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Hydrogen production from biological systems under differentillumination conditions

Federico Rossi 1, Mirko Filipponi*

CIRIAF, University of Perugia, Industrial Engineering Department, Via G. Duranti 67, 06125 Perugia, Italy

a r t i c l e i n f o

Article history:

Received 7 September 2010

Received in revised form

11 March 2011

Accepted 20 March 2011

Available online 20 April 2011

Keywords:

Hydrogen

Chlamydomonas reinhardtii

Artificial illumination

Bioreactor

* Corresponding author. Tel.: þ39 0744 49296E-mail addresses: frossi@unipg.it (F. Ross

1 Tel.: þ39 075 585 38 44.0360-3199/$ e see front matter Copyright ªdoi:10.1016/j.ijhydene.2011.03.124

a b s t r a c t

Hydrogen is a natural by-product of several microbial driven biochemical reactions, mainly

in anaerobic fermentation processes. In addition, certain microorganisms produce

enzymes by which H2 from water may be obtained if an outside energy source, like

sunlight, is provided. Biophotolysis is a biological process which involves solar energy and

algae clusters to convert water into hydrogen. Algae pigments absorb solar energy and

enzymes in the cell act as catalysts to split water into hydrogen and oxygen. There are

many research activities studying hydrogen production from biological systems cyano-

bacteria and green algae and some studies present a complete outline of the main available

pathways to improve the photosynthetic H2 production [1,2].

Efficiency (energy produced from hydrogen divided by solar energy) of such processes

can be estimated up to 10%. This value has to be increased for a large-scale hydrogen

production. The effect of different artificial illumination conditions on H2 production was

studied for green algae cultures (Chlamydomonas reinhardtii). Results will be used to design

a high-efficiency photobioreactor for a large-scale hydrogen production.

Copyright ª 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights

reserved.

1. Introduction energy resources may be exploited, processes occur at atmo-

Hydrogen has received great attention from the international

community during the last decades because of its environ-

mental benefits: it does not evolve the greenhouse gas, its

energy content permass unit is the highest of any known fuel,

it is easily converted into electricity by fuel cells and water is

the only combustion by-product. Currently, 40% of hydrogen

is produced from natural gas, 30% from heavy oils and

naphtha, 18% from coal, 4% from electrolysis and about 1% is

produced from biomass [3].

Today, biological H2 production from algae and bacteria

is becoming important mainly for two reasons: renewable

9.i), filipponi.unipg@ciriaf.i

2011, Hydrogen Energy P

spheric temperature and pressure.

In order to determine the better conditions to carry out this

process, the influence of many different physical and physi-

ological factors has to be considered. Microscopical green

algae such as Chlamydomonas reinhardtii can produce H2 using

sunlight as energy source. Microalgae can use only radiation

with a wavelength between 400 and 700 nm. This part of the

solar spectrum is called Photosynthetic Active Radiation

(PAR). On an energy basis, 43% of the solar radiation is in the

PAR region [4,5].

Hydrogen photoproduction in green algae can be sustained

by depriving the cells of sulphur [3,6]. Sulphur deprivation

t (M. Filipponi).

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 6 ( 2 0 1 1 ) 7 4 7 9e7 4 8 67480

causes a progressive and specific decrease in the photosyn-

thetic O2-evolving capacity of the cells, due to the lack of

photosystem II (PSII) repair function [7]; it has little effect on

cellular respiration and results in culture transition from an

aerobic to an anaerobic state [3,6,8,9].

The establishment of anaerobiosis in a photobioreactor

induces the expression of two [FeFe]-hydrogenases in algal

cells [10,11]. These enzymes redirect the flow of electrons

coming from the photosynthetic electron-transport chain in

thechloroplast fromcarbonfixation towardsproton reduction.

As a result, sulphur-deprived algae produceH2 for several days

[3,6]. During sulphur deprivation, the algal cultures progress

through the following five phases: aerobic, O2-consumption,

anaerobic, H2-production, and termination phases [8].

This protocol is also usually applied in photoheterotrophic

conditions (in the presence of acetate): acetate is sufficient to

obtain anoxic conditions involving a low production of

hydrogen. Therefore, PSII inhibition by sulphur deprivation

could not be necessary to reach anoxic conditions when

cultivated in photoheterotrophic conditions. So, the exact

effects of both sulphur deprivation and acetate are not clear.

Three tests have been conducted to investigate biological

hydrogen production from C. reinhardtii cultures under

continuous artificial illumination. In the first test, lamps with

different emission spectra and different colour temperature

have been employed to determine the best artificial illumi-

nation conditions for H2 production. Influence of growth

medium composition both on hydrogen production and on

algae growth has also been investigated. In the second test,

the effect of growthmedium amount on hydrogen production

has been evaluated. In the third test, the effect of restoring the

culture medium has been investigated.

Artificial illumination is important in order to determine the

best light spectrum in terms ofH2 production: natural lightmay

befilteredorshifted inorder to reproduce thebest spectrum[12].

2. Experimental tests

Three different tests have been performed to value the effect

of different parameters on hydrogen production by C.

reinhardtii:

- the first test (Test 1) has been carried out to evaluate the

influence of both illumination conditions and growth

medium composition on hydrogen production;

- the second test (Test 2) has been carried out to evaluate the

influence of the growth medium amount on hydrogen

production;

- the third test (Test 3) was carried out to determine the effect

of medium restoration on hydrogen production.

Fig. 1 e Experimental setup-three identical samples of

culture in the same photobioreactor.

2.1. Test 1: evaluation of the influence of illuminationconditions and grown medium composition on hydrogenproduction

ThesampleswerepreparedbymakingcellsofC. reinhardtiigrow

on two different culture media: TAP (TriseAcetateePhosphate)

mediumwith sulphates and sulphur-deprived TAPmedium.

The lack of anoxic conditions in cultures grown in TAPwith

sulphate is unfavourable to hydrogen production. So, on equal

terms of illumination, it is expected that the hydrogen

production is much greater in cultures grown on sulphur-free

TAP than in cultures grown on TAP with sulphate. Liquid

cultures were grown at about 28 �C [8] in graduated borosili-

cate glass bottles with n. 5 ground necks closed by plastic

screw taps with apertures with porous buthile/teflon septa.

These septa have low gas permeability and permit to sample

the inner gas from the bottle using a syringe. Each bottle has

been filled with 200 ml of culturemedium and 15 � 106 cells of

C. reinhardtii (1.8 mg Chl./ml).

The choice of this small concentration of C. reinhardtii

allows the light to penetrate deeper into the solution, before

the hydrogen production drops off [13]. It has been also

observed that specific rate of H2 photoproduction increased

with decreasing in cell density [8].

The volume VS of each bottle headspace was 435 cm3.

Bottles have been selected for their high transmittance to

sunlight. In spectral range from approx. 310 to 2200 nm the

absorption of glass is negligible.

The cultures have been maintained under continuous

illumination inside three photobioreactors. Three identical

samples of the same culture (three samples of C. reinhardtii

grown in TAPmediumand three sample of C. reinhardtii grown

in sulphur-deprived TAP medium) were installed inside each

photobioreactor (Fig. 1). A photobioreactor consists of

a parallelepiped box with reflecting walls. In each photo-

bioreactor a different type of lamp was installed.

Two types of vapour fluorescent lamps were used (colour

temperatures of 5600 and 2700 K); a solar simulator (model

91160, Newport) equipped with xenon short arc lamp (colour

temperatures of 5800 K) is also used, in order to study different

illumination conditions. Lamps have been selected consid-

ering their emission spectra and the light-absorption spec-

trum of green algae.

Figs. 2, 3 and 4 show the spectra of the lamps.

A large part of the sunlight spectrum is not absorbed by

green algae as shown in the graph of Fig. 5 that represents the

absorption spectrum of a culture of C. reinhardtii in sulphur-

Fig. 2 e Fluorescent lamp TC [ 2700 K emission spectra

(figure height[ð400 mW=1:000310 nmÞ). Fig. 4 e Xenon lamp TC [ 6800 K emission spectra.

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 6 ( 2 0 1 1 ) 7 4 7 9e7 4 8 6 7481

deprived TAP medium (LAMBDA� 750 UV/Vis/NIR Perkin

Elmer spectrometer).

For this reason, the efficiency of transformation of sunlight

energy into hydrogen energy is theoretically limited. The

maximum absorption occurs into [400, 500] nm and [650,

700] nm ranges. In particular, a fluorescent lamp with 2700 K

colour temperature has been chosen because of its high

emission at 430 nm, corresponding to the lower absorption

wavelength of green algae; the fluorescent lamp with 5600 K

has been chosen because of its selective emission in the [570,

640] nm wavelength range, close to the highest absorption

wavelength of algae. Among artificial light source, Xenon lamp

spectrum matches very well to sunlight. Such lamp has been

selected to simulate sunlight and to verify the effects of illu-

mination with a full spectrum light on hydrogen production.

The amount of hydrogen produced by each culture was

measuredbysamplinggas fromthebottleheadspacewitha gas

tight syringe. A gaschromatograph (model CP 4900, Varian)

with data analysis software (Star 6.41, Varian) was used to

determine the concentration (% vol) of H2 in the headspace of

each bottle. A molecular sieve column (MS-5A) was used to

separate O2, N2 and H2. Signals were generated by the thermal

conductivity detector of the instrument. The signals were

calibrated by injection of known amounts of compounds.

The Test 1 was conducted two times to ensure reliability:

the first one from February 1st, 2010 to March 4th, 2010; the

second one from October 11th, 2010 to November 12th, 2010.

Fig. 3 e Fluorescent lamp TC [ 5600 K emission spectra

(figure height[ð400 mW=1:000310 nmÞ).

Test 1 lasted 32 days and involved 11measurements each time

it was run. Volume of hydrogen concentration measured in

the bottles for each experimental campaign (at the time of

each measurement) is reported in Tables 1 and 2. Data of the

third, fourth and fifth column of both Tables represent the

average hydrogen production rate of the three identical

culture samples inside each of the three photobioreactors,

measured during first campaign. Data of the sixth, seventh

and eighth column represent the average hydrogen produc-

tion rate of the three identical culture samples inside each of

the three photobioreactors, measured during second

campaign. Data of the ninth, tenth and eleventh column

represent the mean values of the two campaigns.

2.2. Test 2: evaluation of the growth medium hydrogenproduction

The Test 2 has been carried out to evaluate the influence of the

growth medium on hydrogen production. The samples were

prepared by growing cells of C. reinhardtii only on sulphur-

deprived TAP medium because such growth medium is the

one which provided the best results during Test 1. Liquid

cultures were grown at about 28 �C in the same graduated

borosilicate glass bottles employed for the first experimenta-

tion. The first half of the bottles have been filledwith the same

amount of culture as in the first experimental campaign

Fig. 5 e absorbance spectrum of C. reinhardtii in sulphur-

deprived TAP medium.

Table 1 e Hydrogen concentration inside bottles holding cultures in TAP with sulphate, at the time of each samplecollection (Test 1).

Measurenumber

Incubationtime(days)

Hydrogen volumeevolume percentage (v/v %)

15 � 106 Chlamydomonas reinhardtii in 200 ml TAP with sulphate

First campaign Second campaign Average values

Fluorescentlamp2700 K

Fluorescentlamp5600 K

Xenonlamp

Fluorescentlamp2700 K

Fluorescentlamp5600 K

Xenonlamp

Fluorescentlamp2700 K

Fluorescentlamp5600 K

Xenonlamp

1 0 0.002 0.002 0.002 0.000 0.000 0.000 0.001 0.001 0.001

2 4 0.085 0.005 0.031 0.069 0.015 0.027 0.077 0.010 0.029

3 5 0.016 0.004 0.033 0.066 0.016 0.029 0.041 0.010 0.031

4 6 0.083 0.040 0.033 0.062 0.002 0.033 0.073 0.021 0.033

5 7 0.013 0.000 0.033 0.059 0.001 0.036 0.036 0.001 0.035

6 11 0.000 0.000 0.040 0.015 0.000 0,039 0.008 0.000 0,040

7 13 0.003 0.000 0.036 0.035 0.000 0.038 0.019 0.000 0.037

8 20 0.006 0.000 0.037 0.011 0.000 0.037 0.009 0.000 0.037

9 22 0.002 0.000 0.022 0.013 0.000 0.029 0.008 0.000 0.026

10 27 0.011 0.000 0.044 0.014 0.000 0.028 0.013 0.000 0.036

11 32 0.018 0.000 0.043 0.015 0.000 0.025 0.017 0.000 0.034

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 6 ( 2 0 1 1 ) 7 4 7 9e7 4 8 67482

(200 ml of culture medium and 15 � 106 cells of C. reinhardtii),

the second half of the bottles have been filled with twice the

amount of culture (400ml of culturemediumand 30� 106 cells

of C. reinhardtii). It is expected that the volume of the culture is

proportional to the amount of H2 produced. The cultures have

been installed in the photobioreactors with the two lamps

which have provided the best results in the first experimental

campaign (the xenon lamp and the fluorescent lamp with

TC ¼ 2700 K). Three identical samples of 200 ml culture and

three identical samples of 400ml culture were installed inside

each photobioreactor.

Test 2 was conducted two times to ensure reliability: the

first one from March 15th, 2010 to April 16th, 2010; the second

one from November 12th, 2010 to December 14th, 2010. Test 2

lasted 33 days and involved 10measurements each time it was

run. Volume hydrogen concentration measured in the bottles

Table 2 e Hydrogen concentration inside bottles holding cultucollection (Test 1).

Measurenumber

Incubationtime(days)

Hydrogen vol

15 � 106 Chlamydomonas

First campaign

Fluorescentlamp2700 K

Fluorescentlamp5600 K

Xenonlamp

Fluoreslam2700

1 0 0.002 0.001 0.000 0.00

2 4 0.147 0.040 0.009 0.29

3 5 0.325 0.056 0.008 0.38

4 6 0.512 0.026 0.009 0.47

5 7 0.809 0.017 0.012 0.46

6 11 0.940 0.001 0.339 0.60

7 13 0.922 0.000 0.649 0.59

8 20 0.851 0.000 1.044 0.29

9 22 0.524 0.000 0.604 0.29

10 27 0.688 0.001 0.810 0.29

11 32 0.658 0.001 0.773 0.29

for each experimental campaign (at the time of each

measurement) are reported in Tables 3 and 4. Data of the third

and fourth column of both Tables represent the average

hydrogenproduction rate of the three identical culture samples

inside each of the two photobioreactors, measured during the

first campaign. Data of the fifth and sixth column represent the

average hydrogen production rate of the three identical culture

samples inside each of the two photobioreactors, measured

during second campaign. Data of the seventh and eighth

column represent the mean values of the two campaigns.

2.3. Test 3: evaluation of the effect of mediumrestoration on hydrogen production

Sustained H2 photoproduction by algal cultures requires the

maintenance of culture anaerobiosis for prolonged periods of

res in TAP without sulphate, at the time of each sample

umeevolume percentage (v/v %)

reinhardtii in 200 ml TAP without sulphate

Second campaign Average values

centpK

Fluorescentlamp5600 K

Xenonlamp

Fluorescentlamp2700 K

Fluorescentlamp5600 K

Xenonlamp

0 0.000 0.000 0.001 0.001 0.000

5 0.038 0.010 0.221 0.039 0.009

5 0.053 0.009 0.355 0.055 0.008

0 0.025 0.010 0.491 0.025 0.009

2 0.016 0.013 0.636 0.017 0.013

2 0.001 0.754 0.771 0.001 0.547

0 0.000 0.685 0.756 0.000 0.667

6 0.000 0.256 0.574 0.000 0.650

5 0.000 0.198 0.410 0.000 0.401

3 0.001 0.193 0.491 0.001 0.502

0 0.001 0.190 0.474 0.001 0.482

Table 3eHydrogen concentration inside bottles holding cultures in 200mlTAP, at the time of each sample collection (Test 2).

Measurenumber

Incubationtime (days)

Hydrogen VolumeeVolume Percentage (v/v %)

15 � 106 Chlamydomonas reinhardtii in 200 ml TAP without sulphate

First campaign Second campaign Average values

Fluorescentlamp 2700 K

Xenonlamp

Fluorescentlamp 2700 K

Xenonlamp

Fluorescentlamp 2700 K

Xenonlamp

1 0 0.000 0.000 0.000 0.000 0.000 0.000

2 3 0.001 0.010 0.015 0.102 0.008 0.056

3 5 0.319 0.001 0.421 0.153 0.370 0.077

4 7 0.542 0.056 0.502 0.298 0.522 0.177

5 11 0.699 0.418 0.615 0.501 0.657 0.460

6 14 0.640 0.638 0.586 0.428 0.613 0.533

7 17 0.529 0.559 0.505 0.213 0.517 0.386

8 24 0.453 0.235 0.435 0.185 0.444 0.210

9 27 0.366 0.154 0.426 0.158 0.396 0.156

10 33 0.383 0.071 0.419 0.151 0.401 0.111

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 6 ( 2 0 1 1 ) 7 4 7 9e7 4 8 6 7483

time. Different ways have been attempted to maintain this

condition like the re-addition of small amounts of sulphate to

the sulphur deprivated medium [14].

Recent studies [15] have demonstrated that re-addition of

acetate to Chlamydomonas cultures increased the final yield of

H2 gas. In fact, the faster consumption of acetate from the

medium by the cell cultures may limit cellular respiration

during H2 photoproduction, possibly increasing the intracel-

lular level of O2 and inhibiting H2 production. For this reason,

the re-addition of acetate back to the medium, at the time of

its expected depletion, partially restores the H2-production

activity of the cells.

In theTest 3 a differentmethod tomaintaina continuousH2

evolution has been proposed. The scope is to regenerate algal

culture and maintain a stable hydrogen production. This

objective can be obtained by removing a part of the catabolites

and adding new metabolites through an alternating restora-

tion of sulphur-deprived TAP medium. The reintegration of

TAP medium in the culture could be combined with a sta-

ble production of hydrogen, overcoming the problem of the

Table 4eHydrogen concentration inside bottles holding culture

Measurenumber

Incubationtime (days)

Hydrogen

30 � 106 Chlamydom

First campaign

Fluorescentlamp 2700 K

Xenonlamp

1 0 0.000 0.000

2 3 0.129 0.032

3 5 1.794 0.055

4 7 1.980 0.761

5 11 0.828 3.400

6 14 0.294 3.559

7 17 0.074 2.966

8 24 0.020 1.619

9 27 0.013 1.031

10 33 0.024 0.360

reduction of hydrogenproduction. The sampleswere prepared

bymaking cells of C. reinhardtii grow at about 28 �C on sulphur-

deprivedTAPmedium. Liquid cultureswere grown in six of the

same graduated borosilicate glass bottles employed for the

first experimentation and subjected to continuous lighting

with a xenon lamp. Eachbottlewas filledwith 200ml of culture

medium and 15 � 106 cells of C. reinhardtii. In one-half of the

bottles, after each fall of the hydrogen production, 100 ml of

algal suspension were replaced with 100 ml of new TAP.

The Test 3 was conducted two times to ensure reliability:

the first one from April 26th, 2010 to May 27th, 2010; the

second one from January 7th, 2011 to February 7th, 2011. Test

3 lasted 32 days and involved 8 measurements each time it

was run. Volume hydrogen concentration measured in the

bottles for each experimental campaign (at the time of each

measurement) are reported in Table 5. Data of the third and

fourth column represent the average hydrogen production

rate of the three identical culture samples inside the photo-

bioreactor, measured during the first campaign. Data of the

fifth and sixth column represent the average hydrogen

s in 400mlTAP, at the time of each sample collection (Test 2).

VolumeeVolume Percentage (v/v %)

onas reinhardtii in 400 ml TAP without sulphate

Second campaign Average values

Fluorescentlamp 2700 K

Xenonlamp

Fluorescentlamp 2700 K

Xenonlamp

0.000 0.000 0.000 0.000

2.111 0.256 1.120 0.144

2.345 1.352 2.070 0.704

2.256 2.985 2.118 1.873

0.854 2.754 0.841 3.077

0.546 2.145 0.420 2.852

0.398 2.014 0.236 2.490

0.214 1.356 0.117 1.488

0.189 1.245 0.101 1.138

0.184 1.225 0.104 0.793

Table 5 e Hydrogen concentration at the time of each sample collection (Test 3).

Measurenumber

Incubationtime (days)

Hydrogen VolumeeVolume Percentage (v/v %)

15 � 106 Chlamydomonas reinhardtii in 200 ml TAP without sulphate e- Xenon lamp

First campaign Second campaign Average values

Without restore With restore Without restore With restore Without restore With restore

1 0 0.004 0.011 0.000 0.001 0.002 0.006

2 4 0.020 0.096 0.095 0.106 0.058 0.101

3 8 0.112 0.220 0.301 0.242 0.207 0.231

4 13 0.676 0.540 0.515 0.594 0.596 0.567

5 15 0.722 0.592 0.524 0.651 0.623 0.622

6 20 0.404 0.744 0.456 0.818 0.430 0.781

7 25 0.263 0.754 0.300 0.854 0.282 0.804

8 32 0.106 0.842 0.254 0.926 0.180 0.884

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 6 ( 2 0 1 1 ) 7 4 7 9e7 4 8 67484

production rate of the three identical culture samples inside

the photobioreactor, measured during the second campaign.

Data of the seventh and eighth column represent the mean

values of the two campaigns.

3. Results and discussion

In the present section plots of hydrogen/oxygen production

versus time are shown for Test 1, Test 2 and Test 3. For each

test, plots represent the average values of the first and second

campaign.

3.1. Test 1 results

Figs. 6 and 7 show the average trends of hydrogen production

versus time for the cultures respectively grown on sulphur-

deprived TAP and in TAP with sulphate. The results confirm

that, on equal terms of illumination, the hydrogen production

ismuch greater in cultures grown on sulphur-free TAP than in

cultures grown on TAP with sulphate. While the O2 concen-

tration decreases over the time in the cultures without

sulphate, O2 concentration is constant (see Fig. 8) in the

cultures with sulphate.

Among the cultures grown in sulphur-deprived TAP, the

samples that showed the best results were the cultures under

fluorescent lampwith TC¼ 2700 K and xenon lamp. In cultures

Fig. 6 e Hydrogen production by cultures in TAP without

sulphate.

under fluorescent lamp with TC ¼ 5600 K no relevant results

were got. In 15 � 106 C. R./200 ml-TAP samples without

sulphate under fluorescent lamp with TC ¼ 2700 K the

concentration of hydrogen increased until the 13th day with

a maximum value of 0.77 percent. From the 13th until the

22nd day, the total amount of hydrogen decreased to 0.41

percent. The higher rate of H2 production (0.13ml/lh) occurred

from the 6th until the 7th day. In 15 � 106 C. R./200 ml-TAP

samples without sulphate under Xenon lamp the concentra-

tion of hydrogen increased until the 20th day with

a maximum value of 0.65 percent. From the 20th until the

22nd day, the concentration of hydrogen decreased to 0.40

percent. The higher rate of H2 production (0.12ml/lh) occurred

from the 7th until the 11th day.

Cultures in TAP with sulphate show an oscillating trend of

the concentration of hydrogen during the first 11 days. In this

period the maximum values reached by the samples under

fluorescent lamp with TC ¼ 2700 K and by the samples under

fluorescent lamp with TC ¼ 5600 K are respectively 0.08 and

0.02 percent. The samples under xenon lamp show an

increasing trend during the whole test period; the maximum

concentration value raised to 0.03 percent after 32 days.

3.2. Test 2 results

Figs. 9 and 10 show the hydrogen production versus time

respectively for 200 ml and 400 ml culture medium. The

comparison with the Test 1 results confirms that, on equal

Fig. 7 e Hydrogen production by cultures in TAP with

sulphate.

Fig. 8 e Concentration of oxygen inside bottles holding

cultures in TAP with and without sulphate.

Fig. 10 e Hydrogen production by cultures in 200 and

400 ml TAP (Xenon lamp).

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 6 ( 2 0 1 1 ) 7 4 7 9e7 4 8 6 7485

terms of TAP, the hydrogen production starts earlier in

cultures grown with the fluorescent lamp than in cultures

grown with the xenon one: for 15 � 106 C. R./200 ml-TAP

samples without sulphate under fluorescent lamp 2700 K, the

higher rate of H2 production (0.16 ml/lh) occurs from the 3rd

until the 5th day, while for the for 15 � 106 C. R./200 ml-TAP

samples without sulphate under Xenon lamp such maximum

value (0.06 ml/lh) occurs from the 7th until the 11th day. In

15 � 106 C. R./200 ml-TAP samples without sulphate under

fluorescent lamp 2700 K the concentration of hydrogen

increased until the 11th day with a maximum value of 0.66

percent. From the 11th until the 33rd day, the hydrogen

concentration decreased to 0.40. In 15 � 106 C. R./200 ml-TAP

samples without sulphate under Xenon lamp the hydrogen

concentration increased until the 11th day with a maximum

value of 0.53 percent. From the 17th until the 33rd day, the

total amount of hydrogen decreased to 0.11 percent.

The results of the second test confirm that 30 � 106 C. R./

400 ml-TAP samples without sulphate under fluorescent lamp

2700 K and under Xenon lamphad twice the amount of culture

medium and Chlamydomonas cells than 15� 106 C. R./200ml-

TAP samples without sulphate and half of the headspace

volume for each bottle. The best results have been provided by

samples under Xenon lamp which show an increasing

Fig. 9 e Hydrogen production by cultures in 200 and 400 ml

TAP (fluorescent lamp 2700 K).

trend until the 11th day and a maximum value of hydrogen

concentration of 3.08 percent. From the 11th until the 33rd

day, the hydrogen concentration decreased to 0.79 percent.

For 30 � 106 C. R./400 ml-TAP samples without sulphate under

fluorescent lamp 2700 K the higher rate of H2 production

(0.42 ml/lh) occurs until the 3rd day, while for the 30 � 106 C.

R./400 ml-TAP samples without sulphate under Xenon lamp

such maximum value (0.14 ml/lh) occurs from the 5th until

the 7th day. Results confirm that hydrogen production starts

earlier in cultures grown with fluorescent lamp than in

cultures grown with xenon one, also doubling the amount of

TAP medium.

3.3. Third test results

In the third experimental campaign, tests have shown that it

is possible to obtain the resumption of hydrogen production

from algal cultures in which new TAP is restored in the

experimental conditions.

Fig. 11 shows the results of hydrogen production in

samples with and without TAP restoring.

New TAP in the cultures allows to keep constant the

metabolism and anaerobic conditions reached during the first

stage of cultivation and needed for the activation of enzymes

Fig. 11 e Hydrogen production by cultures with and

without an alternating restore of TAP.

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 6 ( 2 0 1 1 ) 7 4 7 9e7 4 8 67486

for hydrogen production. The restoration of algal cultures

with new TAP, carried out after the fall of the hydrogen

production, allows to regenerate the culture and, conse-

quently, to increase the hydrogen production.

4. Conclusions

A first experimental campaign was carried out to determine

the hydrogen production obtained by C. reinhardtii grown on

TAP with and without sulphate and under different illumi-

nation conditions. For the same illumination conditions,

results show that the best culture medium is sulphur-

deprived TAP. Best hydrogen production rate was obtained

with fluorescent (colour temperature of 2700 K) and xenon

lamp. The maximum hydrogen concentration (1 vol-

umeevolume percentage) was attained by xenon lamp after

14 days. Samples illuminated by Xenon lamp show also the

higher rate of H2 production (0.27ml/lh). This is themaximum

value of hydrogen evolution rate obtained in the presentwork.

If we compare this value with those obtained in similar

studies [16], we note that it is an order of magnitude lower

than the other values. It can be justified by the use of a quite

small cell concentration (1.8 mg Chl/ml) compared to the

typical concentration used in this studies (about 18 mg Chl/ml).

A second experimental campaign was conducted growing

C. reinhardtii on different amounts of sulphur-deprived TAP

and employing the two lamps which had provided the best

results in the first experimental campaign (xenon lamp and

fluorescent lamp with TC ¼ 2700 K). On the same illumination

and TAP amount, results confirmed the same hydrogen

production of the first campaign. In particular, the hydrogen

production starts earlier and more quickly in cultures grown

with the fluorescent lamp than in cultures grown with the

xenon one but the maximum value of H2 concentration is

slightly higher for cultures grown with xenon lamp. The

results also confirm that a full spectrum light source (xenon

lamp) provides the same (or greater) hydrogen amount as

a light source that emits only in the absorption wavelength

range of green algae; therefore radiation with a spectrum

larger than the algae absorption range has no influence on H2

production.

A third experimental campaign was finally carried out to

determine the effect of an alternating restoring of sulphur-

deprived TAP medium on the hydrogen production obtained

by C. reinhardtii under xenon lamp irradiation. Results show

that the maintenance of algal cultures in constant metabolic

condition through the restoring with TAP produces a constant

increase of hydrogen production.

This information may be useful in the design of algal

H2-production photobioreactor system. The next step of

research is to investigate the hydrogen production of C. rein-

hardtii under sunlight illumination to confirm the production

rate obtained by means of the solar simulator.

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