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
Table o f co nte nts
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
3.1.
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
3.2.2.
Photobioreactor
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
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
3.4.
C onclusions
48
3.5.
A ckn ow le dg em en ts
48
3.6.
References
49
Chapter 4.
Effect of Dynamic Oxygen Concentrations on the growth of
Neocholoris oleoabundans at sub-saturating light conditions
55
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
4.2.2.
Photobioreactor
57
4.2.3.
Light regime
58
4.2.4.
Dry weight concentration
59
4.2.5.
Chlorophyll and Carotenoid determination
59
Table o f con ten ts
4.3.
Results and discussion
4.3.1.
60
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
4.4.
C onclusions
67
4.5.
A ckn ow le dg em en ts
68
4.6.
References
68
Chapter 5.
Effect of Dynamic Oxygen Concentrations on the growth of
Neochloris oleoabundans at high light conditions
73
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
5.4.
C onclusions
81
5.5.
A ckn ow le dg em en ts
81
5.6.
References
82
Chapter 6.
Oxygen production in photobioreactors A look to the
economics
85
6.1.
In tro d u c tio n
6.1.1.
Effects o f oxygen on microaigai growth
85 86
Table o f co nte nts
6.1.2. 6.2.
Effects o f (dynamic) accumulating oxygen in closed photobioreactors
Reducing th e costs fo r degassing w ill reduce th e o verall costs
89 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
6.3.
C onclusions
93
6.4.
References
93
Summary
99
Samenvatting
103
Sumário
109
Acknowledgements
115
About the Author
119
Overview of completed training activities.
121
Table o f con ten ts
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
G eneral in tro d u c tio n & thesis o u tlin e
(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 largescale 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 NADPLight reaction
Dark 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 Ribulose1 ,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
G eneral in tro d u c tio n & thesis o u tlin e
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.,
oxygenase a c tiv ity results in a decrease o f th e
2011). The enhanced
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 3phosphoglycerate 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
In
the
w ater-w ater
cycle
NA D P -
the
photoreduction o f oxygen to w ater takes place in PS I by the electrons generated in PS II (Asada, 1999). In the
w ater-w ater
cycle,
STROMA
C u Z n -S O D '
the
electrons are used again to reduce oxygen, via the reactive oxygen species superoxide and hydrogen peroxide, w ith enzymes,
the
help of the
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
radiais and toxic H20 2. This process
Figure 3 - Formation o f oxygen radicals in the water-w ater cycle during photosynthesis. Adapted fro m Asada (2006)
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
G eneral in tro d u c tio n & thesis o u tlin e
Photoactivation
Photons captured by P680 cause excitation o f P680
STROMA
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 fro m Asada (2006)
Photons
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 nears 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
G eneral in tro d u c tio n & thesis o u tlin e
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. Asada,
K. 2006.
P ro du ction
and
scavenging
o f rea ctive
oxygen
species
in
ch lo ro p la sts and th e ir fu n c tio n s . Plant Physiology, 141(2), 391-396. Asada, K. 1999. The w a te r-w a te r cycle in ch lo ro p la sts: Scavenging o f active oxygens and dissip atio n o f excess p ho ton s. A nnual Review o f Plant Physiology and Plant M o le cu la r Biology, 50(1), 601-639.
9
C hapter 1
Becker, E.W. 1994.
M icroalgae: b io te ch n o lo g y and
m ic ro b io lo g y. C am bridge
U nive rsity Press, C am bridge. Boelee, N.C., T em m in k, H., Janssen, M., Buisman, C.J.N., W ijffe ls , R.H. 2011. N itro ge n and p hosphorus rem oval fro m
m un icip al w a s te w a te r e fflu e n t using
m icroalgal b io film s. W a te r Research, 45(18), 5925-5933. Bosma, R. 2010. T ow ards high p ro d u c tiv itie s o f m icroalgae in p h o to b io re a c to rs , PhD thesis, U nive rsity o f W ageningen, The N etherlands. C arvalho, A.P., M eireles, L.A., M alcata, F.X. 2006. M icroalgal reactors: A review o f enclosed system designs and p erform an ces. B io tech n olog y Progress, 22(6), 14901506. 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. Dismukes, G.C., C arrieri, D., B ennette, N., Ananyev, G .M ., Posewitz, M.C. 2008. A q u a tic p h o to tro p h s : e ffic ie n t a lte rn a tive s to
land-based crops fo r biofuels.
C urren t O p inion in B io technology, 19(3), 235-240. Endo, T., Asada, K. 2008. P hotosystem I and p h o to p ro te c tio n : cyclic e le c tro n flo w and w a te r-w a te r cycle, in: P h o to p ro te c tio n , P h o to in h ib itio n , Gene R egulation, and E n viro nm e nt, (Eds.) B. D em m ig-A dam s, W .W . Adam s, A.K. M a tto o , Vol. 21, Springer. D o rdre cht, pp. 205-221. 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 n viro nm e nta l M o d u la tio n , and E volution. A nnual Review o f Plant Biology, 56, 99-131. 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 In du stria l M ic ro b io lo g y and B io technology, 1-6. G ouveia, L., O liveira, A.C. 2009. M icroalgae as a raw
m a te ria l fo r b iofuels
p ro d u c tio n . Journal o f In du stria l M ic ro b io lo g y & B iotechnology, 36(2), 269-274. Janssen, M . 2002. C u ltiva tio n o f m icroalgae: e ffe c t o f lig h t/d a rk cycles on biomass yield, PhD thesis, U n ive rsity o f W ageningen, The N etherlands.
10
G eneral in tro d u c tio n & thesis o u tlin e
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(3), 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 io tech n olog y and B ioengineering, 108(10), 2390-2402. Ledford,
H.K.,
Niyogi,
K.K. 2005. Singlet oxygen
and
p h o to -o x id a tiv e
stress
m an ag em en t in plants and algae. Plant Cell and E n viro nm e nt, 28(8), 1037-1045. Li, Y., H orsm an, M., W ang, B., W u, N., Lan, C.Q. 2008a. Effects o f n itro g e n sources 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. Li, Y., H orsm an, M ., W u, N., Lan, C.Q., D ubois-Calero, N. 2008b. Biofuels fro m M icroalgae. B io tech n olog y Progress. M ata, T .M ., M a rtin s, A.A., Caetano, N.S. 2010. M icro alga e fo r biodiesel p ro d u c tio n and o th e r a pp lica tion s: A review . R enew able and Sustainable Energy Reviews, 14(1), 217-232. M olina , E., Fernandez, F.G.A., Chisti, M.Y. 2001. T u b u la r p h o to b io re a c to r design fo r algal cu ltures. Journal o f B iotechnology, 92, 113-131. M u ra ta , N., Takahashi, S., N ishiyam a, Y., A llakh ve rd ie v, S.l. 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 (BBA) - Bioenergetics, 1767(6), 414-421. Norsker, N.-H., Barbosa, M.J., V e rm u ë, 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), 2427. O gren, W.L. 1984. P h o to re sp ira tio n : Pathways, re g u la tio n , and m o d ific a tio n . A nnual Review o f Plant Physiology, 35(1), 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 lic a tio n s fo r th e energetics o f pho tosyn th esis. Biochim ica e t Biophysica Acta, 639, 77-98.
11
C hapter 1
Pruvost, J., Van V ooren, G., Cogne, G., Legrand, J. 2009. Investig atio n o f biomass and
lipids
p ro d u c tio n
w ith
N eochloris
o le oabundans
in
p h o to b io re a c to r.
B ioresource Technology, 100(23), 5988-5995. Pruvost, J., Van V ooren, G., Le Gouic, B., C ouzinet-M ossion, A., Legrand, J. 2011. S ystem atic in ve stig a tio n
o f biom ass and lip id
p ro d u c tiv ity by m icroalgae in
p h o to b io re a c to rs fo r biodiesel a p p lica tio n . B ioresource Technology, 102, 150-158. Pulz,
O.
2001.
P h oto 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. Raso, S., van G enügten, B., V erm uë, M ., W ijffe ls , R.H. 2011. Effect o f oxygen c o n c e n tra tio n on th e g ro w th o f N annochloropsis sp. a t low lig h t in te n s ity . Journal o f A pplie d Phycology, 1-9. Raven, J.A., Larkum , A.W .D. 2007. A re th e re ecological im p lic a tio n s fo r th e p roposed e ne rg etic re strictio n s on p h o to s y n th e tic oxygen e v o lu tio n a t high oxygen co nce n tra tio n s? P hotosynthesis Research, 94, 31-42. R ichm ond, A. 1992. Open system s fo r th e mass p ro d u c tio n o f p h o to a u to tro p h ic m icroalgae o u td o o rs : physiological p rinciples. Journal o f A pplie d Phycology, 4(3), 281-286. Stephens, E., Ross, I.L., King, Z., M ussgnug, J.H., Kruse, O., Posten, C., B orow itzka, M .A., Hankam 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 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., Kruse, O., Hankam 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. 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 flu oresce nce to assess th e e x te n t o f p h o to in h ib itio n o f p ho tosyn th esis induced by 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 o f o u td o o r cu ltu re s o f S piru lin a p la te nsis (cyanobacteria). Journal o f Phycology, 34 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
G eneral in tro d u c tio n & thesis o u tlin e
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 oxygen c o n ce n tra tio n , and te m p e ra tu re on th e g ro w th o f C hlorella so ro kin ian a. P h otosyn th etica , 45(2), 309-311. Vacha, F. 1995. The role o f oxygen in pho tosyn th esis. P hotosyn th etica , 31(3), 321334. 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), 287295. 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.
13
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 SUBSATURATING 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 subs 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, largescale 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
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 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
o le oabundans
at
su b-satu ratin g
pressures on th e g ro w th
lig h t
c o n d itio n s
in
a
o f N eochloris
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
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 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
s p e c tro p h o to m e te r set-up to a b so rp tio n
cells
was
m easured
in
a specialized
m in im ize th e e ffe c t o f lig h t sca tte ring on th e
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
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
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
o
2.0
c
max
0) D) > X
o
ö E =L (0
'w 0) c >* W 5 o x: Q_ (0 -s).cos(©) + [r2 - ( r - s ) 2.sin(©)2]
jcos(© + jr)d®
PFD(z) = PFD(z)wall + PFD(z)center
33
i ©
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
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
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 nears 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), 122126. 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 feasible,
how eve r,
proposed. To m ake th e
b o ttle n e cks
still
need
to
be
p ro d u c tio n
o vercom e.
e con om ically
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
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 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.
3.2.2. Photobioreactor 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
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 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 (Lichten th aler,
(UV-1650
1987)
w e re
PC, used
Schimadzu). to
calculate
M o d ifie d
A rn o n 's
c h lo ro p h y ll
and
e qu atio ns 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.
Results and discussion
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 sisirra 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) 1,5
A
Cx(g.Kg"1)
■
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
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 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 (m M )
Po2 (bar)
Pco2
p (d a y 1)
(bar)
± Stdev
Cx (g kg'1) ± Stdev
500 p m o l m"2 s"1
p (d a y 1) ± Stdev
Cx (g kg'1) ± Stdev
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
o
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
^ Q
3
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.
3.5.
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
48
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 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 en w erking sverba n d
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.
3.6.
References
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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.
B ioresource Technology, 102(8), 5129-5137. 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 Dismukes, G.C., C arrieri, D., B ennette, N., Ananyev, G .M ., Posewitz, M.C. 2008. A q u a tic p h o to tro p h s : e ffic ie n t a lte rn a tive s to
land-based crops fo r biofuels.
C urren t O p inion in B io technology, 19(3), 235-240. 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 In du stria l M ic ro b io lo g y and B io technology, 1-6.
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G u illará, R.R.L., R yther, J.H. 1962. Studies o f m a rin e p la n k to n ic d ia to m s. /. C yclotella n an a h uste dt, and D eton ula confervagea (Cleve) gran. Canadian Journal 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 fo r th e role o f th e oxygen-evolving m anganese co m p le x in p h o to in h ib itio n o f P hotosystem II. Biochim ica Et Biophysica A cta-B ioenergetics, 1706(1-2), 68-80. 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 io tech n olog y and B ioengineering, 108(10), 2390-2402. Krieger-Liszkay, A., Fufezan, C., Trebst, A. 2008. Singlet oxygen p ro d u c tio n in p ho tosystem
II and related p ro te c tio n
m echanism . Photosynthesis Research,
98(1-3), 551-564. Ledford,
H.K.,
Niyogi,
K.K. 2005. Singlet oxygen
and
p h o to -o x id a tiv e
stress
m an ag em en t in plants and algae. Plant Cell and E n viro nm e nt, 28(8), 1037-1045. Li, Y., H orsm an, M ., W ang, B., W u, N., Lan, C.Q. 2008. Effects o f n itro g e n sources 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. L ich te nth a le r,
H.K.
1987.
C hlorophylls
and
ca ro ten o id s:
pig m e n ts
of
p h o to s y n th e tic b io m e m bran es. M e th o d s in Enzym ology, 148, 350-382. M olina , E., Fernandez, F.G.A., Chisti, M.Y. 2001. T u b u la r p h o to b io re a c to r design fo r algal cu ltures. Journal o f B iotechnology, 92, 113-131. M u ra ta , N., Takahashi, S., N ishiyam a, Y., A llakh ve rd ie v, S.l. 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 (BBA) - Bioenergetics, 1767(6), 414-421. 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. Pruvost, J., Van V ooren, G., Cogne, G., Legrand, J. 2009. Investig atio n o f biomass and
lipids
p ro d u c tio n
w ith
N eochloris
B ioresource Technology, 100(23), 5988-5995.
50
o le oabundans
in
p h o to b io re a c to r.
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 Raso, S., van G enügten, B., V erm uë, M ., W ijffe ls , R.H. 2011. Effect o f oxygen c o n c e n tra tio n on th e g ro w th o f N annochloropsis sp. a t low lig h t in te n s ity . Journal o f A pplie d Phycology, 1-9. 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. Schenk, P.M., Thom as-H all, S.R., Stephens, E., M arx, U., 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 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 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 flu oresce nce to assess th e e x te n t o f p h o to in h ib itio n o f p ho tosyn th esis induced by 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 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 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 oxygen c o n ce n tra tio n , and te m p e ra tu re on th e g ro w th o f C hlorella so ro kin ian a. P h otosyn th etica , 45(2), 309-311. Valenzuela-Espinoza, E., M illan -N u ne z, R., Trees, C.C., S antam aria-del-A ngel, E., 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. 51
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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. 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), 287295.
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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
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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 1Wetsus, P.O. Box 1113, 8900 CC Leeuwarden, the Netherlands 2 Bioprocess Engineering, Wageningen University, P.O. Box 8129, 6700 EV, Wageningen, 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
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 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.
4.2.2. Photobioreactor 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
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 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.
Results and discussion
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 e xp e rim e n ta l
and run
su b-satu rate d can
be
divid ed
lig h t in
co n d itio n s six
d iffe re n t
was
d e te rm in e d .
phases.
In itially,
The 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
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
2.0
-------p o ; (bar) ------- OD (CU)
1.5 -
■
n (c )ay’ 1)
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
P I - Batch P2
-
C ontinuous
lig h t
-
3 0 /6
-
Po2=0.21 bar P3 - Light regim e
Po2=0.21 bar P4 - Light regim e 3 0 0 /6 -
Po2=0.21 bar P5 - Light regim e
3 0 /6
-
D ynam ic PO2= 0 .2 1/0 .6 3 bar P6 - Light regim e 3 0 0 /6 D ynam ic PO2= 0 .2 1/0 .6 3 bar
Cx ± Stdev
p ± Stdev
Yx,ph ± Stdev
(g L 1)
(d a y 1)
(g m o l-p h '1)
1.05 ± 0 .0 2
N /A
N /A
0.55 ± 0 .0 2
1.14 ± 0 .0 6
1.15 ± 0 .0 4
0.55 ± 0 .0 1
0.80 ± 0 .1 6
0.97 ± 0 .0 3
0.55 ± 0 .0 1
1.09 ± 0 .0 5
1.12±0.02
0.49 ± 0 .0 1
0.82 ± 0 .0 4
0.88 ± 0 .0 2
0.51 ± 0 .0 3
1.07 ± 0 .1 2
1.01 ± 0 .0 7
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
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 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 tlim 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 subs 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 a t s u b -s a tu ra tin g lig h t co nd itio ns
P1 30
P2
P3
P4
Chla+Chlb (mg g'1) 1 Car ( mg g'1)
I
-
P5
P6
es O 20
-
10
-
O +
CS
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
66
rate
was
also
expected
w hen
d yna m ica lly
changing
oxygen
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 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 .
4.5.
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 en w erking sverba n d
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 p p o rt.
4.6.
References
B ritto n , G., Liaaen-Jensen, S., Pfänder, H., Frank, H.A., Young, A.Y., B ritto n , G., Cogdell,
R.J.
1999.
The
P h o to ch e m istry
of
C arotenoids.
K luw er
Academ ic
Publishers. C arvalho, A.P., M eireles, L.A., M alcata, F.X. 2006. M icroalgal reactors: A re vie w o f enclosed system designs and p erform an ces. B io tech n olog y Progress, 22(6), 14901506. Cuaresma Franco, M . 2011. C u ltiva tio n o f m icroalgae in a high irra dian ce area, PhD thesis, W ageningen U niversity, th e N etherlands. 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 Dubinsky, 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 a pplications. A q u a tic M ic ro b ia l Ecology, 56(2-3), 163-176. Falkowski, P.G., Raven, J.A. 2007. A q ua tic p ho tosyn th esis. 2nd ed. Princeton U nive rsity Press, P rinceton.
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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 Fernandez, F.G.A., Sevilla, J.M.F., Perez, J.A.S., G rim a, E.M., Chisti, M.Y. 2001. A irlift-d riv e n e xte rn a l-lo o p tu b u la r p h o to b io re a c to rs fo r o u td o o r p ro d u c tio n o f m icroalgae: assesm ent o f design and p e rfo rm a n ce . Chemical Engineering Science, 56(8), 2721-2732. Foyer,
C.H.,
Bloom ,
A.J.,
Q ueval,
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N octor,
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2009.
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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. Fuentes, M .M .R ., Sanchez, J.L.G., Sevilla, J.M.F., Fernandez, F.G.A., Sanchez-Perez, J.A., G rim a, E.M. 1999. O u td o o r co n tin u o u s c u ltu re o f P o rp h yrid iu m c ru e n tu m in a tu b u la r p h o to b io re a c to r: q u a n tita tiv e analysis o f th e d aily cyclic v a ria tio n o f c u ltu re param eters. Journal o f B iotechnology, 70(1-3), 271-288. 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 ia to m s. /. C yclotella n an a h uste dt, and D eton ula confervagea (Cleve) gran. Canadian Journal fo r M icro b io lo g y, 8, 229-239. Fluertas,
I.E.,
Espié,
G.S.,
Colm an,
B.,
Lubian,
L.M.
2000.
L ig ht-d ep en d en t
b ica rb o n a te upta ke and C 0 2 e fflu x in th e m arine m icroalga N annochloropsis g a d ita n a . Planta, 211(1), 43-49. Kaplan, A., R einhold, L. 1999. C 0 2 c o n ce n tra tin g m echanism s in p h o to s y n th e tic m icroorganism s. A nnual Review o f Plant Physiology and Plant M o le c u la r Biology, 50, 539-+. 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 io tech n olog y and B ioengineering, 108(10), 2390-2402. Lamers, P.P., Janssen, M ., De Vos, R.C.H., Bino, R.J., W ijffe ls , R.H. 2008. Exploring 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 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), 2427. 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), 32863292. 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 d io xide :
Design
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lipids
p ro d u c tio n
w ith
N eochloris
o le oabundans
in
p h o to b io re a c to r.
B ioresource Technology, 100(23), 5988-5995. Raso, S., van G enügten, B., V erm uë, M ., W ijffe ls , R.H. 2011. Effect o f oxygen 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 o f A pplie d Phycology, 1-9. 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), 327332. 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 subs 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
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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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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 Ugwu, C.U., Aoyagi, H., U chiyam a, H. 2007. In flue nce o f Irradiance, dissolved oxygen c o n ce n tra tio n , and te m p e ra tu re on th e g ro w th o f C hlorella so ro kin ian a. P h otosyn th etica , 45(2), 309-311. 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
B ioengineering, 31, 336-344. 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 ioproducts and B io re finin g, 4(3), 287295.
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72
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 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 2 Bioprocess Engineering, Wageningen University, P.O. Box 8129, 6700 EV, Wageningen, 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).
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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 hig h lig h t co nd itio ns 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).
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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
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 hig h lig h t co nd itio ns 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.
Results and discussion
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)
1.5 i
*
Cx(gDW.L*1)
■
n (day-1)
P3
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
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 hig h lig h t co nd itio ns Table 1 - Average biomass concentration and specific growth rate o f Neochloris oleoabundans during each phase o f the experimental run
Batch phase (P I) C ontinuous Light C onstant PO2=0.21 bar (P2) Light Regime 3 0 /6 C onstant PO2=0.21 bar (P3) Light Regime 3 0 /6 D ynam ic PO2= 0 .2 1/0 .6 3 bar (P5) Light Regime 3 0 0 /6 C onstant PO2=0.21 bar (P4) Light Regime 3 0 0 /6 D ynam ic PO2= 0 .2 1/0 .6 3 bar (P6)
Cx ± Stdev
p ± Stdev
(g L"1)
( d a y 1)
-
-
0.50 ± 0 .0 2
1.29 ± 0 .0 8
0.53 ± 0 .0 1
0.84 ± 0 .0 9
0.55 ± 0 .0 2
0.97 ± 0 .1 1
0.52 ± 0 .0 0 5
1.18 ± 0 .1 4
0.54 ± 0 .0 3
1.10 ± 0 .1 0
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 o
0.2 -
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
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 hig h lig h t co nd itio ns 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.
5.5.
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 en w erking sverba n d
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 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 sustainable source o f biofuels? Energy, 44(1), 158-166. 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 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. /. C yclotella n an a h uste dt, and D eton ula confervagea (Cleve) gran. Canadian Journal fo r M icro b io lo g y, 8, 229-239. 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 fo r algal cu ltures. Journal o f B iotechnology, 92, 113-131. 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 d io xide :
Design
and
p e rfo rm a n ce .
Journal
of
Chemical
carbon
T echnology
and
B iotechnology, 33B, 35-58. Raso, S., van G enügten, B., V erm uë, M ., W ijffe ls , R.H. 2011. Effect o f oxygen c o n c e n tra tio n on th e g ro w th o f N annochloropsis sp. a t low lig h t in te n s ity . Journal o f A pplie d Phycology, 1-9.
82
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 hig h lig h t co nd itio ns 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), 23472353. 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 subs a tu ra tin g lig h t in te n sity. B ioresource Technology, 104(0), 565-570. 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 B ioengineering, 42, 891-898. 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. 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 ioproducts and B iorefining, 4(3), 287295.
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C hapter 6
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 1Wetsus, P.O. Box 1113, 8900 CC Leeuwarden, the Netherlands 2 Bioprocess Engineering, Wageningen University, P.O. Box 8129, 6700 EV, Wageningen, 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
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
6 .1 .1 .1 . P h o to re s p ira tio n Figure 1 shows the biochemical
- ' * ■ 2 Ribulose 1,5-bP 2 CO;
reactions
2 0:
involved
in
photorespiratory
the
pathway.
Instead o f tw o molecules o f 3phosphoglycerate 2 Glycolate
(3-PGA),
the
reaction of ribulose bi-phosphate
Glycerate-3P
w ith 0 2 yields one molecule o f 32 NADH
ATP + NADH
2 Glyoxylate
Hydroxypyruvate
PGA and one molecule o f 2phosphoglycolate (2-PG). The full
4 ATP +■4 NADPH
photorespiratory cycle serves as a
Pyruvate NADH
4 GAP
2 Glycine
carbon
recovery
Serine
PGA
tha t
reductive
can
re-enter
cycle. A fte r 2-PG
dephosphorylated Sim plified scheme o f ph otosynthetic (a) and photorespira tory pathw ays (b) adapted fro m the m etabolic Figure
1
system
converting part o f the 2-PG to 3-
-
form ed into
ne tw ork described by Kliphuis e t al. (2011).
glycolate glycine,
and
the is the
is converted 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 increases,
th e oxygenase
th e ra tio b e tw e e n
a c tiv ity
o f th e enzym e
0 2 and C 0 2 in th e m edium 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
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
fo r
N eochloris
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
87
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.
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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
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.
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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 PCo2U 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 lph"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
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
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
91
C hapter 6
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
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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
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 .
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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 flu oresce nce to assess th e e x te n t o f p h o to in h ib itio n o f p ho tosyn th esis induced by 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 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 oxygen species involved in p h o to o x id a tiv e dam age to plants. Plant Physiology, 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, 329(5993), 796-799. 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), 287295. 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.
96
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
97
S um m ary
98
S um m ary
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 nears 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
99
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
100
S um m ary
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
z u urstofspa nn ing (Po2= 0,24; 0,63; 0,84)
bij
d rie
ve rsch ille nd e
p a rtië le
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
lic h tin te n s ite it.
onder V e rd e r
vo lle d ig w e rd e n
g e co n tro le e rd e de
e ffe cte n
co n d itie s op
de
b ij
groei
subverzadigde 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
z o n n e co lle cto r m e t een fa c to r 10 o nd erzoch t.
van
de v e rb lijfs tijd
in
In deze stu die w e rd e n
de 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
104
u it".
De specifieke
groeisn elhe id
w e rd
tijd e n s
de
d rie
ve rsch ille nd e
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
gesloten fo to b io re a c to re n b e re ik t kan w o rd e n .
106
van
N eochloris
oleoabundans
in
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
concentraçôes
cultivadas de
em
o xigénio
fo to b io rre a to re s constantes.
Em
näo
estäo
expostos
fo to b io rre a to re s
a altas
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
sim uladas num
variáveis
e conséquentes
condiçôes
de
luz fo ra m
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
im p le m e n ta çâ o das p rincipáis conclusöes deste estudo.
Foram
a
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 ckn ow le dg em en ts
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
A ckn ow le dg em en ts
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
A ckn ow le dg em en ts
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
W etsus,
m aking
th e
do
her
m aster thesis
ch a ra cte riza tio n
of
at 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
