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Hydrogen production by Rhodobacter sphaeroides O.U.001in a flat plate solar bioreactor

Inci Eroglua,�, Altan Tabanoglua, Ufuk Gunduzb, Ela Eroglua, Meral Yucelb

aDepartment of Chemical Engineering, Middle East Technical University, 06531 Ankara, TurkeybDepartment of Biology, Middle East Technical University, 06531 Ankara, Turkey

a r t i c l e i n f o

Article history:

Received 23 March 2007

Received in revised form

25 September 2007

Accepted 27 September 2007

Available online 26 November 2007

Keywords:

Rhodobacter sphaeroides

Hydrogen production

Solar bioreactor

Outdoor

Polyhydroxybutyrate

Caretenoid

nt matter & 2007 Internane.2007.09.025

thor. Tel.: +90 312 2102609ieroglu@metu.edu.tr (I. E

a b s t r a c t

Rhodobacter sphaeroides O.U.001 can produce hydrogen under anaerobic conditions and

illumination. The objective of this study was to investigate the performance of an 8 l flat

plate solar bioreactor operating in outdoor conditions. Different organic acids were used as

carbon sources (malate, lactate and acetate) and olive mill waste water was used as a sole

substrate source. The consumption and the production of the organic acids were

determined by HPLC. The accumulation of by-products, such as poly-b-hydroxybutyrate

(PHB) and carotenoid, throughout the course of hydrogen production was determined.

The hydrogen production rate was highest (0.01 l/l/h) when malate was the carbon

source. Formate was observed as the fermentation end product. Acetate resulted in low

hydrogen gas production and high PHB accumulation. When acetate was used as the

carbon source, butyrate was produced as a result of fermentation. Promising amounts of

PHB and caretenoid were accumulated during hydrogen production from diluted olive mill

wastewater.

& 2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights

reserved.

1. Introduction

The design of the solar bioreactor plays an important role in

the outdoor conditions, since biological hydrogen production

by photofermentative bacteria (i.e. Rhodobacter species) re-

quires adequate illumination at anaerobic conditions. Detailed

research needs to be carried out to obtain indoor systems that

simulate outdoor situations and can further be transferred

directly to sunlight [1]. Modes of operation, the size and

geometry of the reactor, the position and orientation of the

solar bioreactor are the most important design parameters.

Hydrogen production was achieved by Rhodobacter sphaer-

oides in a 33 l batch solar bioreactor for 25 days from lactate

[2]. Mitsui et al. [3] reported outdoor hydrogen production of

about 90 mmol/h/g Bchl as a maximum from orange proces-

sing wastewater using immobilized Rhodopseudomonas sp. The

tional Association for Hy

; fax: +90 312 2102600.roglu).

duration of hydrogen production was increased by semicon-

tinuous or fed batch outdoor operation. Kim et al. [4] observed

H2 production for 45 days using Rhodobacter sphaeroides B5.

Continuous hydrogen production was observed with ex-

change of half of the medium every fifth day for almost two

months, indicating that there was no significant contamina-

tion of the reactor although no specific sterilization techni-

ques were applied [5].

Outdoor flat bioreactors were used with an increased

surface-to-volume ratio [6] for hydrogen production by

Rhodobacter sphaeroides RV using lactate and propionate as

carbon sources [7]. Tubular solar reactors can utilize irra-

diated light energy efficiently [8]. Rhodopseudomonas palustris

was grown in an outdoor temperature controlled underwater

tubular solar bioreactor [9]. A cone-shaped solar bioreactor

named ‘Heliomites’ was suggested for purple bacteria [10].

drogen Energy. Published by Elsevier Ltd. All rights reserved.

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Nomenclature

ks the substrate consumption rate constant, h�1

kc apparent specific growth rate, h�1

kg specific growth rate constant obtained by logistic

model, 1/h

M instantaneous malate concentration, mol/l

M0 initial molar concentration of malate, mol/l

NB new basal

P moles of hydrogen produced, mol

OMW olive mill wastewater

PHB polyhydroxybutyrate

SB standard basal

R2 the coefficient quantifying goodness-of-fit

rg gas production rate based on culture volume, l/l/h

r1g gas production rate based on bacterial dry cell

weight, l/g/h

t time h

td total gas production time, h

V volume of the culture, l

X cell concentration (dry weight), g/l

X0 initial bacterial concentration obtained by logistic

model, g/l

ðX0Þe experimental initial bacterial concentration, g/l

Xmax maximum bacterial concentration obtained by

logistic model, g/l

ðXmaxÞe experimental maximum bacterial concentration,

g/l

m the specific growth rate, h�1

me the (constant) specific growth rate in the expo-

nential phase, h�1

mmax specific growth rate constant obtained by expo-

nential model, 1/h

Z percent substrate conversion efficiency, %

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When the front surface of the flat panel reactor was inclined

toward the sun, it received the major thrust of solar irradiance

and the back side of the reactor surface was also illuminated

by diffuse and reflected light which may be very effective for

photosynthesis. The reactors were proposed to face the south

with inclinations of 30� and 60� for summer and winter,

respectively [11] and these were taken as a reference for the

simulation of a daily sunlight illumination pattern for photo-

hydrogen production [12]. More solar energy was received

when the reactor was placed in an east–west facing orientation

in the vertical position, and the productivity was higher than

that in a north–south facing orientation [5,13].

The major materials of construction used were glass [14] or

acrylic glass [5]. The depths of the reactors were usually

between 3 and 6 cm in order to eliminate non-homogenous

light intensity distribution [7]. These reactors have, however,

shortcomings in efficient mixing of the cells and substrate.

Stirring is an important factor because it was proved that it

enhances not only total hydrogen evolution but also the

conversion efficiency of the substrate to hydrogen [15].

The wavelength and intensity of light can be changed by

the cooling medium or the culture. Transparent cooling

jackets filled with either water [16–18] or a laser dye can be

used. The optimum laser dye had an absorption range of

420–520 nm, which corresponded to the absorption range of

the carotenoids [10]. Hydrogen production was thought to be

enhanced because the laser dye transformed the absorbed

wavelengths into longer ones, which are more effective in

photosynthesis.

Solar light intensity depends on the location and season,

diurnal variation and the duration of the light/dark cycle.

These all may have an effect on the efficiency of hydrogen

production.

Immobilization of photosynthetic bacteria on agar is

another alternative reactor design for hydrogen production

by solar energy [19].

Solar reactors were usually compared by certain perfor-

mance criteria such as light conversion efficiency [20],

hydrogen production rate, substrate conversion efficiency

[16] and net energy ratio [21]. Previously this group has

reported on the optimization of T, pH, light intensity, the light

source [22] and the carbon to nitrogen ratio [23] for hydrogen

production by Rhodobacter sphaeroides O.U.001. It was found

that use of 15 mM of malate together with 2 mM of Na-

glutamate gave the highest hydrogen production rate in a

standard basal (SB) medium. Also, addition of biotin was

essential [24]. Poly-b-hydroxybutyrate (PHB) and caretenoid

were identified as valuable by-products [25,26]. Industrial

wastes such as dairy plant wastewater [27], sugar refinery

wastewater [18] and olive mill wastewater [17,28,29] could be

used as alternative substrates.

The objective of the present work is to develop a tempera-

ture controlled flat plate solar bioreactor to produce hydrogen

gas by Rhodobacter sphaeroides O.U.001, and to determine the

hydrogen productivity of using malate, acetate, lactate or

olive mill wastewater in outdoor conditions.

2. Materials and methods

2.1. Bacterium

Rhodobacter sphaeroides O.U.001 (DSM 5864) was used. The

preparation of the inoculum was reported previously [16].

2.2. Medium

Malate (7.5 mM) was used as the carbon source and sodium

glutamate (10 mM) was used as the nitrogen source for the

inoculate growth medium.

The liquid culture used for hydrogen production contained

15 mM of malate in an SB medium [30] for Run 1, 15 mM of

malate in the new basal (NB) medium [31] for Run 2, 30 mM of

malate in the SB medium for Run 3, 30 mM of acetate for Run 4

and 20 mM of lactate in the SB medium for Run 5. In all these

experiments the initial sodium glutamate concentration was

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2 mM. The compositions of the basal media are given in

Table 1. Olive mill wastewater was the sole substrate for Run 6;

its properties were given previously [17].

2.3. Solar bioreactor

A flat plate solar bioreactor was manufactured of acrylic sheet

(Plexiglas) of 5 mm thickness and had an illuminated front

area of 0:2 m2. The dimensions of the solar bioreactor were

0:5 m� 0:4 m� 0:04 m. The schematic diagram of the bioreac-

tor is given in Fig. 1. The experimental setup for hydrogen

production is given in Fig. 2. The maximum culture medium

that could be used was 6.5 l. The reactor was inclined 30� and

faced south. It had six outlets at the top: one for collecting the

gas evolved by the culture, one for argon gas exhaust, one for

argon gas flushing through a T-shaped sparger, a septum

containing two outlets for gas sampling, one outlet for

Table 1 – Comparison of the components and theamounts required for 1 l of culture medium used in thestandard basal (SB) medium and the new basal (NB)medium

SB medium NB medium

KH2PO4 500 mg KH2PO4 866 mg

H3BO3 60mg H3BO3 2.8 mg

MgSO4 � 7H2O 200 mg MgSO4 � 7H2O 200 mg

Na2MoO4 � 2H2O 40mg Na2MoO4 � 2H2O 750mg

Biotin 15mg Biotin 0:15mg

Thiamin 250mg Thiamin 0:15mg

Niacin 250mg Niacin 0:45mg

Fe–citrate 5mg FeSO4 � 7H2O 11.8 mg

MnCl2 � 4H2O 100mg MnSO4 � 4H2O 2.1 mg

ZnCl2 70mg ZnSO4 � 7H2O 240mg

CuCl2 � 6H2O 20mg CuðNO3Þ2 � 3H2O 40mg

CaCl2 � 2H2O 50 mg CaCl2 75 mg

NaCl 400 mg NaHCO3 0.1%

NiCl2 � 6H2O 20mg EDTA (disodium salt) 20 mg

CoCl2 � 6H2O 200mg Yeast extract 0.1%

Fig. 1 – Schematic drawing of th

inoculation of the bacteria and a temperature probe entrance.

The reactor had four outlets in its side walls: the first and

third ones from the top were for cooling water coming in and

out, respectively, the second one from the top was for liquid

sampling; and the bottom one was for emptying the reactor.

The reactor was illuminated with sunlight as the sole

source of light energy except during the first night when the

reactor was illuminated with a tungsten lamp ð150 Wm2Þ until

hydrogen production had started. In order to obtain contin-

uous light density data, a Luxmeter (Lutron) was connected to

a computer (PC). It had a sensor placed at the upper left

portion of the solar bioreactor.

A heating blanket was placed on the back face of the

reactor. The temperature was controlled with a temperature

controller (Elimko-E212). It was connected to a temperature

probe (Elimko, Pt-100) that was inserted into the reactor.

A coil that was made of a glass tube (1.70 m long, ID 0.01 m)

was placed into the reactor. Cooling water continuously

flowed through this coil.

Pure argon gas was bubbled through the bioreactor to

obtain an anaerobic atmosphere. The flow of argon gas was

monitored by a rotameter. A cotton filter was used to prevent

contamination.

2.4. Procedure

The cleaning and sterilization of the solar bioreactor were

accomplished by washing the reactor first with detergent and

then with 3% (w/v) H2O2. After each 24 h period the reactor

was emptied and washed with sterile distilled water. Between

and after each of the experiments, the reactor was emptied

while being filled with sterile argon gas, and no further

sterilization was carried out. The sterilized medium was

added to the reactor through the top outlet near the flame.

The temperature of the medium was kept at 32 �C

by turning on the heating blanket and cooling water. The

active culture (20%) of Rhodobacter sphaeroides O.U.001 was

inoculated.

Throughout the experiments, liquid samples were taken

from the sampling ports twice a day in the morning and at

e flat plate solarbioreactor.

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Fig. 2 – Experimental setup for outdoor hydrogen production.

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 E N E R G Y 3 3 ( 2 0 0 8 ) 5 3 1 – 5 4 1534

night. Evolved gas was collected in graduated burettes by the

water displacement method.

2.5. Analysis

Cell growth, pH, GC and HPLC analysis were carried out as

previously described [16]. The culture was centrifuged at

10,000 g at 4 �C for 15 min and the pellet was weighed. Pellet of

1 g was used for carotenoid analysis and the rest was used for

PHB analysis [25,26].

Fig. 3 – Time dependent changes in cell concentration ð�Þ

and total gas production ð�Þ with 15 mM of malate in the

standard basal medium. The shaded regions indicate the

nighttime. The experiment was performed during August

29th–September 8th, in Ankara.

3. Results and discussion

3.1. Hydrogen production with malate

The hydrogen production medium initially contained 15 mM

of malate and 2 mM of Na-glutamate in an SB medium

(Run 1). Illumination of sunlight fits a sinusoidal pattern

with a peak value of approximately 110 klux. The inoculation

was done at 11:00 pm, and the bioreactor was illuminated the

first night.

It was observed that bacteria grew during daytime (Fig. 3).

Cell concentration either remained the same or decreased

during the nighttime period. These results confirmed the

anaerobic photosynthetic growth of Rhodobacter sphaeroides

[24]. In total, 13.1 l of gas (95% H2 and 5% CO2, on an argon-

free basis) was produced as seen in Fig. 3.

It was observed that malate produced formate as a by-

product. In most of the studies carried out in indoor

conditions with column bioreactors [16,23], a formate end

product was also reported in a previous study [15]. Fig. 4

illustrates the variation in malate and formate concentrations

versus time. Malate was consumed continuously, whereas

formate was produced within the first 20 h, and was subse-

quently either consumed or produced. On the first day, the

weather was partially cloudy, so formate production may

have been enhanced. After 120 h, when the malate concen-

tration was very low (less than 2 mM), formate was utilized. It

is not clear whether formate consumption resulted in

hydrogen production, since there are no previously existing

reports regarding this for Rhodobacter sphaeroides.

Table 2 summarizes the results of all runs. As is evident in

the data, the start time for hydrogen production was between

3 and 10 h, which was shorter in outdoor conditions

compared to the 16 h measured at indoor studies [16]. The

highest average hydrogen production rate was obtained in

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Run 1 as 0.010 l/l/h. That was the best obtained at indoor

conditions (0.005 l/l/h). This shows that light/dark cycles

increase the average hydrogen production rate, which makes

the system more promising for outdoor applications.

Malate was consumed completely in this system. The yield

of hydrogen was 4.6 mol of H2 per mole of malate. From the

stoichiometry

C4H6O5 þ 3H2O! 6H2 þ 4CO2. (1)

6 mole of H2 can be produced from 1 mol of malate. However,

some of the carbon is utilized for bacterial growth and

Table 2 – Summary of the results of the experiments

Experiment Run 1 Run 2

Medium type SB NB

Carbon source Malate Malate

Substrate concentration (mM) 15 15

Duration of experiment (days) 9 12

Light density (klux)b 110–115 75–100

Daily average light intensity ðW=m2Þ 495–518 338–450

Day/night periods (h/h) 14/10 11/13

Max. Cell concentration ðg dw=lÞ 0.595 1.159

Final cell amount (g wet weight) 17.44 19.05

Gas production start time (h) 10 3

Total gas produced (l) 13.1 7.2

Rate of hydrogen ðlgas=h lcultureÞ 0.01 0.005

Final pH 7.7 7.7

Substrate conversion (%) 1 1

Yield of hydrogenc 4.6 2.6

Hydrogen production potentiald NA NA

Fermentation product Formate Formate

Yield of fermentation producte 2.87 1.56

Carotenoid (mg/g wet weight) NA 0.30

PHB (mg/g wet weight) 0.09 0.21

Bicarbonate Carbonate (mM) NA 12.7

SB: standard basal medium; NB: new basal medium. NA: not available.a OMW: olive mill waste water, composition was given in [17].b Minimum and maximum light intensities at noon.c Mole hydrogen produced/mole substrate utilized.d Liter hydrogen produced/liter olive mill wastewater utilized.e Mole fermentation product/mole substrate utilized.

Fig. 4 – Time dependent changes in malate consumption ð�Þ

and formate production ð�Þ with 15 mM of malate in the

standard basal medium. The shaded regions indicate the

nighttime. The experiment was performed during August

29th–September 8th, in Ankara.

biosynthesis. The yield of hydrogen from malate obtained in

this experiment is quite acceptable. A total of 1.57 mg of PHB

was produced from 17.44 g of bacteria (wet weight).

The effect of some of the basal medium components on

hydrogen production was investigated in Run 2. The NB

medium was rich in molybdenum, which is one of the critical

elements for nitrogenase, approximately 20 times more than

that in the SB medium (Table 1). The NB medium contained

EDTA that is known to inhibit hydrogenase activity. It is free

of nickel ions which are necessary for the hydrogenase

enzyme synthesis. It also contains 0.01–0.001 times less of

vitamins, namely biotin, thiamin and niacin, compared to the

SB medium. Yeast extract was supplied for growth conditions.

Furthermore, this medium contained a buffer solution

ðKH2PO4=K2HPO4Þ to ensure that the pH was maintained at a

constant value.

The hydrogen production medium contained 15 mM of

malate and 2 mM of Na-glutamate in the NB medium. Almost

the same light intensity distribution profile was observed in

the previous experiment. The inoculation was done during

the daytime, and no tungsten lamp was used.

Fig. 5 presents the bacterial growth curve and the total

hydrogen production obtained in Run 2. The use of the NB

medium resulted in increased cell concentration in compar-

ison with the SB medium. However, a total of 7.2 l of gas

was collected (99% hydrogen and 1% carbon dioxide, on an

argon-free basis). Gas production took place only during

daylight hours.

Run 3 Run 4 Run 5 Run 6

SB SB SB OMWa

Malate Acetate Lactate OMW

30 30 20 4%

12 12 13 9

10–60 80–100 100 90–95

45–270 360–450 450 405–428

11/13 12/12 12/12 14/10

0.588 0.647 0.309 0.368

11.17 20.72 0.61 14.32

15 11 12 20

0.3 5.8 1.0 2.56

0.0003 0.008 0.002 0.003

7.6 9.7 5.3 9.8

1 0.93 0.23 NA

0.6 1.2 0.8 NA

NA NA NA 11.4

Formate Butyrate NA NA

2.06 0.02 NA NA

0.15 0.08 0.04 0.78

6.15 5.40 0.01 1.21

12.0 21.1 0 NA

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Fig. 5 – Time dependent changes in cell concentration ð�Þ

and total gas production ð�Þwith 15 mM of malate in the new

basal medium. The shaded regions indicate the nighttime.

The experiment was performed during October

12th–October 23rd, in Ankara.

Fig. 6 – Time dependent changes in malate consumption ð�Þ

and formate production ð�Þwith 15 mM of malate in the new

basal medium. The shaded regions indicate the nighttime.

The experiment was performed during October

12th–October 23rd, in Ankara.

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Malate and formate concentration profiles are given in

Fig. 6. In this experiment, formate production started after

72 h. This may indicate that, the use of the NB medium

somehow suppresses fermentation. This may have been due

to the presence of the buffer solution ðKH2PO4=K2HPO4Þ.

Malate was consumed continuously. Formate was consumed

during the day and was produced during night. However, after

130 h nearly all of the formate was consumed. It should be

noted that 3 l of gas was produced after 30 h, even though

there was very little malate and formate left.

Table 2 summarizes the results of Run 2. It was observed

that the lag time was further shortened with the use of the NB

medium to 3 h and that the use of the NB medium doubled

the amount of cell concentration compared to the use of

the SB medium. The average hydrogen production rate

(0.005 l/l/h) was less than that of Run 1 (0.010 l/l/h).

Malate was completely consumed in this system and the

yield of hydrogen was 2.6 mol of H2 per mole of malate. More

hydrogen was produced from malate in the SB medium than

in the NB medium. A total of 0.21 mg of PHB and 0.30 mg of

carotenoid was produced per gram of wet weight of bacteria,

and 12.7 mM of bicarbonate and carbonate was present in the

liquid culture at the end of the experiment.

The initial pH of all runs was approximately 7.0. However,

the pH increased to 7.7 and remained constant. The pH

slightly decreased during the night, consistently, and in-

creased during the day. This finding may be a result of the

contribution of the formate produced during the periods of

nighttime hours. Hydrogen production continued upto 200 h.

In the indoor experiments, the pH of the culture increased to

8.5 as a result of NH3 accumulation, and as a consequence,

hydrogen production ceased after 120 h [16].

Miyake et al. [31] used an NB medium with butyrate

and ammonium sulphate for hydrogen production with a

co-culture of Rhodobacter sphaeroides and Clostridium butyri-

cum. Later, Nakada et al. [32] used a aSy basal medium, which

comprises basal medium components combined with ammo-

nium sulfate and yeast extract, and a gL medium, which

comprises basal medium components combined with gluta-

mate and lactate. The use of a NB medium for hydrogen

production with malate did not seem to be feasible.

In Run 3, the hydrogen production medium contained

30 mM of malate and 2 mM of Na-glutamate in an SB medium.

The inoculation was done at 10:00 am. However, due to cloudy

weather conditions, the maximum light intensity was be-

tween 60 and 70 klux during the daytime (Table 2). The

bacterial growth curve was quite similar that in Fig. 3

however, only 0.3 l of gas (95% hydrogen and 5% carbon

dioxide, on an argon-free basis) was produced. The concen-

tration profiles of malate and formate were also quite similar

to those in Fig. 4. Malate was completely consumed. Formate

was produced within the first 40 h and then consumed to

23 mM within 70–100 h and remained at this value. The pH of

the system stayed at 7.5 due to production of the fermenta-

tion end product, formate.

Table 2 summarizes the results of Run 3. Due to the lower

light intensity, the lag time was increased to 15 h. The average

hydrogen production rate was very low (0.0003 l/l/h). The

maximum yield of hydrogen was 0.6 mol of H2 per mole of

malate.

A total of 6.15 mg of PHB and 0.15 mg of carotenoid was

produced per gram wet weight of bacteria and 12 mM of

bicarbonate and carbonate was present in the liquid culture

at the end of the experiment. It should be emphasized that

the bacteria produced PHB instead of hydrogen (nearly 60

times more than in Run 1), indicating the significance of the

shifting between the hydrogen production and the PHB

accumulation metabolism.

3.2. Hydrogen production with acetate

This hydrogen production medium contained 30 mM of

acetate and 2 mM of Na-glutamate in an SB medium. As the

inoculation was done at 7:00 pm, artificial illumination was

present for the first night. The daylight illumination pattern

was similar to that of Run 1 and Run 2. Fig. 7 gives the

bacterial growth curve and total hydrogen production

obtained in Run 4.

When acetate was used as the carbon source, rapid

bacterial growth accompanied rapid hydrogen production

for the first 20 h. Bacterial growth ceased after 30 h, as did the

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Fig. 7 – Time dependent changes in cell concentration ð�Þ

and total gas production ð�Þ with 30 mM of acetate in the

standard basal medium. The shaded regions indicate the

nighttime. The experiment was performed during

September 27th–October 8th, in Ankara.

Fig. 8 – Time dependent changes in acetate consumption ð�Þ

and butyrate production ð�Þ with 30 mM of acetate in the

standard basal medium. The shaded regions indicate the

nighttime. The experiment was performed during

September 27th–October 8th, in Ankara.

Fig. 9 – Time dependent changes in cell concentration ð�Þ

and total gas production ð�Þ in Olive Mill Wastewater

medium. The shaded regions indicate the nighttime. The

experiment was performed during August 21st–August

30th, in Ankara.

I N T E R N AT I O N A L J O U R N A L O F H Y D R O G E N E N E R G Y 3 3 ( 2 0 0 8 ) 5 3 1 – 5 4 1 537

hydrogen production. This may be the result of rapid increase

in the pH of the system to 10. When acetate was used as the

carbon source, it was determined through HPLC analysis that

butyrate was produced in low concentrations as a fermenta-

tion product (Fig. 8). As given in Table 2, the acetate

conversion was 0.93. The yield of hydrogen from acetate

was 1.2 mol of H2 per mole of acetate. The rate of hydrogen

gas production (0.008 l/l/h) was slightly lower than that in Run

1 (0.010 l/l/h).

In total, 5.4 mg of PHB and 0.08 mg of carotenoid were

produced per gram of wet weight of bacteria and 21.1 mM of

bicarbonate and carbonate were present in the liquid culture

at the end of the experiment. Due to the high final pH of the

system, 9.7, all of the carbon dioxide that was produced

remained in the liquid culture as bicarbonate and carbonate,

and thus this way the highest bicarbonate and carbonate

concentration in the culture. The gas composition was found

to be 100% hydrogen on an argon-free basis.

3.3. Hydrogen production with lactate

The hydrogen production medium contained 20 mM of lactate

and 2 mM of Na-glutamate. As the inoculation was done at

11:00 pm, a tungsten lamp was present for the first night. The

illumination pattern was quite similar to that in Run 1, Run 2

and Run 4.

Bacterial growth was limited. Hydrogen production up to 1 l

(96.4% hydrogen and 3.6% carbon dioxide on an argon-free

basis) was observed. Lactate utilization by Rhodobacter sphaer-

oides O.U.001 was limited, and substrate conversion was 0.23.

The medium became acidic after 20 h, such that the pH

dropped to 5.3 and stayed at this value. This may be the

reason of the cessation of bacterial activity. The maximum

cell concentration (0.309 g/l) was very low due to the limited

growth. No fermentation end product was observed. The total

PHB and carotenoid amounts are far below a level sufficient to

be considered significant. Due to the high acidity of the

system, no bicarbonate or carbonate was observed in the

samples.

According to the literature, Rhodobacter sphaeroides RV

instead of Rhodobacter sphaeroides O.U.001 seems to be the

best candidate for hydrogen production systems with lactate

as a carbon source, yielding very high hydrogen production

efficiencies [7,33].

3.4. Hydrogen production with olive mill wastewater

R. sphaeroides O.U.001 was grown in diluted OMW media,

containing 4% OMW, since that percentage represents the

volume ratio of OMW in distilled water. The hydrogen

production potential (HPP), which is defined as the total

hydrogen gas produced per volume of OMW, has a value of

11.4 l hydrogen per liter of OMW (98% H2, 2% CO2 on an argon-

free basis).

As given in Fig. 9, hydrogen was continuously produced

throughout the experiment (2.56 l) during the daytime. Mean-

while, bacterial growth stopped during the periods of dark-

ness. The maximum cell concentration was 0.37 g dry weight

per liter of culture. A total of 1.21 mg of PHB and 0.78 mg of

carotenoid was produced per gram of wet weight of bacteria

(Table 2). Relatively higher amounts of by-products

were accumulated during hydrogen production from olive

mill wastewater, which supports the feasibility of large

scale outdoor biological hydrogen production from a waste

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material. The results of OMW (Run 6) were close to those of

Run 4, because acetate was the major organic acid in the

OMW [29].

Fig. 10 illustrates the light density distribution profiles for

the outdoor experiments performed in Ankara, Turkey,

throughout the summer seasons of the years 2001–2003.

Almost the same illumination profile was observed in Fig.

10a–f with the exception of Fig. 10e. The maximum and

minimum light densities, daily average light intensities and

lengths of days and nights are reported in Table 2.

Fig. 10f shows the illumination pattern of the solar

bioreactor during the time period of 21–30 August 2003. This

experiment was carried out with olive mill waste water. It can

be observed from this figure that, except for the sixth day

(160–180 h) which was partially cloudy, illumination of the

sunlight fits to a sinusoidal pattern with a peak value at

around 95,000 lux ð855 W=m2Þ. During the partially cloudy day

Fig. 10 – Light intensity distribution profiles for outdoor experim

new basal medium (October 12th–October 23rd). (b) 15 mM of ma

8th). (c) 30 mM of acetate in the standard basal medium (Septem

basal medium (September 15th–17th). (e) 30 mM of malate in th

wastewater medium. (August 21st–30th).

variable light intensity was observed due to light scattering

effects. Approximately 14 h light/10 h dark periods were

observed throughout the whole experiment.

4. Kinetic studies

4.1. Logistic model for bacterial growth

By using a logistic model it is possible to model bacte-

rial growth at the exponential phase together with the

stationary phase [16,17]. For the hydrogen production experi-

ments bacterial growth data were found to fit a logistic model;

that is

r ¼ kc � X 1�X

Xmax

� �, (2)

ents performed in Ankara, Turkey. (a) 15 mM of malate in the

late in the standard basal medium (August 29th–September

ber 27th–October 8th). (d) 20 mM of lactate in the standard

e standard basal medium (November 1st–12th). (f) Olive mill

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where r is the rate of bacterial growth ðdX=dtÞ, kc is a constant

resembling the specific growth rate in the linear growth

model (1/h), X is the dry cell weight (g/l), and Xmax is the

maximum of bacterial growth or carrying capacity (g/l). If X is

equal to Xmax, the cell growth rate is zero, if X4Xmax cells

decay and if XoXmax cells grow. Xmax may be equal to the

maximum cell concentration that was observed or it may be

lower or higher. Therefore, it is retained as a model

parameter. By replacing r with dX=dt, and integrating, Eq. (2)

becomes

X ¼Xmax

1þ e�kctðXmax=X0 � 1Þ, (3)

where X0 is the initial bacterial concentration (g/l). Using a

software program for fitting curves, Curve Expert 1.3, plots

and constants (Xmax, X0 and kc) can be obtained. The first

order consumption rate model for 15 mM of malate initially in

the SB medium (Run 1) is given in Fig. 11, and Table 3 gives the

constants of the logistic model obtained for all runs.

The X0 values that are determined by the logistic model are

quite similar to the experimental values. This is also true

of the maximum bacterial concentration results. This

shows that the logistic model simulates the actual growth

data very well.

According to the logistic model, the specific growth rate was

found to be independent of the concentration of the carbon

source: the same specific growth rate values (0.062 and

0:063 h�1) were obtained by doubling the malate concentra-

Fig. 11 – The first order consumption rate model for 15 mM

of malate in the standard basal medium (Run 1).

Table 3 – Kinetic model parameters

Experiment Run 1 Run 2 Run

ðX0Þe 0.14 0.17 0.14

X0 0.11 0.24 0.14

ðXmaxÞe 0.60 1.16 0.59

Xmax 0.60 1.12 0.56

kc ðh�1Þ 0.063 0.033 0.062

R2ðlogisticÞ

0.9776 0.9875 0.987

ks 0.0187 0.0227 0.016

R2ðsubstrateÞ

0.9762 0.9571 0.983

tion. The results are also in accordance with the indoor

experiments [16]. However, when the NB medium was used

instead of the SB medium the specific growth rate constant

decreased from 0.062 to 0:033 h�1. The highest specific growth

rate was obtained when lactate was the carbon source.

However, the carrying capacity was halved (0.30 g/l). This

should be due to the generation of a toxin as a by-product that

limited growth when lactate was used as a carbon source. Use

of acetate resulted in an increased specific growth rate

ð0:18 h�1Þ relative to that of malate ð0:03320:062 h�1

Þ. Due to

rapid growth, the maximum cell concentration was higher

(0.65 g/l) than that obtained for malate. The results of OMW

(Run 6) were close to those in Run 4, because acetate was the

major organic acid in OMW [29]. However, a higher specific

growth rate was obtained in outdoor conditions ð0:16 h�1Þ

compared to that observed in the indoor experiments

ð0:09 h�1Þ [17].

4.2. Malate consumption rate

For the hydrogen production experiments in which malate

concentration data were available, numerical modeling of the

concentration with respect to time was tried as attempted by

Eroglu et al. [23]. The model assumes a first order consump-

tion with respect to malate:

dMdt¼ �ks �M, (4)

where M is the malate concentration (in mM), t is the time

(in h) and ks is the first order rate constant ðh�1Þ. Eq. (4) was

integrated and linearized as follows:

lnM0

M

� �¼ ks � t, (5)

where M0 is the initial concentration of malate, at time zero.

Consumption of malate was observed to be first order as

stated in Eq. (5) and a plot of ln ðM0=MÞ versus t gave a straight

line for all runs (see Fig. 10 for Run 1). The first order rate

constant, ks, was approximately 0:02 h�1 as listed in Table 3.

As can be observed R2 values were close to 1 for all cases. The

first order consumption pattern agrees with the previous

studies when 15 mM of malate was used as a carbon source.

The first order rate constant was reported to be 0:018 h�1

under continuous illumination [23] and 0:021 h�1 within 14 h

3 Run 4 Run 5 Run 6

0.12 0.10 0.09

0.07 0.09 0.08

0.65 0.31 0.37

0.63 0.30 0.35

0.18 0.28 0.16

8 0.9879 0.9858 0.9818

6 – – –

7 – – –

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light and 10 h dark cycles [16] indoor batch operations in a

400 ml column photobioreactor.

5. Concluding remarks

Rhodobacter species are photoheterotrophic bacteria that can

easily switch to fermentative mode and produce organic acids

if the illumination is below a threshold value. When light is

sufficient, then the bacteria shifts its metabolism into a

photofermentation mode in which those organic acids would

be efficiently turned into hydrogen. As a result, biohydrogen

production in an outdoor reactor using sunlight as the sole

source of light energy seems promising.

Hydrogen gas was produced in both the logarithmic phase

and the stationary phase of growth. Photofermentative

growth of Rhodobacter sphaeroides in this outdoor bioreactor

can be explained by a logistic growth model.

It was observed from the experiments that hydrogen

production by Rhodobacter sphaeroides O.U. 001 was strictly

dependent on illumination. Under limited sunlight condi-

tions, i.e. light density o10;000 lux, no hydrogen production

occurred. Rather, fermentation started and the fermentation

end product formate as well as carbon dioxide were produced

when malate was used as the carbon source. When acetate

was used as the carbon source, butyrate was produced as a

result of fermentation. When another acetate-rich mediu-

m—olive mill wastewater—was utilized, promising amounts

of by-product (PHB and carotenoid) formation were observed

which supports the feasibility of biological hydrogen produc-

tion from a waste material.

Compared to indoor conditions, higher amounts of PHB and

carotenoid production were obtained from outdoor condi-

tions. These results revealed that microorganisms tend to

protect themselves from high light intensity by forming

carotenoid pigments and producing PHB as an energy storage

material for dark periods.

Acknowledgments

This study has been supported by the Turkish State Planning

Agency (Project no.: BAP-08-11-DPT.2005K120600), the METU

Research Fund (Project no.: AFP 99-06-02-13) and the Eur-

opean Union 6th Framework Integrated Project (HYVOLU-

TION).

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