x p e rim e n t w ith d yna m ica lly changing
oxygen co n ce n tra tio n s are show n. D uring Phase P5 dyna m ic oxygen co n d itio n s
w e re a pplied and th e re a cto r was o p e ra te d a t a lig h t regim e o f 30 m in utes lights
"O n " and 6 m in u te s lights "O ff". The oxygen was a llo w e d to build up fro m
Po2=0.21 bar to PO2=0.63 bar fo r 30 m in u te s and th e n was fo rc e d to decrease to
s ta rtin g level d urin g 6 m in u te s o f degassing. A t th o se co n d itio n s th e specific
g ro w th rate was 0.97 ± 0.11 d a y '1 (Table 1) and show ed a hig he r specific g ro w th
rate th a n th e one m easured in th e re fe ren ce e x p e rim e n t (Phase P3).This shows
th a t th e dyna m ic change in oxygen co n ce n tra tio n s as such, does n o t c o n trib u te to
th e decrease o f th e g ro w th ra te a t th e high lig h t c o n d itio n used. Phase P6 was
p e rfo rm e d using an exposure tim e a t high oxygen c o n c e n tra tio n o f Pq2=0.63 bar
fo r 300 m in, fo llo w e d by 6 m in utes fo r degassing and th e lig h t supply c o n tro lle d
using th e 3 0 0 /6 lig h t regim e. The specific g ro w th o f th e algae m easured was 1.10
± 0.1 d a y 1. W ith no sig n ifica n t decrease in g ro w th rate co m p ared w ith th e
re fe ren ce e x p e rim e n t (Phase P4), th is re su lt c o n firm s once again th a t th e
decrease in th e specific g ro w th rate o f N eochloris o le oabundans is n o t caused by
th e b u ild -u p o f th e oxygen c o n c e n tra tio n b u t th a t th e lig h t regim e a pplied is m ore
im p o rta n t fo r th e g ro w th o f th e algae.
78
Table 1 - Average biomass concentration and specific growth rate o f Neochloris

oleoabundans during each phase o f the experimental run
Cx ± Stdev p ± Stdev
(g L"1) ( d a y 1)
Batch phase (P I) - -
C ontinuous Light -
0.50 ± 0 .0 2 1.29 ± 0 .0 8
C onstant PO2=0.21 bar (P2)
Light Regime 3 0 /6 -
0.53 ± 0 .0 1 0.84 ± 0 .0 9
Light Regime 3 0 /6 -
0.55 ± 0 .0 2 0.97 ± 0 .1 1
D ynam ic PO2= 0 .2 1/0 .6 3 bar (P5)
Light Regime 3 0 0 /6 -
0.52 ± 0 .0 0 5 1.18 ± 0 .1 4
Light Regime 3 0 0 /6 -
0.54 ± 0 .0 3 1.10 ± 0 .1 0
D ynam ic PO2= 0 .2 1/0 .6 3 bar (P6)
W h e n co m p aring th e specific g ro w th ra te a t c o n s ta n t lo w oxygen c o n c e n tra tio n

(P o2=0.21 bar) and a pplying a lig h t regim e 3 0 /6 (P3), w ith th e one o b ta in e d at
co n tin u o u s lig h t and co n sta n t PO2=0.21 bar (P2), th e d ark p erio d th a t algae w e re
expe rie ncing resu lted in a decrease o f th e specific g ro w th rate fro m 1.29 ± 0.08
day"1 d o w n to 0.84 ± 0.09 day"1. A t co n sta n t lo w oxygen c o n c e n tra tio n (PO2=0.21),
lig h t regim e 3 0 0 /6 (P4) and co n sta n t oxygen c o n c e n tra tio n (PO2=0.21 bar),
co n tin u o u s lig h t (P2) th e decrease in g ro w th rate is m uch sm aller. A t a ligh t
regim e 3 0 0 /6 th e algae w e re exposed to d ark p eriods less o fte n , co m p ared w ith
lig h t regim e 3 0 /6 resu ltin g in th e observed decrease in th e specific g ro w th rate.
The dyna m ic oxygen c o n d itio n s fo r th e b o th lig h t regim es (3 0 /6 and 3 0 0 /6 ) (P5

and P6) did n o t give rem arkab le changes in th e specific g ro w th rate co m p ared
w ith th e co rresp on din g references (P3 and P4) g ro w th rates o b ta in e d a t co n sta n t
oxygen co n c e n tra tio n (PO2=0.21 bar). W hen co nside rin g th e p ro d u c tiv ity in closed
p h o to b io re a c to r system s th e se results show th a t th e lig h t in p u t plays a fa r m ore

im p o rta n t role th a n th e oxygen b uild-up , as long as oxygen is rem ove d a t a reg ula r
basis.
79
Chapter
5.3.2. Chlorophyll and carotenoid content

Being exposed to average high lig h t c o n d itio n s co m b in e d w ith high oxygen
c o n c e n tra tio n th e fo rm a tio n o f ROS and sing let oxygen is bound to occur.
M icroalgae have d eveloped p h o to a da ptive and p h o to p ro te c tiv e m echanism s to
deal w ith such u nfa vora ble p h o to -o x id a tiv e stress c o n d itio n s, to p ro te c t th e ir
p h o to s y n th e tic apparatus (A m ara e t al., 2012; D ubinsky & S tam bler, 2009). A t
e levated oxygen c o n ce n tra tio n s in th e p h o to b io re a c to r was th u s expected to
induce extra ca ro te n o id p ro d u c tio n to p ro te c t th e cells by q uenching th e
e le ctron s and act as a n ti-o x id a n t against th e ROS fo rm e d . (D em m ig-A dam s &
Adam s, 2002)
0.4
0.3 -
-Q
£
o
+
re
Z 0.2 -
o
03
o
0.1 -
0 . 0 -I T T T T T ----------------
P2 P3 P4 P5 P6
Figure 2 - Ratio o f to ta l carotenoid and chlorophyll a+b content in

Neochloris oleoabundans a t the different phases.
P= Phase (see Figurel fo r the description o f the different phases)
Figure 2 show s th a t th e ra tio o f ca ro ten o id s and c h lo ro p h y ll rem ained stable fo r

all th e phases. The expected change in p ig m e n ta tio n at d yna m ica lly changing
80
oxygen co n ce n tra tio n s was n o t observed, in d ic a tin g th a t th e cells did n o t show

any o xid a tive stress responses a t th e a pplied oxygen and lig h t co nd itio ns.
5.4. Conclusions
The e ffe c t o f dyna m ic oxygen co n ce n tra tio n s and lig h t/d a rk regim e o f high ligh t
on g ro w th o f N eochloris o le oabundans was e valuated. As in th e previous w o rk
u n d e r lo w lig h t in te n sity, th e gradual increase o f th e p a rtia l oxygen c o n c e n tra tio n
up to 0.63 bar, fo llo w e d by p e rio d o f rapid degassing did n o t b ring sig n ifica n t
decrease in th e specific g ro w th rate. A d d itio n a lly w h e n th e algae w e re exposed
fo r 300 m in u te s a t high oxygen co n c e n tra tio n , th e re was no s ig n ific a n t change in
th e specific g ro w th ra te o f th e algae N eochloris oleoabundans. These results
in d ica te th a t th e algae do n o t experience th e expected p h o to -o x id a tiv e in h ib itio n
caused by high oxygen co n c e n tra tio n in co m b in a tio n w ith high ligh t, as long as th e
oxygen is rem ove d via reg ula r degassing. But th e y also show th a t th e te m p o ra ry
exposure to a ccum ula ting oxygen co n ce n tra tio n s in th e solar receiver has less
im p a ct on th e g ro w th rate th a n th e residence tim e o f th e algae in th e dark zone o f
th e degasser. This indicates th a t th e num ber o f degassers in large-scale
p ro d u c tio n o f algae can be reduced w ith o u t severe loss o f biom ass due to
p h o to re s p ira tio n and p h o to -o x id a tio n . M o re o v e r, decreasing th e num ber of
degasser w ill lead to increased p ro d u c tiv ity , as th e algae spend re la tiv e ly sm aller
tim e in th e d a rk zone o f th e degasser.
discussions and th e ir fin a n cia l su p p o rt.
81
C hapter 5
5.6. References
A m aro, H .M ., M acedo, A.C., M alcata, F.X. 2012. M icroalgae: An a lte rn a tiv e as

H um an N u tritio n . Science, 298 (5601 ), 2149 - 2153
D ubinsky, Z., Stam bler, N. 2009. P h otoa cclim atio n processes in p h y to p la n k to n :

m echanism s, consequences, and app lica tion s. A q u a tic M ic ro b ia l Ecology, 56(2-3),
163-176.
Gong, Y.M ., Jiang, M.L. 2011. Biodiesel p ro d u c tio n w ith m icroalgae as fe ed sto ck:
fro m strains to biodiesel. B io tech n olog y Letters, 33(7), 1269-1284.
G u illard, R.R.L., R yther, J.H. 1962. Studies o f m a rin e p la n k to n ic d iatom s. /.

Kliphuis, A.M.J., Janssen, M ., van den End, E.J., M arte ns, D.E., W ijffe ls , R.H. 2011.
Light re sp ira tio n in C hlorella so ro kin ian a. Journal o f A pplie d Phycology, 1-13.
L ich te nth a le r, H.K. 1987. [34] C hlo ro ph ylls and ca ro ten o id s: Pigm ents of
p h o to s y n th e tic b io m e m bran es, in: M e th o d s in Enzym ology, (Ed.) R.D. Lester
Packer, Vol. V o lu m e 148, A cadem ic Press, 350-382.
M o lin a , E., Fernandez, F.G.A., Chisti, M.Y. 2001. T ub ula r p h o to b io re a c to r design

N orsker, N.H., Barbosa, M.J., V erm uë, M .H ., W ijffe ls , R.H. 2011. M icroalgal
p ro d u c tio n - A close loo k a t th e econom ics. B io tech n olog y Advances, 29(1), 24-27.
Pirt, S.J., Lee, Y.K., W alach, M.R., W a tts Pirt, M., Balyuzi, H .H.M ., Bazin, M.J. 1983.
A tu b u la r b io re a c to r fo r p h o to s y n th e tic p ro d u c tio n o f biom ass fro m carbon
d io xide : Design and p e rfo rm a n ce . Journal of Chemical T echnology and
B iotechnology, 33B, 35-58.
82
Singh, R.N., Sharma, S. 2012. D eve lop m e nt o f su ita b le p h o to b io re a c to r fo r algae

p ro d u c tio n - A review . R enew able and Sustainable Energy Reviews, 16(4), 2347-
2353.

Sousa, C., C om padre, A., V erm uë, M .H., W ijffe ls , R.H. 2013a. Effect o f oxygen at
lo w and high lig h t in te n sitie s on th e g ro w th o f N eochloris oleoabundans. Algal
Research, 2(2), 122-126.
Sousa, C., Valev, D., V e rm u ë, M .H ., W ijffe ls, R.H. 2013b. Effect o f D ynam ic Oxygen
C on cen tratio ns th e g ro w th of N eocholoris oleoabundans. A ccepted fo r
p u b lica tio n .
T orzillo , G., Carlozzi, P., Pushparaj, B., M o n ta in i, E., M aterassi, R. 1993. A tw o -

plane tu b u la r p h o to b io re a c to r fo r o u td o o r c u ltu re o f Spirulina. B io tech n olog y and
Tredici, M.R., Z itte lli, G.C. 1998. Efficiency o f su n lig h t u tiliz a tio n : T ub ula r versus
fla t p h o to b io re a c to rs . B io tech n olog y and B ioengineering, 57(2), 187-197.
W ijffe ls , R.H., Barbosa, M.J. 2010. An o u tlo o k on m icroalgal b io fu els. Science,

329(5993), 796-799.

o f b ulk chem icals and biofuels. Biofuels, B ioproducts and B iorefining, 4(3), 287-
295.
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84
Oxygen p ro d u c tio n in p h o to b io re a c to rs - A lo o k to th e econom ics
Chapter 6 . O x y g e n p r o d u c t io n in p h o t o b io r e a c t o r s
A LOOK TO THE ECONOMICS

Claudia Sousa 2, N iels-H enrik N orsker2,3, M aria Barbosa3' 4, M arian V e rm u ë 2'3,
Rene H. W ijffe ls 2,3
the Netherlands
3 Wageningen UR, AlgaePARC, www.AlgaePARC.com
4 Food and Biobased Research, Wageningen UR, Bornse Weilanden 9, 6708 WG,
Wageningen, the Netherlands
6.1. Introduction
N a tu ra lly o il-ric h m icroalgae like N eochloris o le oabundans fo rm an a ttra c tiv e
ren ew a ble source fo r b io fu e l p ro d u c tio n . The o u td o o r large-scale p ro d u c tio n is
te ch n ica lly fe asib le b u t still faces m a jo r challenges conce rn ing th e e con om ic
fe a s ib ility and th e energy balance (Cheng & T im ilsina, 2011; N orsker e t al., 2011;
Stephens e t al., 2010a; Stephens e t al., 2010b). .
For c u rre n t o u td o o r p ro d u c tio n tw o m a jo r algae c u ltiv a tio n system s can be

d istin gu ishe d; open and closed systems. Open racew ay ponds are th e m ost used
system s w o rld w id e , because th e y are cheap in inve stm e n ts and o p e ra tio n costs.
In a d d itio n , th e energy balance fo r biom ass p ro d u c tio n in th ese system s is
p ositive (N orsker e t al., 2011). The m a jo r d ra w b a ck is th a t th e y are vu ln e ra b le fo r
c o n ta m in a tio n and can o n ly be used fo r p ro d u c tio n o f fa s t g ro w in g algae o r algae
species th a t can g ro w u n d e r e xtre m e co n d itio n s, like high salt o r high pH, th a t
p re ve n t invasions by co n ta m in a n ts (A m aro e t al., 2012; Pulz, 2001). A n o th e r
disadvantage is th a t th e y o p e ra te a t lo w biom ass densities w hich m ake harvesting
o f th e cells energy d em an din g and co stly (Salim e t al., 2012).
In closed p h o to b io re a c to r system s (PBR) hig he r biom ass densities can be achieved

w ith sm aller risk fo r co n ta m in a tio n , b u t so fa r, no p o sitive energy balance is
achieved in th ese system s and th e costs fo r p ro d u c tio n are still to o high
85
C hapter 6
(Dism ukes e t al., 2008; N orsker e t al., 2011; W ijffe ls & Barbosa, 2010; W ijffe ls et
al., 2010). Especially th e high e nergy in p u t re q u ire d fo r m ixing s till fo rm s a m a jo r
b o ttle n e c k (N orsker e t al., 2011). The m ixing in closed PBR's is needed to p ro vid e
th e algae w ith s u ffic ie n t lig h t and carbon d io xide and to rem ove th e
p h o to s y n th e tic a lly pro du ced oxygen. If n o t rem ove d, th e a ccum ula ting oxygen
w ill in h ib it th e g ro w th o f th e algae via p h o to re s p ira tio n and w ill cause p h o to -
o xid a tive dam age as a re su lt o f p h o to in h ib itio n , especially a t high lig h t in te n sity.
There are ind icatio ns, h ow eve r, th a t th e energy needed fo r m ixing can be reduced
considerably. (N orsker e t al., 2011). Sousa e t al. (2012) proved th a t a d d itio n o f
e xtra b ica rb o n a te to th e m ed iu m is a good m e th o d to o vercom e p h o to re s p ira tio n
and by d oing so th e algae can w ith s ta n d high oxygen co n c e n tra tio n s a t lo w ligh t
in te n sitie s (N orsker e t al., 2011; Sousa e t al., 2012). This im plies th a t less energy
w ill be needed fo r degassing to rem ove th e surplus o f oxygen.
It was also fo u n d th a t algae can in fa c t w ith s ta n d lon ge r exposure to e levated

oxygen co n ce n tra tio n s, as long as th e oxygen is fre q u e n tly rem ove d (Sousa e t al.
2013b). This m eans th a t th e energy needed fo r degassing can be fu rth e r reduced.
Here, an o ve rvie w o f th e a b o ve -m e n tio n e d studies on e ffe cts o f oxygen on th e
algal g ro w th and th e p roposed m eth od s to reduce th e energy in p u t fo r rem oval o f
th e oxygen w ill be p ro vid e d . The e ffe cts o f im p le m e n tin g th e p roposed m eth od s
on th e overall energy re q u ire m e n ts and costs w ill be calculated, to v e rify if indeed
th ese m eth od s w ill c o n trib u te to a p ositive energy balance and a considerable
re d u ctio n o f th e algal biom ass cost.
6.1.1. Effects of oxygen on microalgal grow th

In closed p h o to b io re a c to rs th e oxygen p roduced d u rin g pho tosyn th esis
accum ulates to high co n ce n tra tio n s and induces processes like p h o to in h ib itio n
and p h o to re s p ira tio n , b o th leading to a decrease o f th e yield on lig h t o f th e
m icroalgae (Torzillo e t al., 1998).
86
6 .1 .1 .1 . P h o to re s p ira tio n
- ' * ■ 2 Ribulose 1,5-bP Figure 1 shows the biochemical

reactions involved in the
2 CO; 2 0:
photorespiratory pathway.
Instead o f tw o molecules o f 3-
phosphoglycerate (3-PGA), the
2 Glycolate Glycerate-3P reaction of ribulose bi-phosphate
w ith 0 2 yields one molecule o f 3-
2 NADH ATP + NADH
PGA and one molecule o f 2-
2 Glyoxylate Hydroxypyruvate
4 ATP +■4 NADPH
phosphoglycolate (2-PG). The full
photorespiratory cycle serves as
Pyruvate a carbon recovery system
NADH
converting part o f the 2-PG to 3-
4 GAP 2 Glycine Serine PGA tha t can re-enter the
reductive cycle. A fte r 2-PG is
dephosphorylated and the
Figure 1 -Sim plified scheme o f ph otosynthetic (a) and
form ed glycolate is converted
photorespira tory pathw ays (b) adapted fro m the m etabolic
ne tw ork described by Kliphuis e t al. (2011). into glycine, glycine is
decarboxylated and deaminated
fo r fu rth e r serine synthesis and glycerate form ation. The transport o f glycerate and its
phosphorylation to 3-PGA completes the photorespiratory pathway (Foyer et al., 2009; Tchernov et
al., 2008; Tural & Moroney, 2005; W ingler et al., 2000). During photorespiration, C 02 and
amm onium (NH4+) are lost and th e ir re-fixation requires additional ATP and NADPH. This means that
less energy is available fo r growth and the biomass yield on light energy w ill decrease when
photorespiration occurs (Kliphuis et al., 2011). The photorespiratory pathway thus has an influence
on the photosynthetic yield which can be defined as the am ount of C 02 fixed per am ount o f light
energy absorbed and, as such, w ill directly influence the productivity of microalgae cultures.
A t su b-satu ratin g lig h t in te n sitie s th e g ro w th rate o f m icroalgae decreases due to

p h o to re s p ira tio n (Figure 1). If th e ra tio b e tw e e n 0 2 and C 0 2 in th e m edium
increases, th e oxygenase a c tiv ity o f th e enzym e Rubisco associated w ith
re sp ira tio n increases w h ile its carboxylase a c tiv ity associated to pho tosyn th esis
ceases. This p h o to re s p ira tio n e ffe c t was indeed fo u n d fo r N eochloris
o le oabundans cu ltu re d a t su b -sa tu ra tin g lig h t in te n s ity upon increasing th e
oxygen co n c e n tra tio n in th e m ed iu m fro m 0.21 to 0.84 p a rtia l pressure (Sousa e t
al., 2012). To o vercom e th e in h ib ito ry e ffe c t o f oxygen, th e C 0 2 c o n c e n tra tio n was
increased by th e a d d itio n o f e xtra N aH C 03 to th e c u ltu re m ed iu m . Once th e
b ica rb o n a te (N aH C 03) co n ce n tra tio n and th e c o rresp on din g C 0 2 c o n c e n tra tio n
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C hapter 6
w e re increased, th e specific g ro w th ra te o f th e algae aug m e nted again. This

show s th a t th e negative e ffe c t oxygen can be o vercom e by re s to rin g th e 0 2/ C 0 2
ra tio by an increase o f th e carbon d io xide p artial pressure. By increasing th e
N aH C 03 c o n te n t in th e m ed iu m , th e oxygen is th u s a llo w e d to a ccum ula te u n til
p a rtia l pressure o f 0.84 bar is reached, w ith o u t c o m p ro m isin g on p ro d u c tiv ity .
A d d itio n o f extra N aH C 03 could th e re fo re reduce th e energy needed fo r rem oval
o f th e oxygen by degassing.
6 .1 .1 .2 . P h o to in h ib itio n an d p h o to -o x id a tiv e d a m a g e
A t high and o ve r-sa tu ra tin g lig h t in te n sitie s a d d itio n a l p h o to in h ib itio n e ffects

w e re expected. A t th o se co n d itio n s th e fo rm a tio n o f oxygen radicals and o th e r
rea ctive oxygen species (ROS) such as H 20 2 happens (M u ra ta e t al., 2007). In
a d d itio n , hig hly reactive singlet oxygen is fo rm e d via p h o to -a c tiv a tio n
(T rian ta ph ylid es e t al., 2008) and causes p h o to -o x id a tiv e dam age, re su ltin g in a
loss o f p h o to s y n th e tic a c tiv ity and d eath o f cells. The specific g ro w th rate o f
N eochloris oleobundans cu ltu re d at n e a r-sa tu ra tin g lig h t c o n d itio n s indeed
d ra m a tic a lly decreased fro m 1.36 day"1 a t co n sta n t Po2 o f 0.21 to 0.68 day"1 a t Po2
o f 0.84. C on trary to w h a t happened a t su b-satu ratin g lig h t inten sitie s, an increase

o f th e PCo2 fro m 0.007 to 0.02 bar a t Po2 o f 0.84 bar did n o t have any p ositive
e ffe c t on th e overall g ro w th o f th e algae. A t th ese high lig h t co n d itio n s,
p h o to in h ib itio n seem to d o m in a te th e p h o to re s p ira tio n e ffe cts and th e overall
in h ib ito ry e ffe c t o f oxygen could n o t be o vercom e by a d d itio n o f e xtra carbon
dioxide.
One should realize th a t m icroalgae cu ltu re d in closed p h o to b io re a c to rs a t high

cell densities m ainly e n co u n te r lo w lig h t co n d itio n s. In p a rtic u la r in vertica l
stacked tu b u la r p h o to b io re a c to rs o r in ve rtica l fla t-p a n e l reactors, th e lig h t is
d ilu te d . W h e n th e algal biom ass d e n sity is high enough to p re v e n t lig h t co n d itio n s
th a t evoke a d d itio n a l, p h o to -in h ib itio n effects, th e in h ib ito ry e ffe c t o f oxygen can
be o vercom e by extra N aC 03 a d d itio n to th e m ed iu m . D uring th e s ta rt-u p o f a
c u ltu re , w h e n th e c u ltu re is still d ilu te d , h ow eve r, p h o to -o x id a tiv e dam age is
bound to occur and a d d itio n o f extra N aH C 03 w ill n o t help to reduce th e
in h ib ito ry effects.
88
6.1.2. Effects of (dynam ic) accum ulating oxygen in closed

photobioreactors
In closed tu b u la r p h o to b lo re a c to r (PBR) th e algae do n o t experience a co n sta n t
oxygen p a rtia l pressure b u t th e y are su b je ct to changing oxygen c o n ce n tra tio n s.
The algae experience an Increase In oxygen c o n c e n tra tio n along th e length o f th e
tubes, and th is m ay Induce re d u c tio n o f th e g ro w th ra te along th e tu b e s (Ugwu e t
al., 2008). In a d d itio n , a degassing u n it Is placed a t th e end o f th e tu b e to rem ove
th e a ccum ula ted oxygen w h ile w h ich th e algae are d e p rive d fro m th e ligh t. This
dyna m ic change In oxygen co n ce n tra tio n In th e tu b e s and th e lig h t co n d itio n s
e n co u n te re d d u rin g 6 m in utes degassing w e re sim u la te d In a fu lly c o n tro lle d
closed p h o to b lo re a c to r and th e g ro w th rate o f N eochloris oleobundans at
tu rb ld o s ta t co n d itio n s was m easured. This g ro w th rate was co m p ared w ith th e
g ro w th rate o f th e algae th a t w e re subjected to a te n tim e s lon ge r exposure tim e
(300 m in utes) to high oxygen co n ce n tra tio n s. Surprisingly, th e algae did n o t su ffe r
fro m th e extension o f th e residence tim e a t high oxygen c o n ce n tra tio n s. The
specific g ro w th rate was n o t a ffe cte d by th e d yna m ica lly changing oxygen
c o n ce n tra tio n , b u t o nly by th e fre q u e n t exposure to dark p eriods e n co u n te re d
d u rin g degassing. A sig n ifica n t decrease o f th e algae specific g ro w th was observed
w h e n a pplying a lig h t regim e o f 30 m in utes lig h t "O n " and 6 m in u te s lights "O ff".
The results o f th is stu dy show th a t It Is possible to Increase th e residence tim e In

th e solar receiver considerably. This can be done by decreasing th e v e lo c ity In th e
tu b e s and by reducing th e n u m b e r o f degassing units. Decreasing th e v e lo c ity In
th e tu b e s w ill re su lt In substa ntia l energy and costs savings In design and
o p e ra tio n o f plants fo r m icroalgae p ro d u c tio n w h ile reducing th e a m o u n t o f
degassing u nits to m in im ize th e tim e o f darkness to w h ich th e algae are exposed,
m ay re su lt In hig he r p ro d u c tiv ity The tu rb u le n c e created by th e v e lo c ity Is
Im p o rta n t to avoid th e risk o f algae d e p o sitio n In th e tu b e w alls w hich can lead to
b lo fo u lln g . W h e n reducing th e ve lo city, th e cost w ill be reduced b u t one should
be aw are th a t th e risk o f b lo fo u lln g w ill Increase.
89
C hapter 6
6.2. Reducing the costs for degassing will reduce the overall
costs
The p resented studies in d ica te th a t th e energy re q u ire m e n ts fo r rem o vin g oxygen
by degassing in closed p h o to b io re a c to rs can be s u b s ta n tia lly reduced. A t co n sta n t
su b -sa tu ra tin g lig h t in te n sitie s it is possible to o p e ra te a t oxygen c o n c e n tra tio n o f
4 tim e s air sa tu ra tio n by re sto rin g th e C 0 2/ 0 2 ra tio th ro u g h increasing PCo2-
U nder dyna m ic oxygen co n ce n tra tio n s, th e in h ib ito ry e ffe c t o f oxygen on th e
specific g ro w th rate o f N eochloris oleoabundans was n o t fo u n d . M o re o v e r; th e
residence tim e o f th e algae a t high oxygen co n c e n tra tio n could be increased up to
10 tim e s w ith o u t scrutin izin g th e g ro w th rate. In fa c t, th e exposure o f th e algae
cells to d ark periods in th e degasser had a bigger negative im p a c t th a n th e
exposure to high oxygen co n c e n tra tio n as such.
6.2.1. Biomass productivity costs - base case

From th e analysis o f th e above m e n tio n e d studies, it was concluded th a t th e
energy in p u t fo r m ixing and degassing in tu b u la r p h o to b io re a c to r systems could
co nside rab ly be reduced. The e ffe cts o f reducing th e energy in p u t on th e overall
energy balance as w e ll as on th e o verall p ro d u c tio n costs w e re e valuated using
th e e con om ic m odel deve lo pe d by N orsker e t al. (2011). The m odel was
deve lo pe d to calculate th e energy and costs associated to m icroalgal biomass
p ro d u c tio n in th e N ethe rla nd s fo r th re e d iffe re n t system s a t 100 ha scale. One o f
th e system s was th e h o rizo n ta l tu b u la r p h o to b io re a c to r. This analysis resu lted in a
cost price o f 4.15 € per Kg o f biomass, and a negative n e t energy balance (25.5 MJ
kgDW "1) fo r p ro d u c tio n o f algae biom ass in th ese system s. All calculations w e re
based on th e assum ptio n th a t th e p h o to s y n th e tic e fficie n cy in th e tu b u la r system
was 3% resu ltin g in a areal p ro d u c tiv ity o f 41.41 to n ha"1 yr"1 (N orsker e t al., 2011).
This value w ill be used as o u r base case.
6.2.2. Effect o f increasing th e C 0 2/ 0 2on biomass production costs

In th e m odel th e areal p ro d u c tiv ity was used as in p u t p a ra m e te r. The areal
p ro d u c tiv ity was calculated using th e m easured biom ass yield on lig h t YXiPh (g m o l-
ph"1) th a t w as m easured in th e p revious studies (Sousa e t al., 2012), co m b in e d
w ith th e ye a rly solar in p u t o f lig h t in th e N etherlands. The European Database o f
90
D aylight and Solar R adiation re p o rts a to ta l solar ra d ia tio n o f 3 .6 2 x l0 13 J h a 'V e a r'1

in th e N ethe rla nd s and 42.3% o f th is solar irra dian ce can be used fo r
p ho tosyn th esis (PAR, 400-700 nm w ave len gth ), resu ltin g in an average irra dian ce
o f 1 .5 3 x l0 13 J ha"1 year"1 on PAR ligh t. Assum ing an average w a ve le n g th o f 550
nm , th e average energy c o n te n t o f one m ole o f p h o to n s in th e PAR range o f lig h t
is 2.18x10s J m ol-ph "1. This m eans an average ye arly p h o to n flu x o f a b o u t 7 .0 2 x l0 7
m oles o f PAR p ho ton s per ha. In o u r lab-scale e xp e rim e n ts a t su b-satu ratin g lig h t
co n d itio n s and a p a rtia l oxygen pressure o f 0.21 bar, a biom ass yield YXiPh o f 1.04 g
m ol-ph "1 was fo u n d (Sousa e t al., 2012). C om bined w ith th e average ye a rly p h o to n
flu x o f 7.02 X IO 7 m oles o f PAR p h o to n s per ha, th is can be tra n s la te d in an aerial
p ro d u c tiv ity o f 73 to n .h a ^.y e a r"1. It is o p p o rtu n e to m e n tio n th a t th e specific
g ro w th rates re p o rte d by Sousa e t al. (2012) are co m p arab le w ith th e g ro w th
rates fo u n d by Pruvost e t al. (2009). The biom ass yields m easured by Sousa e t al.
(2012) w e re nevertheless hig he r th a n th o se fo u n d by Pruvost e t al. (2009). This
show s th a t N. o le oabundans e xhibits high biom ass yields w h e n g ro w n on a m arine
salt w a te r m ed iu m o r on a fre s h w a te r BBM m ed iu m as used by Pruvost and
cow o rke rs. This o u tco m e is n o t su rp rising since N. o le oabundans (UTEX 1185) has
been isolated fro m an arid soil (Guiry, 2011). It is th e n necessary to ta ke th is in
c o n sid e ra tio n w he n loo king a t th e results o f th is study. The areal p ro d u c tiv itie s
calculated fro m th e m easured biom ass yields g re a tly exceed th e value used in th e
base case. The values fo r th e areal p ro d u c tiv itie s w e re s u b s titu te d in th e
e con om ic m odel and th e biom ass p ro d u c tio n costs and energy w e re calculated
fo r an algal p ro d u c tio n fa c ility w ith 100 hectares o f tu b u la r p h o to b io re a c to rs . It is
necessary to ta ke in to a ccou nt th e m easured biom ass yield on lig h t energy. The
biom ass yield on lig h t energy m easured a t low p a rtia l oxygen pressure was 1.04 g
m ol-ph "1 (P o2=0.21 bar | PCo2=0.007 bar) b u t d ro p p e d to 0.73 g m ol-ph "1 a t high
p a rtia l oxygen pressure (PO2=0.84 bar | PCo2=0.007 bar). Upon a d d itio n o f N aH C 03

it increased again to 0.92 g .m o l-p h"1 (PO2=0.84 bar | PCo2=0.02 bar) resu ltin g in an
areal p ro d u c tiv ity o f 65 to n .h a '^ y r'1. To achieve th e p ro d u c tiv ity o f 65 to n ha"1 yr"1
u n d e r a p a rtia l oxygen pressure o f 0.84 bar (400% air s a tu ra tio n ) th e C 0 2 p artial
pressure was increased fro m 0.007 bar to 0.02 bar by increasing th e N aH C 03
c o n c e n tra tio n in th e m ed iu m (Sousa e t al., 2012). The a d d itio n a l costs fo r th e
e xtra C 0 2 should be ta ke n in to a ccou nt w he n d e te rm in in g th e o verall costs. The
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o verall biom ass p ro d u c tio n cost using 3 tim e s m o re carbon d io xide b u t w o rk in g at

Po2=0.84 bar w ith N eochloris o le oabundans w ith a biom ass yield on lig h t energy
u n d e r th is circum stances o f 0.92 g m ol-ph "1 is 3.60 € per kg. This is 13% less th a n
calculated fo r th e base case (4.15 € per kg). The n e t e nergy balance in these
circum stances im p ro ve d b u t is still negative (-6.73 MJ kgDW"1).
6.2.3. Biomass productivity costs - increase th e length o f the tubes

/ decrease the velocity in the tubes.
In th e lab-scale e xp e rim e n ts in w hich w e m im icked th e d yna m ica lly changing
oxygen co n c e n tra tio n th a t th e algae experience in th e tu b u la r system , w e
observed th a t th e g ro w th rate was h a rd ly a ffe cte d and th a t th e algae are able to
w ith s ta n d 10 tim e s lon ge r residence tim e s in th e tu b e s a t e levated oxygen
co n ce n tra tio n s. The residence tim e in th e tu b e can be increased by lo w e rin g th e
v e lo c ity o r by increasing th e length o f th e tubes. Both m eth od s w ill lead to a
decrease in th e a m o u n t o f energy needed fo r m ixing and degassing and in th e
to ta l costs fo r algae p ro d u c tio n . Decreasing th e v e lo c ity in th e tu b e s w ill increase
th e tim e w h ich th e algae are in th e solar receiver and w ill a llo w th e oxygen to
build up to h ig he r values. Increasing th e length o f th e tu b e s w ill as w e ll increase
th e tim e in th e solar receiver and a llo w th e oxygen to b u ild -u p to high
co n ce n tra tio n s. Taking in co n sid e ra tio n th a t th is w ill n o t a ffe c t th e g ro w th o f
N eochloris oleoabundans as seen by th e w o rk on dyna m ic oxygen c o n c e n tra tio n s
w e calculated th e biom ass p ro d u c tio n cost u n d e r th ese circum stances. W hen
increasing th e len gth o f th e tu b e s w h ile keeping th e same v e lo c ity in th e tubes,
th e biom ass p ro d u c tio n cost h ardly changed. The biom ass p ro d u c tio n cost o nly
decreased fro m 4.15 € per kg to 4.12C per kg. A lth o u g h Sousa e t al (2013b) state
th a t a lO x increase o f th e tim e to w h ich th e algae are exposed to sun is possible,
th e v e lo c ity in th e tu b e s was o n ly decreased fro m 0.5 m s"1 to 0.25 m s"1 as a to o
high re d u ctio n o f th e v e lo c ity m ig h t re su lt in p ro ble m s such as b io film fo rm a tio n
on th e re a c to r w alls. Decreasing th e ve lo c ity by a fa c to r o f tw o results in a
decrease on th e biom ass p ro d u c tio n cost o f 4.15 € to 2.84 € per kg. This 32 %
re d u ctio n on th e cost o f biom ass p ro d u c tio n is m ainly due to th e re q u ire d a m o u n t
o f circu la tio n pum ps and decreased energy re q u ire m e n t. In th e base case analysis
a negative n e t energy balance (-25.5 MJ kgDW"1) was fo u n d . In th e p re sen t case
92
th e decrease o f th e v e lo c ity im p lica te s a d ra stic re d u c tio n o f th e energy re q u ire d

fo r re circu la tio n resu ltin g in a p ositive n e t energy balance (+4.2 MJ kgDW "1).
The e va lu a tio n o f th e dyna m ic oxygen co n c e n tra tio n s in an o u td o o r p ilo t scale

p h o to b io re a c to r to va lid a te th e results fo u n d a t lab-scale w o u ld be an in te re s tin g
fo llo w up to th is research. It w o u ld be in te re s tin g to in ve stiga te to w h ich e x te n t
th e ve lo c ity in th e tu b e s can be decreased to m axim ize th e energy balance
w ith o u t excessive p ro ble m s w ith b io fo u lin g . In th e e x p e rim e n ts a t dynam ic
oxygen co n ce n tra tio n s, th e d ark p eriods a t w hich th e algae w e re exposed w e re
linked to th e decrease o f th e specific g ro w th rate. It w o u ld be in te re s tin g to stu dy
th e e ffe c t on th e energy balance o f a co m b in a tio n o f a decrease o f th e v e lo c ity in
th e tu b e s and o f an increase in th e degassing capacity o f th e degasser to reduce
th e d ark tim e a t w h ich th e algae are exposed.
6 .3 . Conclusions
The tw o m eth od s a d o p te d to o vercom e th e negative e ffe c t o f oxygen in
m icroalgal cu ltu re s did re su lt in a decrease in biom ass p ro d u c tio n costs. A t
c o n sta n t oxygen c o n ce n tra tio n , w ith expense o f using m ore carbon d io xide th e
biom ass p ro d u c tio n cost w e re reduced by 13 % and did im p ro v e th e n et energy
balance, a lth o u g h it is still negative.
U nder dyna m ic oxygen co n c e n tra tio n th e ve lo c ity can be decreased fro m 0.5 m s"1
to 0.25 m s"1 w hich results in a 32% saving on th e biom ass p ro d u c tio n cost b u t
m o re im p o rta n t, it w ill re su lt in a p ositive energy balance fo r tu b u la r
p h o to b io re a c to rs . This e va lu a tio n was done w ith o u t ta k in g in co nside ratio n
m eth od s to avoid b io film d e p o sitio n on th e p h o to b io re a c to rs w alls, w h a t w o u ld
be le ft here as a suggestion fo r fu rth e r research. Evaluation o f th e dynam ic
oxygen co n ce n tra tio n s in an o u td o o r p ilo t scale p h o to b io re a c to r in o rd e r to
v a lid a te th e results fo u n d a t lab-scale w o u ld be an in te re s tin g fo llo w up to th is
research.
6 .4 . R eferences
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Cheng, J.J., T im ilsina, G.R. 2011. Status and barrie rs o f advanced b io fu e l

te chn olo gie s: A review . R enew able Energy, 36(12), 3541-3549.
Foyer, C.H., Bloom , A.J., Q ueval, G., N octor, G. 2009. P h o to re s p ira to ry

m e ta b o lism : genes, m uta nts, energetics, and red ox signaling. A nnual Review o f
Plant Biology, 60(1), 455-484.
G uiry, W ., 2011. In G uiry, M .D. and G uiry, G.M . AlgaeBase. W o rld -w id e e le ctro n ic
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Norsker, N.-H., Barbosa, M.J., V e rm u ë, M .H., W ijffe ls , R.H. 2011. M icroalgal

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Pulz, O. 2001. P h o to b io re a cto rs: p ro d u c tio n system s fo r p h o to tro p h ic

m icroorganism s. A p plie d M ic ro b io lo g y and B iotechnology, 57, 287-293.
Salim, S., V erm uë, M .H ., W ijffe ls, R.H. 2012. Ratio b etw e e n a u to flo c c u la tin g and
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Sousa, C., C om padre, A., V e rm u ë, M .H., W ijffe ls , R.H. 2013a. Effect o f oxygen at
lo w and high lig h t in te n sitie s on th e g ro w th o f N eochloris oleoabundans. Algal
Research, 2(2), 122-126.
Sousa, C., Valev, D., V erm uë, M .H ., W ijffe ls, R.H. 2013b. Effect o f D ynam ic Oxygen
C on cen tratio ns th e g ro w th of N eocholoris oleoabundans. A ccepted fo r
p u b lica tio n .
Stephens, E., Ross, I.L., King, Z., M ussgnug, J.H., Kruse, O., Posten, C., B orow itzka,
M .A., H ankam er, B. 2010a. An e con om ic and te ch n ica l e v a lu a tio n o f m icroalgal
b iofuels. N at Biotech, 28(2), 126-128.
Stephens, E., Ross, I.L., M ussgnug, J.H., W agner, L.D., B orow itzka, M .A., Posten, C.,
Kruse, O., H ankam er, B. 2010b. Future prospects o f m icroalgal b io fu e l p ro d u c tio n
system s. T rends in Plant Science, 15(10), 554-564.
Tchernov, D., Livne, A., Kaplan, A., Sukenik, A. 2008. The k in e tic p ro p e rtie s o f
rib u lo s e -l,5 -b is p h o s p h a te carboxylase/oxygenase m ay explain th e high a p p a re n t
p h o to s y n th e tic a ffin ity o f N annochloropsis sp. to a m b ie n t ino rg a nic carbon. Israel
Journal o f Plant Sciences, 56(1-2), 37-44.
T orzillo, G., B ernardini, P., M asojidek, J. 1998. O n-line m o n ito rin g o f c h lo ro p h yll
high oxygen co n c e n tra tio n and lo w te m p e ra tu re and its e ffe c t on th e p ro d u c tiv ity
o f o u td o o r cu ltu re s o f S pirulina p la te nsis (cyanobacteria). Journal o f Phycology, 34
5 0 4 -5 1 0
148(2), 960-968.
Tural, B., M o ro n e y, J.V. 2005. R egulation o f th e expression o f p h o to re s p ira to ry

genes in C hlam ydom onas re in h a rd tii. Canadian Journal o f Botany, 83(7), 810-819.
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Ugwu, C.U., Aoyagi, H., Uchiyam a, H. 2008. P h o to b io re a cto rs fo r mass c u ltiv a tio n
o f algae. Bioresource Technology, 99(10), 4021-4028.
W ijffe ls , R.H., Barbosa, M.J. 2010. An o u tlo o k on m icroalgal b io fu els. Science,

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295.
W in g le r, A., Lea, P.J., Quick, W.P., Leegood, R.C. 2000. P h o to re s p ira tio n : m e ta b o lic
p athw ays and th e ir role in stress p ro te c tio n . Philosophical Transactions o f th e
Royal Society B-Biological Sciences, 355(1402), 1517-1529.
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Su m m a r y
P h o to tro p ic m icroalgae are regarded as a p ro m ising fe e d sto ck fo r sustainable

biodiesel p ro d u c tio n , as m icroalgae can use n a tu ra l s u n lig h t as lig h t source and
are able to u tilize C 0 2 fro m flu e gases and n u trie n ts (P, N) fro m w aste stream s. To
m ake large-scale o u td o o r m icroalgae p ro d u c tio n in closed p h o to b io re a c to rs
e co n o m ica lly fe asib le and sustainable, th e costs fo r m ixing and degassing should
be reduced and th e overall energy balance should becom e p ositive. The m ixing is
needed fo r e ffe ctive and e ffic ie n t supply o f ligh t, p ro visio n o f carbon d io xide and
degassing is needed fo r th e rem oval o f p h o to s y n th e tic a lly g e n erated oxygen.
This thesis focused on th e e ffe c t o f th e a ccu m u la tio n o f oxygen on th e g ro w th o f

th e oleaginous m icroalga N eochloris o le oabundans a t d iffe re n t lig h t inten sitie s.
These studies show a t w h a t co n ce n tra tio n s oxygen becom es to x ic fo r th e algae at
th e d iffe re n t lig h t co n d itio n s e n co u n te re d d u rin g o u td o o r c u ltiv a tio n . This reveals
w h e n th e oxygen should be rem ove d fro m th e p h o to b io re a c to r and th u s th e need
fo r degassing.
The d iffe re n t oxygen levels reached in o u td o o r tu b u la r p h o to b io re a c to rs have

been im posed on N eochloris o le oabundans w h ile c u ltu re d in a fu lly c o n tro lle d
s tirre d ta n k rea ctor, o p e ra te d in tu rb id o s ta t. U nder c o n tin u o u s illu m in a tio n o f
200 p m o l m"2 s"1 (sub -sa tu ra ting lig h t in te n s ity ) th e g ro w th ra te o f N eochloris
o le oabundans a t th e th re e oxygen p a rtia l pressures (Po2= 0.24; 0.63; 0.84) was
1.38; 1.36 and 1.06 day"1 re sp ective ly was m easured. A t th e oxygen p artial
pressure o f 0.84 bar th e carbon d io xide p a rtia l pressure ( P Co2) was increased fro m
0.007 to 0.02 bar to see if th e in h ib itin g e ffe c t o f p h o to re s p ira tio n could be
o vercom e. Indeed, th e a d d itio n o f carbon d io xide resu lted in an increase o f th e
g ro w th rate fro m 1.06 to 1.36 d a y '1. The increase o f specific g ro w th rate
co n firm e d th a t p h o to re s p ira tio n was ta kin g place and th a t th is negative e ffe c t can
be o vercom e by re sto rin g th e C 0 2/ 0 2 ra tio . In Chapter 3 th e e ffe c t o f p artial
oxygen pressure on g ro w th o f N eochloris oleoabundans was stu die d a t near-
s a tu ra tin g lig h t in te n s ity in a fu lly -c o n tro lle d p h o to b io re a c to r. A t th e p artial
oxygen pressures te ste d (Po2= 0.24; 0.42; 0.63; 0.84 bar), th e specific g ro w th rate
was 1.36; 1.16; 0.93 and 0.68 d a y '1, respectively. An increase o f th e P C02 fro m
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S um m ary
0.007 to 0.02 bar a t Pq2 o f 0.84 bar a t th ese hig he r lig h t inten sitie s, h ow eve r, did
n o t show any p ositive e ffe c t on th e overall g ro w th o f th e algae, c o n tra ry to w h a t

happens at su b -sa tu ra tin g lig h t inten sitie s. These results in d ica te th a t at
s a tu ra tin g lig h t in te n s ity th e in h ib ito ry e ffe c t o f oxygen by p h o to re s p ira tio n
c a n n o t be o vercom e and th a t p h o to in h ib itio n e ffe cts prevailed. The ch lo ro p h y ll
c o n te n t o f N eochloris oleoabundans g ro w n a t 200 p m o l m"2 s'1 is a b o u t 1.9 tim e s
h ig he r th a n w he n cu ltiv a te d a t 500 p m o l m"2 s"1, w hereas th e ca ro te n o id c o n te n t
was a b o u t 1.6 lo w e r, b o th d e m o n s tra tin g p h o to a c c lim a tio n effects. The elevated
oxygen co n c e n tra tio n in th e g ro w th m ed iu m as such do n o t a ffe c t th e p ig m e n t
c o n te n t a t sub- and near sa tu ra tin g lig h t co n d itio n s. This indicates th a t elevated
oxygen co n ce n tra tio n s in th e m e d iu m do n o t c o n trib u te to m ore p h o to o xid a tive
dam age a t th e h ig he r lig h t co n d itio n s used, b u t th a t oxygen o n ly in h ib its th e
g ro w th via p h o to re sp ira tio n effects.
M icroalgae g ro w n in closed p h o to b io re a c to rs do n o t experience c o n s ta n t high

co n ce n tra tio n s o f oxygen. In closed tu b u la r p h o to b io re a c to rs , th e oxygen
co n ce n tra tio n s increase o ve r th e tu b e s due to p ho tosyn th esis and drops in th e
degasser w h e re th e surplus o f oxygen is rem oved. In a d d itio n , th e algae are
exposed to th e lig h t w h ile residing in th e tu b e s and are exposed to darkness in th e
degasser. In C h a p te r 4, th e d yna m ica lly changing oxygen co n c e n tra tio n s and
su bse qu en t lig h t c o n d itio n s w e re sim u late d in a CSTR a t fu lly c o n tro lle d
co n d itio n s a t su b-satu ratin g lig h t in te n s ity and th e e ffe c t on th e g ro w th o f
N eochloris o le oabundans was studied at n e a r-sa tu ra tin g lig h t in te n s ity . In
a d d itio n , th e e ffe c t o f a 10 tim e s e lo n g a tio n o f th e residence tim e a t in th e solar
rece ive r was inve stiga te d. In th is study, 3 d iffe re n t lig h t regim es w e re used:
co n tin u o u s lig h t; 30 m in u te s lig h t on fo llo w e d by 6 m in u te s lig h t o ff and 300
m in u te s lig h t on fo llo w e d by 6 m in utes lig h t o ff. The specific g ro w th rate
m easured a t co n sta n t lo w oxygen co n c e n tra tio n PO2=0.21 bar d u rin g th ese 3 lig h t
regim es w e re ; 1.14 ± 0.06 day"1; 0.80 ± 0.16 day"1 and 1.09 ± 0.05 day"1
respectively. The e ffe c t o f d yna m ica lly changing oxygen co n c e n tra tio n s fro m
Po2=0.21 bar to PO2=0.63 bar fo llo w e d by su bse qu en t degassing to PO2=0.21 bar
d u rin g th e d ark p e rio d resu lted in sim ila r specific g ro w th rates. The decrease o f
th e algae specific g ro w th observed w h e n a pplying d iffe re n t lig h t regim es, shows
th a t th e exposure o f th e algae cells to d ark periods in th e degasser has bigger
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n egative im p a ct th a n th e te m p o ra ry exposure to a ccum ula ting oxygen

c o n ce n tra tio n s in th e solar receiver. In c h a p te r 5 a s im u la tio n o f th e dynam ic
oxygen co n ce n tra tio n s fe lt by th e m icroalgae in tu b u la r p h o to b io re a c to rs u nd er
high lig h t in te n sitie s was studied as a fo llo w up o f c h a p te r 4. W h e n th e algae w e re
exposed to co n sta n t oxygen co n c e n tra tio n and c o n s ta n t high lig h t th e specific
g ro w ra te was 1.29 ± 0.08 day"1. Using a lig h t regim e o f 30 m in utes o f lig h t ON
fo llo w e d by 6 m in u te s lights OFF and degassing resulted in a specific g ro w th rate
o f 0.84 ± 0.09 day"1, th e e lo n g a tio n o f th e tim e (lights ON) to 300 m in u te s resulted
in 1.18 ± 0.05 d a y '1. W h e n d yna m ica lly changing oxygen c o n c e n tra tio n s w e re
a pplied, sim ila r specific g ro w th rates w e re o b ta in e d . These results in d ica te th a t
th e algae do n o t experience th e expected p h o to -o x id a tiv e in h ib itio n caused by
high oxygen co n c e n tra tio n in co m b in a tio n w ith high ligh t, as long as th e oxygen is
rem ove d via reg ula r degassing. The te m p o ra ry exposure o f th e algae to th e
darkness in th e degasser has m ore im p a ct on th e p ro d u c tiv ity as it has u n d e r low
light.
The rem oval o f oxygen in a degasser n o t o n ly requires energy b u t it also reduces

th e o verall p ro d u c tiv ity , as p h o tosyn th esis ceases w h e n th e algae reside in th e
d ark zone o f th e degasser. C h a p te r 6 is a general discussion a b o u t th e
im p le m e n ta tio n o f o u r m ain fin d in g s o f th is thesis. The e ffe cts o f reducing th e
energy in p u t fo r degassing and m ixing on th e o verall energy balance as w e ll as on
th e overall p ro d u c tio n costs w e re e valuated using an e con om ic m odel. The m odel
was used to calculate th e energy and costs associated to m icroalgal biomass
p ro d u c tio n in th e N etherlands fo r th re e d iffe re n t system s a t 100 ha scale. The tw o
m eth od s a d o p te d to o vercom e th e negative e ffe c t o f oxygen in m icroalgal
cu ltu re s did re su lt in a decrease in biom ass p ro d u c tio n costs. M o re o v e r, it show ed
th a t using o u r fin d in g s a p ositive energy balance fo r o u td o o r p ro d u c tio n o f
N eochloris o le oabundans in closed p h o to b io re a c to rs can be reached.
101
S a m e n va ttin g
102
S a m e n va ttin g
Sa m e n v a t t in g
F o to tro fe m icroalgen w o rd e n ais ve elbe love nd e g ro n d s to f v o o r duurzam e

b io d ie se lp ro d u ctie beschouw d, aangezien m icroalgen z o n lic h t ais lic h tb ro n , C 0 2
u it rookgassen en n u trië n te n (P, N) u it a fva lstro m e n kunnen g eb ru iken . Om de
o u td o o r-m ic ro a lg e n p ro d u c tie in g esloten fo to b io re a c to re n econom isch a ttra c tie f
en duurzaam te m aken, m oe ten de kosten v o o r h et m engen en h e t ontgassen
om laag w o rd e n g e b ra ch t en de algehele energiebalans m o e t p o s itie f w o rd e n . Het
m engen is nodig om h e t lic h t e ffe c tie f en e ffic ië n t te ve rsp re ide n en v o o r het
to e d ie n e n van ko o lsto fd io xid e . H et ontgassen is nodig om h e t fo to s y n th e tis c h
g ep ro d u ce e rd e z u u rs to f te v e rw ijd e re n .
D it p ro e fs c h rift ric h t zich op de e ffe cte n van de o p h o p in g van z u u rs to f op de groei

van de ve t-o p h o p e n d e m icroalgen N eochloris o le oabundans bij ve rsch ille nd e
lic h tin te n s ite ite n . Deze studies laten zien bij w elke co nce n tra tie s z u u rs to f toxisch
w o rd t v o o r de algen bij ve rsch ille nd e lic h tin te n s ite ite n die de algen g ed uren de de
o u td o o r-p ro d u c tie o n d e rvin d e n . D it g e e ft aan w a n n e e r de z u u rs to f v e rw ijd e rd
m o e t w o rd e n u it de fo to b io re a c to r en dus w a n n e e r ontgassen nodig is.
De ve rsch ille nd e niveaus van lic h tin te n s ite ite n die in buisvorm ig e o u td o o r-
fo to b io re a c to re n gehaald w o rd e n , w e rd e n N eochloris o le oabundans opgelegd
te rw ijl deze g e kw e e kt w e rd in een vo lle d ig g e c o n tro le e rd e en goed g e roe rd e
ta n k re a c to r in tu rb id o s ta t. O n de r co n tin u e b e lic h tin g van 200 p m o l m 2 s'1
(subverzadigde lic h tin te n s ite it) w e rd bij d rie ve rsch ille nd e p a rtië le
z u urstofspa nn ing (Po2= 0,24; 0,63; 0,84) een specifieke groeisn elhe id van 1,38;
1,36 en 1,06 dag"1g em ete n . Bij de p a rtië le zu u rstofspa nn ing van 0,84 bar w e rd de
p a rtië le ko o lsto fd io xid e sp a n n in g ( P Co2) ve rh oo gd van 0,007 t o t 0,02 bar om te
te ste n o f de re m m e n d e e ffe cte n van de fo to re s p ira tie o v e rw o n n e n kunnen
w o rd e n . De to e vo e g in g van ko o ls to fd io x id e re s u lte e rd e inderdaad in een
to e n a m e van de specifieke g ro eisn elhe id van 1,06 t o t 1,36 dag"1. De to e n a m e in
de specifieke g ro eisn elhe id b eve stigt d a t fo to re s p ira tie plaatsvond en d a t h et
negatieve e ffe c t van fo to re s p ira tie o ve rw o n n e n kan w o rd e n d o o r de C 0 2/ 0 2
ve rh o u d in g in h e t m ed iu m te h erstelle n. In h o o fd s tu k 3 w e rd h et e ffe c t van de
p a rtië le zu urstofspa nn ing op de g ro ei van N eochloris o le oabundans g ete st o n d e r
103
S a m e n va ttin g
b ijna-verzadigde lic h tin te n s ite it in een co m p le e t g e co n tro le e rd e fo to b io re a c to r.

Bij de p a rtië le zu urstofspa nn ing en (Po2= 0,24; 0,42; 0,63; 0,84 bar) die g ete st
w e rd e n , was de specifieke g ro eisn elhe id 1,36; 1,16; 0,93 en 0,68 dag"1. Een
ve rh og ing van de PCo2 van 0,007 t o t 0,02 bar b ij een P02 van 0,84 bar b ij deze
h ogere lic h tin te n s ite it had geen p o s itie f e ffe c t op de to ta le g ro ei van de algen in
te g e n ste llin g t o t w a t er bij subverzadigde lic h tin te n s ite ite n gebeurde. Deze
re su lta te n geven aan d a t bij verzadigde lic h tin te n s ite ite n h e t re m m e n d e e ffe c t
van de fo to re s p ira tie n ie t m ee r o ve rw o n n e n kan w o rd e n en d a t fo to in h ib itie -
e ffe cte n de overha nd hebben. H et ch lo ro fy lg e h a lte van N eochloris oleoabundans
die g e kw e e kt was bij 200 p m o l m"2 s"1, was 1,9 keer hoger dan h e t g e h a lte van de
cellen die b ij 500 p m o l m"2 s"1 w aren g e kw ee kt, te rw ijl h et ca ro te n o ïd e g e h a lte 1,6
keer lager was; beide zijn u itin g e n van fo to a c c lim a tis a tie -e ffe c te n . De ve rh oo gd e
z u u rs to fc o n c e n tra tie in h e t g ro e im e d iu m had geen e ffe c t op h e t p ig m e n tg e h a lte
b ij sub- and b ijna-verzadigde lic h tin te n s ite ite n . D it to o n t aan d a t ve rh oo gd e
z u u rsto fco n ce n tra tie s in h et m ed iu m geen e xtra fo to -o x id a tie v e schade b ij de
hogere lic h in te n s ite it veroorzaken, m aar d a t z u u rs to f alleen re m m e n d w e rk t via
fo to re s p ira tie .
M icro alge n die in g esloten fo to b io re a to re n g roeien, o n d e rv in d e n geen co nsta nte

hoge co n ce n tra tie s van zu urstof. In g esloten buisvorm ig e fo to b io re a c to re n n e e m t
de zu u rs to fc o n c e n tra tie g e le id e lijk to e o ve r de lengte van de buis to e d o o r
fo to s y n th e s e om in de ontgasser snel a f te nem en ais h e t o verscho t aan z u u rs to f
a c tie f v e rw ijd e rd w o rd t. D aarnaast zijn de algen aan h e t lic h t b lo o tg e s te ld te rw ijl
ze in de buisvorm ig e re a c to r b lijve n en zijn ze aan c o m p le te d u iste rn is
b lo o tg e ste ld in de ontgasser. In H o o fd s tu k 4 w e rd e n de dynam isch ve ra n d e re n d e
z u u rsto fco n ce n tra tie s en de daarm ee gepaard gaande lic h tc o n d itie s gesim uleerd
in een CSTR onder vo lle d ig g e co n tro le e rd e co n d itie s b ij subverzadigde
lic h tin te n s ite it. V e rd e r w e rd e n de e ffe cte n op de groei van N eochloris
o le oabundans bij b ijna-verzadigde lic h tin te n s ite ite n bestudeerd.
D aarnaast w e rd h e t e ffe c t van een verlen g in g van de v e rb lijfs tijd in de

z o n n e co lle cto r m e t een fa c to r 10 o nd erzoch t. In deze stu die w e rd e n d rie
ve rsch ille nd e lichtreg im e s g e b ru ik t: co n tin u licht, 30 m in u te n "lic h t aan" gevolgd
d o o r 6 m in u te n "lic h t u it" en 300 m in u te n "lic h t aan" gevolgd d o o r 6 m in u te n
" lic h t u it". De specifieke groeisn elhe id w e rd tijd e n s de d rie ve rsch ille nd e
104
S a m e n va ttin g
lichtreg im e s en b ij de co nsta nte lage z u u rs to fc o n c e n tra tie van PO2=0.21 bar
g em ete n en was re sp e ctie ve lijk 1,14 ± 0,06 dag"1; 0,80 ± 0,16 dag"1 en 1,09 ± 0,05
d a g '1. Het e ffe c t van de dynam isch ve ra n d e re n d e z u u rs to fc o n c e n tra tie s van
Po2=0,21 bar to t PO2=0,63 bar, gevolgd d o o r h e t ontgassen t o t PO2=0,21 bar
tijd e n s de d on ke re p erio d e, re su lte e rd e in dezelfde specifieke g ro eisn elhe id. De
verlaging van de specifieke groeisn elhe id van de algen w e rd vastgesteld w a n n e e r
ve rsch ille nd e lic h t regim es g e b ru ik t w e rd e n . U it de re s u lta te n bleek d a t het
tijd e lijk b lo o ts te lle n van de algen aan d on ke re p eriodes in de ontgasser een g ro te r
n e g a tie f e ffe c t h e e ft dan h et tijd e lijk b lo o ts te lle n aan o p lo p e n d e
z u u rsto fco n ce n tra tie s in de zo nn eco lle cto r.
In h o o fd stu k 5 w e rd de sim u la tie van de dynam ische z u u rs to fc o n c e n tra tie s die

o n d e rvo n d e n w o rd e n d o o r de m icroalgen in buisvorm ig e fo to b io re a c to re n o n d e r
hoge lic h tin te n s ite it bestu de e rd , ais ve rvo lg op h o o fd s tu k 4. W a n n e e r de algen
aan co nsta nte zu u rsto fco n ce n tra tie s en aan co nsta nte hoge lic h tin te n s ite ite n
b lo o tg e ste ld w are n , was de specifieke groeisn elhe id 1,29 ± 0,08 d ag'1. W a n n e e r
een lich tre g im e van 30 m in u te n "lic h t aan" gevolgd d o o r 6 m in u te n "lic h t u it" en
ontgassen to e g e p a st w e rd , re su lte e rd e d it in een specifieke g ro eisn elhe id van
0,84 ± 0,09 d ag '1, en de verlen g in g van de tijd ("lic h t aan") t o t 300 m in u te n
re su lte e rd e in een g ro eisn elhe id van 1,18 ± 0,05 d a g 1. W a n n e e r de algen w e rd e n
b lo o tg e ste ld aan dynam isch ve ra n d e re n d e z u u rs to fc o n c e n tra tie , w e rd dezelfde
g ro eisn elhe id g em ete n . Deze re su lta te n to n e n aan d a t algen de fo to o x id a tie v e
in h ib itie n ie t ervaren d o o r hoge z u u rs to f co n ce n tra tie s in c o m b in a tie m e t hoge
lic h tin te n s ite it, zolang de zu u rs to f re g elm atig v e rw ijd e rd w o rd t d o o r m id de l van
ontgassen. De tijd e lijk e b lo o ts te llin g van de algen aan de d u iste rn is in de
ontgasser h e e ft m ee r e ffe c t op hun p ro d u c tiv ite it dan de b lo o ts te llin g aan lage
lic h tin te n s ite ite n . H et v e rw ijd e re n van zu u rs to f in een ontgasser v e rb ru ik t n ie t
alleen energie, m aar h e t re d u ce e rt ook nog de to ta le p ro d u c tiv ite it, o m d a t de
fo to s y n th e s e o p h o u d t w a n n e e r de algen in de d on kere zone van de ontgasser
v e rb lijve n .
H o o fd s tu k 6 is een algem ene discussie o ve r de im p le m e n ta tie van onze

h o o fd b e vin d in g e n u it d it p ro e fsch rift. De e ffe c te n van h e t reduceren van de
g e b ru ikte energie v o o r h et ontgassen en m engen op de to ta le energiebalans en
de to ta le p ro d u ctie ko ste n w e rd e n in een econom isch m odel geëvalueerd. Dit
105
S a m e n va ttin g
m odel w e rd g e b ru ik t om de energie en de kosten b ijb e h o re n d aan de

m icro a lg e n b io m a ssa p ro d u ctie in N ederland v o o r d rie ve rsch ille nd e system en op
een schaal van 100 ha te berekenen. De tw e e m eth od e s die overge no m e n w erd e n
om de negatieve e ffe cte n van z u u rs to f in m ic ro a lg e n c u ltu re n te o v e rw in n e n ,
resu lte e rd e n in een verlaging van de b io m a ssa prod uctie kosten . Bovendien to o n d e
de analyse aan d a t w a n n e e r onze b evindingen g e b ru ik t w o rd e n , een positieve
energiebalans voor de o u td o o r-p ro d u c tie van N eochloris oleoabundans in
gesloten fo to b io re a c to re n b e re ik t kan w o rd e n .
106
S a m e n va ttin g
107
Sum ario
108
S um ario
SUMÁRIO
As m icroalgas fo tó tro fic a s sao consideradas urna fo n te p rom issora de m a té ria -

p rim a para a produçao sustentável de biodiesel, devido ao uso da luz solar natural
com o fo n te energética e utilizaçao do C 0 2 p ro v e n ie n te de gases de co m b ustä o e
n u trie n te s (P, N) o b tid o s pela degradaçao biológica de residuos. Para que a
p ro du çao em larga escala de m icroalgas em fo to b io rre a to re s seja
e co n ó m ica m e n te viável e sustentável, os custos da m istura e desgaseificaçao
deveräo ser m inim izados e o balanço e ne rg ético global deve ser p ositivo . A
m istu ra é necessária para o fo rn e c im e n to e fic ie n te de luz e d ió xid o de carbono,
e n q u a n to a desgaseificaçao é necessária para a rem oçao de oxigénio gerado
fo to s s in te tic a m e n te .
O estud o e fe c tu a d o centra-se no im p a cto da acum ulaçao de o xigénio no

crescim en to da m icroalga oleaginososa N eochloris o le oabundans sob d ife re n te s
intensidades de luz. A investigaçao efectuada d e m o n s tro u quais as concentraçôes
de o xigénio que se to rn a m tóxicas para as algas, ñas d ife re n te s condiçôes de luz
que podem ser ve rificadas no cu ltivo ao ar livre. Foi ainda id e n tific a d o o tim in g
ideal para a rem oçao de oxigénio do fo to b io re a c to r, bem com o a consequente
necessidade de desgaseificaçao da cu ltura.
Foram te sta d o s d ife re n te s níveis de o xigénio que sao a ting id os na p roduçao de

m icroalgas N eochloris oleoabundans em fo to b io rre a to re s tu b u la re s dispostos ao
ar livre, num re a cto r ta n q u e p e rfe ita m e n te a gitado em condiçôes co ntrola da s e
regim e de tu rb id o s ta to . Sob ilum in a çao co n tin u a de 200 m"2 s"1 (in te nsid ad e de luz
su b -satu ran te ), a taxa específica de crescim en to da N eochloris o le oabundans fo i
de 1,38; 1,36 e 1,06 dia"1, m edida a tré s d ife re n te s pressöes parciais de oxigénio
(Po2= 0.24; 0.63 e 0.84 resp ectiva m e nte). À pressäo parcial de oxigénio de 0,84
bar, a pressäo parcial de d ió xid o de carbono ( P Co2) fo i a um en ta da de 0,007 para

0,02 bar, visando te s ta r se o e fe ito in ib id o r da fo to rre s p ira ç a o poderia ser
superado. V e rifico u-se que a adiçao de d ió xid o de carbono resu ltou num a u m e n to
da taxa específica de crescim en to, de 1,06 para 1,36 dia"1. O a u m e n to da taxa
específica de crescim en to co n firm o u que a fo to rre s p ira ç a o ¡nibe o crescim ento, e
que esse e fe ito negativo pode ser superado re s ta ura nd o a relaçao C 0 2/ 0 2.
109
Sum ario
No cap ítu lo 3 descreve-se o e fe ito da pressäo parcial de oxigénio no crescim en to

da m icroalga N eochloris o le oabundans a urna inten sid ad e da luz n ea r-sa tu ra n te
num fo to b io re a c to r em condiçôes controla da s. Ás pressöes parciais de oxigénio
testadas (P q 2 = 0,24, 0,42, 0,63, 0,84 bar), a taxa específica de crescim en to fo i de
1,36, 1,16, 0,93 e 0,68 dia"1, resp ectiva m e nte. Um a u m e n to de PCo 2 de 0,007 para
0,02 bar sob Pq2 0,84 bar a esta inten sid ad e de luz m ais fo rte , nao m ostro u
q u a lq u e r e fe ito p o sitivo sobre o crescim en to global das algas, ao c o n trá rio do que
acontece em intensidades sub-saturantes. Estes resultados indicam que a
intensidades de luz saturantes, o e fe ito in ib itó rio do oxigénio p o r fo to rre s p ira ç a o
näo pode ser superada e que os e fe ito s da fo to in ib iç a o prevalecem . O te o r de
c lo ro fila de N eochloris o le oabundans cu ltiva da sob 200 p m o l m '2 s"1 é cerca de 1,9
vezes m a io r do que qua nd o cu ltiva da a 500 p m o l m"2 s"1, e n q u a n to que o te o r de
c a ro ten ó id es é cerca de 1,6 m en or; d e m o n s tra n d o e fe ito s de fo to a c lim a ta ç â o . A
elevada co ncentraçâo de o xigénio no m eio de cu ltu ra , p o r si só, nao afecta o te o r
em p ig m e n to s sob condiçôes de luz sub-saturantes e nea r-saturan te s . Isto indica
que concentraçôes elevadas de o xigénio nao c o n trib u e m para o a u m e n to de
danos fo to -o x id a tiv o s no m eio, nas condiçôes de luz m ais elevadas utilizadas, mas
que o oxigénio apenas ¡nibe o crescim en to p o r m eio de e fe ito s de fo to -re s p ira ç â o .
As m icroalgas cultivadas em fo to b io rre a to re s näo estäo expostos a altas

concentraçôes de o xigénio constantes. Em fo to b io rre a to re s tu b u la re s , as
concentraçôes de o xigénio a um en ta m ao longo dos tu b o s devido à a ctividade
fo to s s in té tic a , d im in u in d o no desgaseificador, onde o excedente de oxigénio é
re m o vid o . Além disso, as algas estäo expostas à luz e n q u a n to no in te rio r dos
tu b o s, e no escuro qua nd o no desgaseificador. No ca p ítu lo 4, as concentraçôes de
oxigénio d in á m ica m e n te variáveis e conséquentes condiçôes de luz fo ra m
sim uladas num CSTR a condiçôes co ntrola da s sob inten sid ad e de luz sub-
s a tu ra n te e near- sa tu ra n te , te n d o sido e studado o e fe ito sobre o crescim en to da
N eochloris oleoabundans. A d icio n a lm e n te , fo i avaliado o e fe ito de um
a lo n g a m e n to de 10 vezes o te m p o de perm anéncia no re c e p to r solar. Neste
estud o fo ra m u tiliza do s 3 regim es d ife re n te s : de luz co n tin u a : 30 m in u to s de luz
seguidos de seis m in u to s de escuro, e 300 m in u to s de luz seguidos de seis
m in u to s de auséncia de luz. A taxa específica de crescim en to m edida à
concentraçâo de oxigénio co nsta nte de PO2=0.21 bar d u ra n te os 3 regim es de luz
110
S um ario
fo ra m resp e ctiva m e n te de 1,14 ± 0,06 dia"1; 0,80 ± 0,16 d ia '1 e 1,09 ± 0,05 dia"1. O
e fe ito de a lteraçâo dinám ica de co nce ntra çâo de oxigénio, de PO2=0.21 bar para
Po2=0.63 bar, fo i seguido de subséquente desgaseificaçao para PO2=0.21 d u ra n te o
p eríod o de auséncia de luz, te n d o resu ltad o em taxas específicas de crescim en to

sem elhantes. A d im in u iça o da taxa especifica de crescim en to observada quando
aplicados regim es de luz d ife re n te s d em o n stra que a exposiçao das algas a
períodos de escuridäo no desgaseificador te m m a io r im p a cto negativo do que a
exposiçao te m p o rá ria a altas concentraçôes de o xigénio no re c e p to r solar. No
cap ítu lo 5 descreve-se o estud o de urna sim ulaçao das concentraçôes de oxigénio
dinám icas sentida pelas m icroalgas em fo to b io rre a to re s tu b u la re s sob
intensidades de luz elevadas. Q uando as algas fo ra m expostas a co ncentraçâo de
oxigénio co nsta nte e luz elevada co nsta nte, a taxa específica de crescim en to fo i
de 1,29 ± 0,08 dia"1. Num regim e de luz de 30 m in u to s seguidos de 6 m in u to s de
luz desligada e desgaseificaçao, a taxa de crescim en to observada fo i de de 0,84 ±
0,09 dia"1. O p ro lo n g a m e n to de te m p o (luzes acesas) para 300 m in u to s resu ltou
em 1,18 ± 0,05 dia"1. Q uando im postas concentraçôes de o xigénio dinám icas,
fo ra m , o btid as taxas específicas de crescim en to sem elhantes . Estes resultados
indicam que as algas näo e xp e rim e n ta m a inibiçao fo to -o x id a tiv a esperada,
causada pela alta co nce ntra çâo de oxigénio em com binaçao com a luz elevada,
desde que o oxigénio seja re m o vid o através de desgasificaçao regular. A
exposiçao te m p o rá ria das algas ao escuro no desgaseificador te m m ais im p acto
sobre a p ro d u tivid a d e , ta i com o aconteceu sob intensidades de luz mais baixas.
A rem oçao de oxigénio no desgaseificador näo só re q u e r energía com o ta m b é m

reduz a p ro d u tiv id a d e global, sendo que a fo to ssíntese cessa qua nd o as algas se
e n co n tra m na zona escura do desgaseificador. No ca p ítu lo 6 d¡scute-se a
im p le m e n ta çâ o das p rincipáis conclusöes deste estudo. Foram avaliados os
e fe ito s da reduçâo da energía necessária para a desgaseificaçâo e m istura no
balanço global de energía, bem com o sobre os custos globais de produçâo,
u tiliza n d o um m o d e lo económ ico. O m o d e lo fo i usado para calcular a energía e os
custos associados à p ro du çâo de biomassa de m icroalgas nos Países Baixos, em
tré s sistem as d ife re n te s, a urna escala de 100 ha. As duas m eto do log ía s utilizadas
para superar o e fe ito n egativo do o xigénio em cu ltu ra s de m icroalgas resultaram
num a d im in u içâ o dos custos de pro du çâo de biom assa. Os resultados alcançados
lii
Sum ario
neste estud o d e m o n stra m ser possível a tin g ir um balanço e ne rg ético p o sitivo na

p ro du çao de m icroalgas N eochloris o le oabundans em fo to b io rre a to re s dispostos
ao ar livre.
112
S um ario
113
A ckn ow le dg em en ts
114
A cknow ledg em ents
And n ow is th e tim e fo r th e ve ry last th in g b e fo re p rin tin g th e thesis:

a ckno w le dg m e n ts. I once to ld a frie n d I w o u ld like to have his courage w he n
w ritin g th e a ckno w le dg m e n ts, sim p ly sta tin g a h e a rtfe lt: "T hank you e v e ry o n e !" -
Yet, I could n o t resolve m yself to do so, as I do w ish to th a n k each one personally,
n am ing all th o se w ith o u t w h o m th is thesis w o u ld n o t have been possible. But
th e n again, by n am ing some, I risk n o t nam ing m any w h o c o n trib u te d to m ake th is
possible - if you are one o f th e m : th a n k YOU ve ry m uch!
M y fir s t w o rd s go to Rene: th a n k yo u ! I o w e you m y d eepest g ra titu d e fo r all th e

s u p p o rt and e n co u ra g e m e n t along th ese 4 years o f ups and dow ns. Y our clear
m ind and p o sitive a ttitu d e (co n tra stin g m y pessim ism ) are am ong th e decisive
d rivin g forces to co m p le te th is w o rk . M arian , you a rrived h a lf w a y along th is
p ro je c t as m y supervisor, and y o u r enthusiasm , su p p o rt, m e n to rin g and help w e re
a key in g re d ie n t on th is thesis. M arcel, I also w a n t to th a n k you fo r th e firs t tw o
years you w e re m y supervisor. You p resented me to algae and you are th e one
w ith w h o m I learned th e m ost d urin g m y algae jo u rn e y .
N ext to m y supervisors, I w a n t to th a n k W etsus fo r th e o p p o rtu n ity I was given to

w o rk in such a big and w o n d e rfu l e n v iro n m e n t. W etsus was m y guest h om e fo r
six m o n th s in 2 0 0 6 /2 0 0 7 and I cam e back fo r a longer stay in 2008. I w a n t to
a cknow ledge W etsus d ire c tio n and th a n k fo r th e chance to p e rfo rm m y PhD thesis
th e re . In p a rticu la r, I w ish to th a n k Gert-Jan fo r th e o p p o rtu n ity he gave me. To all
th e m em be rs o f th e algae te a m , th a n k you fo r th e fr u itfu l discussions d u rin g
th e m e m eetings. To m y p a rtn e rs in crim e w ith in th e te a m : Ana M arta , Sina, Anja,
Ellen, Lenneke, Anne and Zlatica, th a n k you all.
I also w o u ld like to th a n k m y stu de nts, Krystian, Ana C om padre, D im ita r and

Agnes fo r th e ir w o rk and c o n trib u tio n to th is thesis.
To all th e frie n d s and colleagues th a t had to deal w ith m y m o rn in g m oo d: Ana

M a rta , Justina, Tom , Piotr, A driaan, A strid , Adam , Alexandra, Bruno, Bob, Dries, I
th a n k you so ve ry m uch fo r all th e g re a t m om e n ts w e had in th e o ffic e w e shared
a t som e p o in t. For all th e lau gh te r, dancing, shouting... I really had fu n a t th e
115
o ffic e ! Ana, tu näo só p a rtilh a ste e s crito rio com igo (logo no inicio) com o ta m b é m
com eçaste ao m esm o te m p o que eu. O brigada p o r to d o s os m o m e n to s que
p a rtilh a m o s: risos, choros, alegrias, frustra çôe s, saudades do nosso Portugal...
som os tä o d ife re n te s ! mas m esm o assim conseguim os cria r urna am izade que
fica rá para o resto das nossas vidas.
I w a n t to th a n k th e w h o le W etsus sta ff. W e all co m p la ine d a lo t a b o u t e verythin g,

b u t you guys rock! The W etsus la b o ra to ry and fa c ilitie s w o u ld ju s t n o t w o rk o u t
w ith o u t you. P a rticularly I w a n t to a cknow ledge W im . Y our e n o rm o u s patience
and p ro fessionalism are a dm ira ble. I o fte n w o n d e r h ow you p u t up w ith all th e
PhD stu d e n ts a ro u n d you...
Bart, Ingo, Sandra, Natasja, Elmar, Petra, Agata, , Lena, Taina, Perry, Tim , Camiel,
Luew ton, Paula, Philipp, N adine, C hristina, Urania, Lucia, Elsemiek, Johannes k.,
Kam uran, Jos, M a rtin a , am ong m any o th e rs w h o m I apologize n o t to m e n tio n .
You are m y W etsus fa m ily and a bunch o f crazy peo ple I w ill neve r fo rg e t. I also
w a n t to a cknow ledge e ve ryb o d y a t Bioprocess Engineering in W ageningen, w h o
alw ays m ade m e fe ei w e lco m e and w e re alw ays ready to help.
To m y p a ra n ym p h s A lexandra and Pedro, th a n k you so m uch fo r accepting m y

in v ita tio n and helping m e a t th e end o f th is road. T hank you fo r y o u r frie n d s h ip !
Bruno, B rigitte, Bastien, Em eline, A delin e e t Clém ence, m erci p o u r to u s ces bons
m o m e n ts passés en France e t p o u r to u jo u rs m e fa ire s e n tir la bienvenue.
A b ilio e Alcina, M anuela e V ito r, C ristina, Fernanda e G eovandro, vocês sao os

m elho res irm aos e cunhados do m u n d o ! O brigada p o r sem pre me apo ia rem e
d e m o n stra re m que estao lá para m im para o bem e para o m al... Téte, mana,
fazes-m e urna fa lta in d e scritive l! O brigada p o r tu d o !
Joao e Isabel Basto, obrigada pelo apo io e ajuda nesta ú ltim a fase da m inha tese.
M axim e, th e re are no w ords... y o u r u n co n d itio n a l help and s u p p o rt w e re /a re

tre m e n d o u s ly im p o rta n t. Thank you fo r e v e ry th in g ! I did n o t m anage to fin ish
b e fo re you... [Ja m m e rl]
Este é o parág ra fo m ais d ifícil de escrever p orq u e nao existem palavras que
possam descrever a g ra tid a o que te n h o para com os m eus país. Papá e m am a,
vocês sao o meu m u n d o e o vosso apoio, a m o r e com preensäo ¡ncondicionais
116
poderá jam ais ser agradecida. Todas as m inhas alegrias, tristezas, preocupaçoes,
nervosism os, inseguranças, extases fo ra m urna p artilh a co nsta nte e nunca vos
poderei agradecer o su ficie n te p o r me o u vire m , a poiarem mas ta m b é m p o r me
saberem dizer nâo e m o stra re m -m e quando estava errada. Esta tese näo é m inha,
é nossa! Obrigada p o r tu d o !
117
A b o u t the a u th o r
118
A b o u t th e a u th o r
A bout the A uth o r
Cláudia Sousa was born on th e 21st o f O cto b er

1980 in Felgueiras, P ortugal. A fte r High school in
h er h o m e to w n , she sta rte d studies in biological
eng in ee rin g w ith th e U nive rsity o f M in h o , in
Braga, Portugal. D uring her last year, she w e n t to
th e N ethe rla nd s to do her m aster thesis at
W etsus, m aking th e ch a ra cte riza tio n of an
e xp e rim e n ta l set-up fo r stu dyin g m em b ra n e fo u lin g . A fte r g e ttin g her MSc.
d ip lo m a in Portugal, she cam e back to th e N etherlands to s ta rt her PhD p ro je c t in
c o lla b o ra tio n b e tw e e n th e algae th e m e o f W etsus and th e Bioprocess Engineering
g ro u p o f W ageningen U niversity.
119
T ra inin g a ctivitie s
120
T ra inin g a c tivitie s
O v e r v ie w o f c o m p l e t e d t r a in in g a c t iv it ie s .
Discipline specific activities

Advanced Course on M icro b ia l Physiology and F erm e n ta tio n T echnology (2010)
B io re actor Design and O p e ra tio n (2010)
Solar Biofuels fro m M icro orga nism s (2009)
W etsus th e m e m eetings (2009, 2010, 2011 & 2012)
General courses
W o rk in g Safely in Laboratories (2011)
PhD scie n tific w ritin g (2010)
PhD p re se n ta tio n skills (2009)
VLAG PhD w e e k (2009)
Conferences
1st In te rn a tio n a l Algal C onference, A m ste rd am , The N etherlands (2008)
4th Congress o f th e In te rn a tio n a l Society fo r A pplie d Phycology (ISAP), Halifax,
Nova Scotia, Canada (2011)
W etsus congress (2009, 2010, 2011 & 2012)
Optionals
PhD excursion Spain (2012)
PhD excursion USA (2010)
W etsus w a te r challenge (2009)
P re pa ra tion p ro je c t proposal (2008)
Teaching
Bioprocess design - w o rk in g classes
121

Oxygen Accumulation in Photobioreactors

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Oxygen accumulation

This research was co nd ucte d u n d e r th e auspices o f th e G raduate School VLAG

Cláudia Alexandra da Fonseca e Sousa

PhD thesis W ageningen U niversity, W ageningen, NL (2013)

Chapter 1. General introduction & thesis outline 1

1.2. Large-scale o u td o o r c u ltiv a tio n 2

1.3. P hotosynthesis, p h o to re s p ira tio n & p h o to in h ib itio n 3

1.4. Thesis o u tlin e 7

Chapter 2. Growth of the microalgaeNeochloris oleoabundans at high

2.1. In tro d u c tio n 16

2.2. M a te ria l and m eth od s 17

2.2.1. Cultures and medium 17

2.2.3. Dry weight concentration 19

2.2.4. Specific absorption coefficient 20

2.2.5. Photosynthesis-irradiance curve (PI- curve) 21

2.3. Results and discussion 21

2.3.1. Light regime 21

2.3.2. Growth and productivity 23

2.3.3. Implications on reactor design 26

A p pe n dix 2A. D is trib u tio n o f in cid e n t p h o to n flu x d en sity o ve r p h o to b io re a c to r

A p pe n dix 2B. C alculation o f lig h t g ra d ie n t in p h o to b io re a c to r 33

A p pe n dix 2C. Specific a b so rp tio n sp ectrum N. oleoabundans 34

Chapter 3. Effect of Oxygen at low and high light intensity on the

3.1. In tro d u c tio n 38

3.2. M a te ria l and m eth od s 40

3.2.1. Cultures and medium 40

3.2.3. Dry weight concentration 41

3.2.4. Chlorophyll and carotenoids 41

3.3. Results and discussion 42

3.3.1. Controlled cultivation o f algae at high and low light intensity 42

Chapter 4. Effect of Dynamic Oxygen Concentrations on the growth of

4.1. In tro d u c tio n 56

4.2. M ate ria ls and m eth od s 57

4.2.1. Cultures and medium 57

4.2.3. Light regime 58

4.2.4. Dry weight concentration 59

4.2.5. Chlorophyll and Carotenoid determination 59

4.3. Results and discussion 60

4.3.1. Effect o f the applied light-regime and the dynamically changing 0 2 on

4.3.2. Effect on biomass yield on photons 63

4.3.3. Chlorophyll and carotenoid content 64

4.3.4. Final remarks 66

Chapter 5. Effect of Dynamic Oxygen Concentrations on the growth of

5.1. In tro d u c tio n 74

5.2. M a te ria l and m e th o d 75

5.2.1. Culture and photobioreactor system 75

5.2.2. Light regime 76

5.2.3. Off-line analysis o f samples 76

5.3. Results and discussion 77

5.3.1. Effect o f dynamic 0 2 and light regime on algal growth 77

5.3.2. Chlorophyll and carotenoid content 80

Chapter 6. Oxygen production in photobioreactors A look to the

6.1. In tro d u c tio n 85

6.1.1. Effects o f oxygen on microaigai growth 86

6.1.2. Effects o f (dynamic) accumulating oxygen in closed photobioreactors 89

6.2. Reducing th e costs fo r degassing w ill reduce th e o verall costs 90

6.2.1. Biomass productivity costs - base case 90

6.2.2. Effect o f increasing the C02/ 0 2 on biomass production costs 90

About the Author 119

Overview of completed training activities. 121

Chapter 1. G e n e r a l in t r o d u c t io n & th esis o u t lin e

N owadays, a lo t o f research is done on large-scale m icroalgal p ro d u c tio n using

Figure 1 - Schematic representation o f the environmental factors influencing microalgae

N eochloris o le oabundans is m e n tio n e d as one o f th e m o st p ro m ising strains o f