hydrogen production with the microalga chlamydomonas reinhardtii grown in a compact tubular...
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i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 7 ( 2 0 1 2 ) 1 6 9 5 1e1 6 9 6 1
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Hydrogen production with the microalga Chlamydomonasreinhardtii grown in a compact tubular photobioreactorimmersed in a scattering light nanoparticle suspension
Luca Giannelli, Giuseppe Torzillo*
Istituto per lo Studio degli Ecosistemi (ISE) del Consiglio Nazionale delle Ricerche (CNR), Sede di Firenze, Via Madonna del Piano, 10,
I-50019 Sesto Fiorentino, Firenze, Italy
a r t i c l e i n f o
Article history:
Received 12 June 2012
Received in revised form
10 August 2012
Accepted 23 August 2012
Available online 25 September 2012
Keywords:
Hydrogen
Chlamydomonas reinhardtii
Photobioreactors
Nanoparticles
* Corresponding author. Tel.: þ39 055 522599E-mail address: [email protected] (G. Tor
0360-3199/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.ijhydene.2012.08.1
a b s t r a c t
A new photobioreactor design (110 l) for the biological production of hydrogen with the
microalga Chlamydomonas reinhardtii is presented. The photobioreactor (PBR) was made up
of 64 tubes (i.d., 27.5 mm, length, 2 m) arranged on an 8 � 8 square pitch cell connected by
64 U-bends for a total length of 133 m. The PBR was contained in a rectangular parallele-
piped tank (2.5 � 2 � 2 m) made with isotactic polypropylene, except for the opposite
square faces which were made of transparent Plexiglas. The tubes were immersed in
a thermostatic water bath and continuously illuminated with artificial light. The culture
was circulated with a peristaltic pump. To attain a uniform distribution of light to the cells,
we used a suspension of silica nanoparticles that scattered the light supplied by the light
bulbs (2 � 2000 W) from the opposite square sides of the photobioreactor. Growth exper-
iments carried out with C. reinhardtii CC124 strain, showed a 23% net increase in the final
chlorophyll concentration when the nanoparticle suspension was used. Hydrogen
production with the C. reinhardtii strain CC124 was investigated with the new photo-
bioreactor design and carried out using a direct inoculum of sulfur-limited cultures having
a residual sulfate concentration below 1 mg l�1. The mean hydrogen output was
3121.5 � 178.9 ml. The reactor fluid dynamic was investigated, and a tri-dimensional light
profile inside the PBR is reported.
Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
reserved.
1. Introduction after the discovery by Melis et al. [3] who demonstrated the
Energy and environmental concerns prompt researchers to
explore new clean energy sources. An attractive and “green”
contribution to resolving this problem could be the photobi-
ological hydrogen production using some species of micro-
algae and cyanobacteria which are able to produce H2 when
grown under suitable conditions [1,2]. Among microalgae,
Chlamydomonas reinhardtii has gained increasing importance
2; fax: þ39 055 5225920.zillo).2012, Hydrogen Energy P03
possibility of achieving sustained hydrogen production by
means of sulfur deprivation. Sulfur deprivation causes
a progressive decrease in the photosynthetic O2-evolving
capacity of the cells, due to the lack of photosystem II (PSII)
repair function [3,4]. When the photosynthesis rate drops
below the level of respiration, the culture in sealed photo-
bioreactors becomes anaerobic in a short period of time [3].
Under these conditions C. reinhardtii is able to synthesize an
ublications, LLC. Published by Elsevier Ltd. All rights reserved.
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[FeeFe]-hydrogenase which combines electrons and protons
coming both from the water splitting complex and the
fermentation of starch accumulated during the aerobic phase,
to produce significant amounts of H2 for several days [5e9].
Until today, the production of hydrogen with C. reinhardtii
has been mostly carried out at laboratory scale [10e12], while
there is still little information on the scale-up of the process
[13,14]. One of the most important technical barriers to the
scale-up of hydrogen production with C. reinhardtii is the
capability to design suitable photobioreactors in order to
obtain a uniform illumination of the cultures. Several design
modifications have been proposed, most of them aimed at
improving light distribution on the surface of the cultures, but
no optimal configuration has been developed yet. One of the
key problems related to the illumination of microalgal
cultures is that light is not a miscible nutrient as are other
substrates, but rather a spatial external physics-dependent
self-distributing element. Light penetration in dense cultures
of microalgae is subjected to rapid attenuation due to scat-
tering and absorption from the cells [15,16] thus partitioning
the culture in several compartments with two extremes:
a highly illuminated external algal layer in which cultures are
very often subjected to light irradiance superior to that
required for saturation, and an inner onewhere cells are in the
dark. Between the two layers is an intermediate zone where
light becomes increasingly limited [17]. To attenuate the
effects of this physical constraint that actually acts as a strong
drawback in algal biotechnology, different techniques have
been proposed so far, such as fast mixing in order to induce
rapid light/dark cycles within the culture depth [11,18e20],
ultra-dense thin-layer cultures flowing on tilted smooth or
corrugated surfaces (cascades) [21e23], high intensity tangent
light dilution/scattering devices [24e26], and application of
scattering materials in the culture suspension [27]. All these
strategies aim at improving light conversion efficiency by
“diluting” the incident light over a larger culture surface and to
reduce, as much as possible, the amount of time the cells
spend in the dark i.e., deep layers of the culture and dark parts
of the reactors. In order to benefit from the light dilution
effect, sunlight impinging on a given ground area, should be
spread over the PBR surface area, and this can be accom-
plished by increasing the illuminated surface of the PBR. In
principle, the average daily sunlight irradiance recorded on
a horizontal surface should be reduced by a factor corre-
sponding to the ratio between the reactor surface and the
ground area it occupies (AR/AG) [28], such that the incident
light on the PBR surface falls below the photosynthetic light
saturation level of the culture. The optimum value of the AR/
AG ratio, therefore, will depend on the algal strain and the
place where the PBR operates.
Another important technical limitation to the scale-up of
the photobioreactor for hydrogen production, based on sulfur-
deprivation protocol [3], is the necessity to perform repetitive
washing of cells in sulfur-free medium by centrifugation. This
procedure has several disadvantages: it is costly, time
consuming and energetically inefficient, and it increases the
risk of culture contamination [29]. To circumvent this
problem, dilution methods to deprive C. reinhardtii cultures of
sulfur have been proposed [29,30]. The aim of this work was to
present a new design for a compact 110-l tubular
photobioreactor for a larger scale hydrogen production which
was immersed in a scattering light silica nanoparticle
suspension to attain an effective light diluting system. This
approach allowed an increase of the surface to volume ratio
(SVR) of the photobioreactor as well as the surface to footprint
ratio (SFR). Both factors are fundamental in improving the
performance of the photobioreactors. Moreover, a direct
inoculum of sulfur-starved cultures, eliminating the necessity
of a centrifugation step, has been tested on a larger scale.
2. Materials and methods
2.1. Algal strain and culturing conditions
C. reinhardtii CC124 cells were grown for three days (72 h) in
TAP (TriseAcetateePhosphate) medium in 5 � 400 ml
columns (5-cm optical path) to a final chlorophyll concentra-
tion of about 35 mg l�1, and immersed in a temperature
regulated water bath at 28 � 0.5 �C. Mixing of the cultures was
achieved by bubbling a mixture of air and CO2 (v/v, 97/3). A
mean incident photon flux density (PFD) of 70 mmol m�2 s�1
was supplied on both sides of the cultures using cool white
lamps (OsramDulux L 55W,Osram, Italy). Light irradiancewas
measured using a flat quantum-radio photometer sensor
connected to an LI-250A light meter (LI-COR, Biosciences,
Lincoln, NE, USA).
2.2. Inoculum scale-up
The cultures were first grown in TriseAcetateePhosphate
medium (TAP) in 5 glass columns, and thereafter were diluted
with fresh medium in five glass bottles (working volume, 8 l;
light-path 35 cm) using TAP and TAP-S respectively for growth
and hydrogen production experiments in the 110-l PBR.
Culture mixing was achieved by bubbling a mixture of air and
CO2 (v/v, 97/3). The cultures were irradiated with
300 mmol photonsm�2 s�1 from one side. After four days (96 h)
of growth, the cultures (about 40 l) were used to directly
inoculate the tubular PBR (110 l). Fresh sterile TAP-S prepared
with reverse osmosis water (ROW) was used to fill up the
photobioreactor. Sterilization of a large volume of culture
medium was carried out by means of both filtration and UV
treatments. Both sulfate concentration in the culture medium
and starch accumulation in the cells were measured at
intervals of about 12 h in order to identify the right conditions
for the final inoculation in the 110 l tubular PBR, which was,
usually carried out when the concentration of sulfur in the
medium dropped below 1 mg l�1, and the chlorophyll
concentration of the culture was about 15 mg l�1.
2.3. Analytical procedures
Chlorophyll concentration was determined spectrophoto-
metrically in 90% acetone [31]. During the hydrogen produc-
tion phase, the total biogas output was measured with an
automated software interface according to Ref. [11], and its
composition assessed by gas chromatography (Clarus 500,
Perkin Elmer, Waltham, Massachusetts) using a packed
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column (Carbosieve S-II Spherical Carbon, Supelco, Bellefonte,
Pennsylvania) and N2 as the carrier gas.
Biomass starch content was quantified according to Ref.
[32]. The solid starch granules were solubilized by boiling the
pellet for 10 min in distilled water. The calibration curve was
obtainedwith reagent grade anhydrous starch (Sigma Aldrich,
Milan, Italy).
The dissolved sulfate concentration was measured by
turbidimetry according to Ref. [33]. Cells were removed by
centrifugation and the culture medium was concentrated
10e20 times through evaporation, and the precipitate was
re-solubilized in 5-ml of distilled water. The calibration
curve was obtained following the same procedure used for
sulfate determination of the concentrated sample. The
method was validated with good results by following the
sulfate concentration changes during the growth curve of C.
reinhardtii cultures and compared to those reported in
Ref. [34].
2.3.1. Hydrogen collection and measurementH2 produced by the cultures in the 110 l PBR was measured
according to the system previously described in Ref. [14]. A
software for automatic culture control and data acquisition
Fig. 1 e Enclosed tubular photobioreactor used for the experime
the 110-l PBR. (b) Frontal view of the tubular PBR set in a contain
Detail of the PBRmade up 8 tube layers and connected each othe
the reactors showing the tube layers with opposite inclination
was used (Chemitec, Florence, Italy). Raw data recorded
during the experiments were then processed using a Linux-
based software written by us. Data of H2 gas measurements
were stored at 1-min intervals, and represented the average of
values recorded during a 3-s interval.
2.4. The photobioreactor
The pilot scale enclosed PBR with a culture hold-up volume of
110 l was composed of a grid of Plexiglas tubes made up of 64
elements (i.d.¼ 27.5mm; o.d.¼ 32mm; L¼ 2m) arranged on an
8 � 8 square pitch cell (equally distributed on a 110 � 110 cm
surface) and 64-U-bends to form a 133-m long circuit. A general
viewof the110 lPBR isshown inFig.1a.Tubeswereconnectedto
transparent U glass by means of transparent Plexiglas barrel
fittings, and were supported by a framemadewith transparent
Plexiglas which helped keep the tube array in a horizontal
position. The PBR was entirely contained in a rectangular
parallelepiped container made by isotactic polypropene (PP-H)
walls (2.5 � 2 � 2 m), except for the two opposite square trans-
parent faces which were made of Plexiglas panels (wall thick-
ness of 20 mm) (Fig. 1b). Culture tubes were immersed in
a scattering lightnanoparticle suspension (D-50: 0.54mm,99.0%;
nts of H2 production with C. reinhardtii. (a) General view of
er filled with a light scattering nanoparticle suspension. (c)
r by U-bends to form a 133-m long circuit. (d) Frontal view of
to facilitate culture draining.
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Fig. 2 e Time course of pH response to the injection of
a concentrated HCl solution at the entrance of the liquid
into the PBR. The automatically generated dimensionless
DpH trace plotted against elapsed time; the dashed line
represents the 0.05 DpH (t0) condition used to calculate the
mixing time.
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SkySpring Nanomaterials, Inc. Westhollow, Houston, USA). To
facilitate the draining of the tube layers were set slightly tilted
(Fig. 1c and d). At the end of the circuit the culture flowed into
a 2.2 l transparent Plexiglas cylindrical degasser (i.d. 10 cm,
height 40 cm, working volume 2.04 l). The degasser contained
several hose-fittings for fresh medium addition, air bubbling,
and for culture/biogas collecting and sampling. During the H2
production experiments, thehead space of the degasser, i.e. the
volume above the culture level, was about 0.35 l (i.e., 15% of to
total volume of the degasser). The surface to volume ratio
calculated as ratio between the surface area of tubes (circum-
ference) and the volume was 121.8 m�1. The culture was circu-
lated with a peristaltic pump (Model RM3, Valisi Pumps, Milan,
Italy). To prevent any risk of sulfate release to the medium,
a special elastomeric hose was used (Agi Pumps, Milan).
Culture temperature was kept constant during the exper-
iments using a flat plate heat exchanger (Mod. WP24-30 [CG1,
CG2], GEA WTT GmbH, Germany) connected to a heat pump
(Brat FF 0051, Climaveneta, Bassano del Grappa, Italy). The PBR
was operated by the control system previously described in
Ref. [11,14] where temperature, redox potential and dissolved
oxygen were recorded simultaneously. The thermostatic
liquid adopted was a water suspension of silica nanoparticles
used to scatter incident light incoming from two HQI-T 2000
W/D/I E40 4X1 lamps (OSRAM, Milan).
The PBR was equipped with a hydraulic-driven vertical
shutter used to regulate the amount of incident light delivered
to the cultures.
Table 1 e Mean head losses and Reynolds numbers asa function of the circulating fluid speed. Experimentswere carried out in triplicate using tapwater. Data are themean ± SD.
Culture speed(ms�1)
Reynolds number(e)
Head losses(Pa)
0 0 0
0.0927 � 0.006 2355 � 152 1516 � 174
0.1344 � 0.003 3416 � 76.2 2948 � 87
0.1330 � 0.016 3380 � 406 2893 � 627
0.2124 � 0.012 5397 � 305 6788 � 706
0.2373 � 0.015 6030 � 381 8328 � 973
0.2549 � 0.017 6475 � 432 9503 � 1175
0.2890 � 0.010 7343 � 254 12,006 � 774
0.3158 � 0.007 8022 � 178 14,163 � 587
2.5. Fluid dynamic reactor characterization
Three relevant fluid dynamic parameters were measured to
characterize the reactor; that is: the mixing time, the resi-
dence time and the head losses.
2.5.1. Mixing time measurementMixing time (tmix) and mean circulation time (tc) were evalu-
ated by pulsing a fixed amount (25 ml) of concentrated
hydrochloric acid inside the reactor and following the pH
changes over the time. The recorded data traces were elabo-
rated using a Linux software programwritten by us to execute
an algorithm in which the spikes representing the minimum
pH value were isolated by using the mathematical definition
of maximum/minimum over an interval. The isolated
minimum points were then automatically adjusted by the
value of the unaffected fluid flowing in the tubes (identifiable
as a maximum in the function) to calculate the DpH values to
be plotted against time (Fig. 2).
The mixing time (i.e. 95% response time) was assumed
equal to the time where the following condition was met for
the first time:
DpH ðtmixÞ � 0:05$DpH ðt0ÞThe time elapsed between one of the minimum pH spikes
and the following represents, of course, the value of the mean
circulation time (tc) which can be calculated easily using the
aforementioned software as a by-product. The parameters
calculated through the automated software approach were:
tmix ¼ 26:2 h
tc ¼ 10:98 min
2.5.2. Head loss for water recycleFrictional head losses have been calculated by direct flow
measurements on the reactor where the pump speed was
increased stepwise until its maximum was reached (nine
steps). The results were elaborated using well established
values for the concentrated head loss coefficients, that is, 1.25
for the bends, 1 for the PBR inlet, 0.5 for the PBR outlet, and
0.25 for the valve [35]. Churchill’s correlation was used for the
calculation of the friction factor [36]. The achieved results are
shown in Table 1. The culture speed selected for the culture
recycle was m ¼ 0.21 m s�1 which was a good compromise
between the necessity to reach a sufficient culture turbulence
(Re > 4000), and to minimize the head losses and cell damage.
During these field measurements, using the calculated fluid
speed and the tubes’ length, a second control value for the tcparameter could be calculated and used to verify the reliability
of the results obtained by the automated software and the
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accuracy of the implemented algorithm. The formula used for
the direct calculation is:
tc ¼�64$LPipe þ 5
�$u ¼ 10:44 min
whereu is theculturespeed,andLPipe, the lengthofasingle tube.
The result deviates only by 4.9% (still within experimental error
boundaries) acting as positive validation for the automated
software calculations and thus for the calculated tmix value.
2.6. Calculation of light absorption by the culture
The fraction of the total incident PFD absorbed by the culture,
was obtained using the information gathered with measure-
ments of light attenuation inside the nanoparticle suspension.
Given the light as a function of the axial coordinate (x), the
total diffused light captured by each infinitesimal surface
element of the pipes can be used to calculate a point-by-point
integral on the overall length. The scheme of the integration
grid used for the calculations is shown in Fig. 3.
The formula used to calculate the total absorbed photons
was characterized by two contributions: the diffused light
absorbed by the pipe walls, and the direct light harvested by
the pipe sections perpendicular to the incident light (i.e. the
transparent section of the curves).
ITot ¼ IPipes þ ICurves (1)
IPipes ¼ SPipes$PFDPipes (2)
For the calculations, a uniform value of
1000 mmol photons m�2 s�1 was adopted for the incident radia-
tion on both illuminated surfaces which represents the surface-
averagedvalueof the lightmeasurementsheldoneach face.The
PFD decrease along thewhole length of the PBR is describedwell
by using the results obtained through the light extinction
experiments; that is, the value of the total incident light on the
pipe surface can be expressed as a point function of the x coor-
dinate. PFDPipes canbe calculated applying thebasicdefinition of
the integral operator to this function, thus obtaining the Equa-
tion (3). The value for the total irradiated surface of the pipes
used in the equation could be calculated directly by the external
diameter and the pipe length, multiplied by the total number of
pipes (64), giving a total surface of 13.4 m2. The integral part,
subdivided in n sections of small length (0.01 m) can be solved
Fig. 3 e Scheme of a sample pipe section of the tubular PBR
(half of the total length) where the integration grid for the
calculation of the total incident PFD is represented. The
PFD function along the x axis is represented by the Rn
values (diffused radiation intensity on the nth surface).
together with the values of diffused radiation on each section
(Rn), obtaining an integral averaged PFD of
63.8 mmol photonsm�2 s�1. Using these calculated numbers, the
total contribution from the scattered light amounts to:
Ipipes ¼ Spipes,
Z
L
RðxÞdx ¼ Spipes,Xni¼1
Ri þ Ri�1
2ðxi � xi�1Þ
¼ 854:64 mmol s�1 (3)
The second scattering component coming from the curves in
thepipes couldbedirectly estimatedbycalculating the incident
light on a given surface as reported in equation (2). The value of
the illuminated surface used in this equationwas derived from
thesectionof thecurves (0.05m)where theculture isexposedto
a direct radiation of 360 mmol photonsm�2 s�1 (as calculated by
the light extinction measurements). Using these values, the
amount of absorbed photons from direct radiation is easily
attained:
ICurves ¼ SCurves$PFDCurves ¼ 0:321$360 ¼ 115:56 mmol s�1 (4)
3. Results
3.1. Nanoparticle light scattering and attenuationcharacterization
To ensure uniform illumination of the cultures despite the fact
that the tube grid was oriented in the same direction as the
incident radiation, the use of scattering particles was
investigated.
Fig. 4 e Light scattering (A) and extinction (B) plots along
the PBR depth. Four different concentrations were tested:
10 mg lL1 (circles), 20 mg lL1 (squares), 40 mg lL1 (triangles)
and 60 mg lL1 (diamonds).
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Nanoparticle light attenuation and scattering were studied
as a function of their concentration (10, 20, 40 and 60mg l�1) to
find the most effective one to diffuse light along the perpen-
dicular direction to incident light. Measurements were carried
out in an 80 l capacity, rectangular vessel having the same
light path length (2.0 m) as those in the real PBR to interpolate
the results of the direct measurements without introducing
any extinction or scattering models. The light attenuation
curves and the perpendicular scattered radiation intensity
plotted against the reactor depth are depicted in Fig. 4.
The concentration of nanoparticles, which proved to be
more effective for perpendicular light scattering, was
60 mg l�1; using this concentration the maximum incident
light recorded on the tube surface was 1.5 fold higher than
that achieved with 40 mg l�1 nanoparticles. Despite their high
scattering effect, the asymptotical light intensity, i.e. that
recorded far away from the illuminated surfaces, was too low
to ensure proper illumination of the PBR’s deepest parts. On
the contrary, the highly light permeable suspension of
10 mg l�1 showed a perpendicular scattering component
insufficient to sustain a satisfactory culture growth with
a maximum incident light of 6 mmol photons m�2 s�1 in
correspondence to a culture depth of 10 cm. A reasonable
compromise between these two situations was achieved with
the concentration of 40 mg l�1 nanoparticles. This concen-
tration was adopted for the following growth experiments
since it allowed the best ratio between light penetration to
perpendicular scattering to be achieved.
Combining the data acquired by these experiments with
those of incident light on the PBR’s illuminated surfaces an
accurate profile of the light intensity inside the nanoparticle
suspension was produced (Fig. 5). The light impinging on the
Fig. 5 e Tri-dimensional light extinction profile inside the PBR. T
the illuminated surfaces. The intensity peak included in this sam
due to reflection phenomena inside the illuminating device. Th
visualize the homogeneity of the achieved light distribution.
PBR’s transparent surfaces was characterized by a non-
homogeneous distribution around an average value of
1000 mmol photonsm�2 s�1 as a consequence of unpredictable
reflection phenomena inside the illuminating device hosting
the lamps. This behavior, however, does not represent
a problem when nanoparticles are involved but can create
a highly heterogeneous illumination pattern when only water
is used as the thermostatic liquid. As shown by the iso-curves
in Fig. 5 when nanoparticles were used, highly uniform illu-
mination can be attained. The incident light intensity of
1000 mmol photonsm�2 s�1 is decreased to one third in the first
20 cm of the reactor not occupied by culture tubes, and is then
maintained close to 100 mmol photons m�2 s�1 i.e., below the
level of photosynthesis saturation for C. reinhardtii, for the
entire length of the tubes in the grid. For this reason, this
system proves to be very effective in diluting excessive inci-
dent light and should be tested under natural over-saturating
sunlight conditions.
3.2. Growth experiments
Three different illumination intensities were tested to assess
the PBR performance with C reinhardtii cultures with and
without the addition of nanoparticles in the water bath in
which culture tubes were submerged.
The lowest light irradianceof500þ500mmolphotonsm�2 s�1
was testedwithoutnanoparticles only as itwas relatively low to
be used for light scattering, i.e. to promote light dilution. Under
these illumination conditions, C. reinhardtii cultures grown in
full medium (TAP), the chlorophyll concentration increased up
to 32 mg l�1 within about 4 days of continuous illumination.
Doubling the light intensitywithout theuseof thenanoparticles
he data refer to a 50-cm section located around the center of
ple section shows the unevenness of the incident radiation
e bottom of the plot represents the iso-curves useful to
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Fig. 6 e Algae growth curves expressed as a function of the
total biomass chlorophyll content. Key: circles, 500D 500
mmol photons mL2 sL1 control (without nanoparticles);
triangles, 1000D 1000 mmol photons mL2 sL1 control;
diamonds, 1000D 1000 mmol photons mL2 sL1 with
nanoparticles.
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provoked a clear increase in algal growth, measured as an
increase in the chlorophyll concentration, up to 64.6mg l�1. The
addition of the nanoparticles in the thermostatic fluid
compartment caused a sharp increase in chlorophyll, up to
79.5mg l�1 (Fig. 6).Themajorcontribution to thegrowthattained
with the use of nanoparticles was observed within the first 4
days of growth, thereafter, with the increased photolimitation,
growth almost leveled in both the cultures. From these experi-
ments it was possible to conclude that cultures irradiated with
1000þ 1000 mmol photonsm�2 s�1 (i.e., suppliedonboth sidesof
the PBR), the addition of nanoparticles promoted a 23% increase
ingrowthcompared to theculture irradiatedwith thesame light
irradiance but without nanoparticles.
3.3. Sulfur consumption kinetics
Sulfur deprivation of cells was achieved with substantial
modification to the standard protocol [3], that is, through
culture dilution and consumption by cells.
Fig. 7 e Sulfate consumption of a hydrogen production
experiment, from the inoculum in the bubble column
reactors to the final exhaust culture. The different phases
of the experiment have been highlighted in-graph with
simple sketches for a direct visualization of the different
steps involved and the corresponding sulfate
concentration. Time reported indicates the overall duration
of the experiment since the initial setup.
The decay of sulfur concentration during the growth was
monitored daily in order to determine the desired concen-
tration of residual sulfur to be used for experiments of
hydrogen production in the 110-l PBR. The results of the
experiment are shown in Fig. 7.
The cultures were grown in TAP medium for 72 h of
continuous light (70 mmol photons m�2 s�1), in 5 � 400 ml
columns. During this time they reduced the sulfate concen-
tration from 51 mg l�1 to 21.6 mg l�1. Thereafter, the cultures
were diluted in TAP-S, in four 8-l bottles. In these vessels,
cultivation proceeded for an additional 92 h of continuous
illumination. Due to the scale factor between the first two
steps of the culture inoculum preparation and growth in TAP-
S medium, the sulfate concentration at hour 72 dropped by
one order of magnitude, i.e. to 3.93 mg l�1. During the
following hours of cultivation, the sulfate concentration
declined at a constant rate. The last section of Fig. 7 shows an
unexpected increase in the sulfate concentration from
0.75mg l�1 to 3.2mg l�1after the direct inoculum of the culture
from the four 8-l bottles inside the large scale PBR (110 l). Such
an unexpected increase in the sulfate concentration was due
to the quality of water (ROW) used for the preparation of the
culturemediumwhichwas found to contain a fixed amount of
dissolved sulfate that resulted mainly from equilibrium
phenomena across the membrane of the reverse osmosis (RO)
apparatus.
During growth of the culture in 8 l bottles starch began to
accumulate in the cells (Fig. 8) while the concentration of
sulfate decreased to below 1 mg l�1.
3.4. Hydrogen production experiments in the 110 l PBR
To achieve a more uniform illumination, the hydrogen
production experiment using the C. reinhardtii cultures were
carried out with the PBR immersed in the nanoparticle
suspension. The initial chlorophyll concentration used during
the experiments was attained by five 8-l bottles (about 40 l
total) for the inoculation stage. The inoculum was poured
directly inside the PBR and diluted with fresh TAP-S medium
until the PBR was filled up (110 l). The average hydrogen
production obtained in these experiments (3 replicates) was
3.12 � 0.17 l, which corresponded to a mean rate of
0.61 ml l�1 h�1 (Fig. 9). The value of biogas shown in Fig. 9,
represents pure hydrogen which was present in the biogas
Fig. 8 e Starch accumulation curve during the cultivation
inside the 8-l bottles (time between 72 and 168 h).
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Fig. 9 e Hydrogen production attained with C reinhardtii
cultures grown in the 110-l PBR using artificial light
(1000 D 1000 mmol photons mL2 sL1) supplied in on both
sides of the PBR. The continuous line indicates the
hydrogen production by culture exposed to scattered light
by nanoparticle suspension in which PBR was immersed,
while broken line indicates the hydrogen production
achieved by the control culture exposed to the same
incident light intensity without nanoparticles. For clarity,
the oxygen accumulated during the initial phase (20 h) has
been omitted, as well as the aerobic stage of cultivation
(first 20 h).
i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 7 ( 2 0 1 2 ) 1 6 9 5 1e1 6 9 6 116958
accumulated over the experiments, calculated by multiplying
the total biogas volume by the final H2 concentration obtained
through gas chromatography analysis. As expected, corre-
sponding H2 production obtained with similar cultures
exposed to same light irradiance without nanoparticles was
lower, about 1870 ml, corresponding to an H2 production rate
of 0.42 ml l�1 h�1 (Fig. 9).
3.5. Light-to-hydrogen conversion efficiency
Assuming that one mole of photons in the PAR radiation
carries an average energy content of 209 kJ mol�1 and that the
lower heating value of hydrogen is 0.01294 kJ ml�1 [34], the
light-to-hydrogen conversion efficiency in the high concen-
tration experiments has been calculated according to the
following formula (total hydrogen volume expressed in ml
and total production time in h):
h ¼ VH2$DHcomb;H2
ITot$10�6$3600$t$Ephot
$100
¼ 3121:5$0:01294970:2$10�6$3600$25$209
$100
¼ 0:213
(5)
4. Discussion
4.1. Light intensity management
In this work we have tested the use of a nanoparticle
suspension as a highly scattering medium enabling light
beams parallel to the PBR tubes to be dispersed along all
possible plains and act as an effective light diluting system.
This configuration allowed us to increase the light dilution
ratio, which, eassuming that all the impinging light is in the
end absorbed by the culturee, can be calculated as the ratio
between the surface of the opposite transparent faces of the
PBR, 1.6m2 each, and the total surface of tubes 13.4m2 (that is,
13.4 m2/(1.6 m2 � 2) ¼ 4.18). This means that the incident light
on the tube surface (cross section of the PBR) is reduced by
a factor of about 4. However, this ratio would indicate mainly
a mere geometric reduction of incident light, if no scattering
materials were suspended in the water bath in which the
photobioreactor was immersed. In fact, owing to the geometry
of the PBR with tubes which were parallel to the main light
direction, and without any scattering, the amount of light
absorbed by the culture would mostly be the light impinging
on the curves whichwere situated perpendicularly to the light
direction. Actually, scattering effect was achieved even
without the nanoparticles suspension, as shown by the
growth achieved during the experiments, nevertheless the
nanoparticles suspension proved capable of increasing the
PBR productivity by 23%.
This newmethod for light dilution showsat least twomajor
advances over state of the art PBR designs. The first one is
represented by the way light is distributed to the culture. As
shown in Fig. 5, an incident light of 2000 W supplying
1000 mmol photons m�2 s�1 on each of the two transparent
faces of the reactor, is roughly one order of magnitude higher
than the photosynthesis saturation of C. reinhardtii [11]; with
nanoparticles, the lightwas reducedbelow the saturation level
for C. reinhardtii (around 100 mmolm�2 s�1), thus improving the
light conversion efficiency. In addition to the reduced incident
light intensity, there was the evident advantage of a more
uniform illumination achievable along the entire length of all
the PBR’s tubes, even if tangential illumination is applied. This
feature can allow a highly efficient scale-up of the PBR by
increasing the number of tubes (i.e., by decreasing the pitch
size) without altering the footprint. The second major
advancement is represented by the fact that with the use of
nanoparticles it is virtually possible to obtain, by changing
their concentration, an unlimited range of incident light
intensities for growing anumber of photosynthetic organisms,
illuminated with both artificial and solar light. This is espe-
cially useful for anoutdoor, direct solar light drivenproduction
where the high direct radiation intensity may cause over-
saturation with loss of efficiency, or even photoinhibition
which may damage the culture [14,37]. With the PBR design
described here, even direct incident sunlight can be profitably
“diluted” within the limit for photosynthesis saturation
(around 200 mmol photons m�2 s�1).
4.2. Sulfur starvation protocol
With few exceptions [13,38], the hydrogen production through
sulfur-starvation approach has always been conducted using
the two-step protocol [3]. The maximum value of starch
accumulated by the cells after 48 h of illumination was
96.82 � 0.14 mg l�1 which can be considered an adequate
amount to start hydrogen production. Moreover, according to
Ref. [30], the threshold of residual sulfur concentration
allowing the highest hydrogen production corresponds to
40 mM, and this value was used in this study to calculate the
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i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 7 ( 2 0 1 2 ) 1 6 9 5 1e1 6 9 6 1 16959
length of the starvation time. It is well known that metabolic
shift between sulfur starvation and normal growth is quite
fast due to the sulfur-scavenging enzymeArs and the speed of
the sulfate-permease I and II [39] therefore, inoculating the
PBR with a sulfur-starved culture still carrying amounts of
sulfur above that critical threshold causes the risk of a rapid
depletion of starch reserves before the onset of hydrogen
production. In fact, there is a general consensus that a low
starch content in correspondence to the start of the hydrogen
production phase usually leads to a scarce overall production
[6,8,40,41]. This is why the amount of starch accumulated in
the cells was measured at time intervals so as to calculate, as
precisely as possible, the right time to transfer the inoculum
into the PBR.
4.3. Hydrogen production and efficiency
The production recorded in these experiments, when
normalized on the culture volume, represents a notable 1.7-
fold increase compared to that achieved by the same strain
in a previous experiment carried out in a 50-l tubular PBR
where we obtained about 850 ml of hydrogen (i.e.,
0.17 ml l�1 h�1) [14]. As the inoculum technique was different,
a direct quantification of the nanoparticle effect on the
hydrogen production performance of the two PBR designs
cannot be easily achieved. Nevertheless, it is conceivable that
as the sulfur starvation of cultures attained by dilution is ex-
pected to yield a lower hydrogen amount compared to that
attained through cell washing in TAP-S medium [30], the
increased hydrogen production achieved using the former
should be expected to be lower than optimal. For this reason,
the factor responsible for the augmented production can be
attributed to the introduction of the nanoparticle suspension.
This explanation is further supported by the lower amount of
H2 attained with the control culture (without scattering light
nanoparticles). Hydrogen production efficiency under sulfur-
limited conditions has been widely investigated and re-
ported to be low, due to the downregulation of the PSII by
sulfur starvation. Many workers aimed at maximizing this
efficiency through cell immobilization or enhanced mixing
conditions [10e12,42], reporting values ranging between 1%
and 2% [41,42], or much lower in the case of direct solar light
use, about 0.05% [14]. This work aimed at circumventing the
problem of light saturation by using a nanoparticle suspen-
sion as a high light dilution matrix. The artificial light of an
intensity close to 1000 mmol photons m�2 s�1 per side was
adequate to ensure sustainable hydrogen production in a 110 l
PBR. The calculated efficiency was 0.213% which was more
than 2-fold higher than that achieved in our previous experi-
ments [14], but still almost one order lower than that attained
in laboratory cultures, meaning that other limiting factors
may have played an important role. Among them, the overly
length mixing time characteristic of the 110-l tubular reactor
(ca 24 h) which increased the hydrogen saturation level of
cultures [14]. Recent results [43], have shown a direct inhibi-
tion effect of high hydrogen concentrations in the PBR head
space on hydrogen photoproduction activity in laboratory
algal grown cultures and clearly demonstrated that hydrogen
output in C. reinhardtii depended significantly on the level of
partial pressure of H2 in the PBR gas phase. Increasing the gas
phase to liquid phase volume by a factor of 4 resulted in
a 100% increase in the hydrogen output. These findings are
important for the optimal design of the PBR for hydrogen
production. However, in the case of tubular photobioreactors,
characterized by a perfect plug flow regimen inside the tubes,
the increase of the liquid/head space interface has little or no
effect on the gas removal due to the extremely high ratio
between the residence time inside the circuit compared to
that in the degasser. Increasing the flow rate can help reduce
both the mixing and residence time thus helping to lower the
hydrogen/culture contact. However, replacing the curves with
properly designed manifolds conveying the gas toward the
degasser could be the best solution to the gas removal
problem in the tubular reactors.
Nevertheless, the total volume collected over the experi-
ments was considerably higher (3121.5 � 178.9 ml, i.e.,
0.60ml l�1 h�1) than that reported up to nowwith C. reinhardtii
and it is conceivable that the better performance attained
compared to a previous experiment in a 50-l PBR [13] was due
to the more uniform illumination pattern promoted by the
nanoparticle light scattering. However, notwithstanding this
considerable increase in the production rate, there is still a gap
of more 50% in the production rate between laboratory
cultures (1-l PBR) and scaled PBR (up to110-l volume) which
has to be bridged [11].
While preparing this manuscript, another interesting
application involving scattering nanoparticles to improve
light distribution was proposed [27]. As a scattering matrix,
sponges coated with scattering SiO2 were used, directly
immersed in the cultures. However, direct immersion of
scattering material in the culture may reduce the working
volume of the photobioreactor and increase the risk of cell
sticking on the scattering surfaces. Moreover, scattering
material directly immersed in the culture is expected to be
more effective with diluted cultures which allow the light to
deeply penetrate the cultures, and this contrasts with the
necessity to operate with dense cultures to reduce harvesting
costs. In our case, the nanoparticle suspension was physically
and spatially separated from the culture itself. This solution
was chosen to facilitate photobioreactor cleaning and
management. The compact PBR design with light scattering
nanoparticles presented here can represent a basic platform
that can be expanded by implementing a solar tracking light
system able to concentrate light and deliver it to the two faces
of the PBR. Concentrated solar light could be properly diluted
with an appropriate concentration of nanoparticles to attain
a highly illuminated and controllable outdoor closed solar
light-driven production system. This design can be extended
to include the production of high value substances produced
by microalgae.
5. Conclusions
This paper proposes a new method to avoid the light satura-
tion effect by using nanoparticles for light scattering. The
nanoparticles allowed a dilution of light by a factor of four,
which strongly reduced the risk of oversaturation of the
photosynthetic electron transport chain. Cultures grown in
tubes submersed in nanoparticles showed higher growth
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i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 7 ( 2 0 1 2 ) 1 6 9 5 1e1 6 9 6 116960
compared to the ones grown in the absence of nanoparticles.
The final target of this photobioreactor design is the exposure
of the culture to solar light harvested through solar tracking
mirrors delivering solar light to the culture through two light
ducts, thus replacing the artificial light supply. Another
important advancement reported in this work was the elimi-
nation of the centrifugation step which is mandatory for
developing hydrogen production on a large scale. The meth-
odology proposed was based on careful tracking of the sulfur
concentration during the scale-up of the inoculum.
Acknowledgments
This work wasmainly supported by the MIUR (Italian Ministry
of University and Research) through the project “Metodologie
Innovative per la Produzionedi Idrogenoda Processi Biologici”,
Fondo Integrativo Speciale per la Ricerca, FISR, contract
number 1756. Partial support was also provided by the joint
project “Microalgal biohydrogen production: From laboratory
to outdoor photobioreactors” in the framework of the Bilateral
Scientific Agreement between the National Research Council
of Italy and the TUBITAK (Turkey). The authors thank Mr.
Edoardo Pinzani for technical assistance. Special thanks are
due to Mr Marcello Diano from M2M Engineering (Caserta,
Italy) for the 3D representation of the 110-l photobioreactor.
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