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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: torzillo@ise.cnr.it (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.

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 116952

[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.

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

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).

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

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

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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