Optimization of Photo-Hydrogen Production by Immobilized Rhodopseudomonas Faecalis RLD-53

doi:10.4236/nr.2011.21001

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Natural Resources, 2011, 2, 1-7

doi:10.4236/nr.2011.21001 Published Online March 2011 (http://www.scirp.org/journal/nr)

Optimization of Photo-Hydrogen Production by

Immobilized Rhodopseudomonas Faecalis RLD-53

Bing-Feng Liu, Guo-Jun Xie, Wan-Qian Guo, Jie Ding, Nan-Qi Ren

State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin, China

E-mail: [email protected]; [email protected]

Received December 7th, 2010; revised January 10th, 2011; accepted January 17th, 2011

ABSTRACT

In this work, the optimization of hydrog en production by photo-fermentation bacteria immobilized on agar gel granu le

was systematic investigated in batch culture. Experiment focus on the effect of some important affecting factors on

photo-hydrogen production. Results indicated that immobilized Rhodopseudomonas faecalis RLD-53 exhibited the

highest hydrogen yield of 3.15 mol H2/mol acetate under follow optimal condition: agar granule diameter of 2.5 mm,

inoculum age of 24 h, agar concentration of 2%, biomass of 4 mg/ml in agar and light intensity o f 9000 lux. More im-

portantly, immobilized photo-fermentation bacteria not only can enhance hydrogen production but can increase ac-

ids-tolerance capa city, even at pH 5.0 hydrogen also was produced, and th us hopefully immob ilized photo-fermen tation

bacteria can be applied in the combination of dark and photo-fermentation for hydrogen production with high yield.

Keywords: Hydrogen Production, Photo-Fermentation, Agar Gel, Immobilized Rhodopseudomonas Faecalis,

Acids-Tolerance Capa city

1. Introduction

The shortage of fossil fuels, the pollution of global

environment and emissions of greenhouse gas are at-

tracting more and more attention of researches. But, up to

now, the major source of energy is still supplied from

fossil fuels. Hence, we urgent need to develop new and

renewable energy to replace fossil fuels. Bio-hydrogen is

a clean, environmental friendly and recycle energy car-

rier and recognized as a promising substitute of fossil

fuels in future. At present, two main pathways, dark and

photo-fermentation, were used for bio-hydrogen produc-

tion [1]. Recently, bio-hydrogen production by photo-

fermentation has exhibited great potential. In particular

dark-fermentation process produced short chain acids,

which can be utilizing by photo-fermentation process for

additional hydrogen production. Thus, the combination

of dark and photo fermentation for hydrogen production

can achieve a high overall hydrogen yield, which hope-

fully close to theoretically maximum value of 12 mol

H2/mol hexose. The overall hydrogen yield of 13.7 mol

H2/mol-sucrose (equivalent to 6.85 mol H2/mol hexose)

was obtained using sequential dark and photo-fer

mentation from beet molasses [2]. Previous our work

showed that the two-step of dark and photo fermentation

reached a nice hydrogen yield of 6.32 [3] and 5.37 [4]

mol H2/mol glucose in batch experiment. Other some

reports also demonstrated that the total hydrogen yield of

sequential dark and photo-fermentation was obviously

higher than that of a single dark fermentation process

using single substrate as sole carbon source [5-8].

The immobilization technology for dark fermentation

hydrogen production has been widely studied [9,10].

Usually, bacteria main were immobilized on carrier such

as polyacrylamide [11], polyvinyl alcohol [12], or agar

gel [13], etc. Nearly all their work showed that immobi-

lized dark fermentative bacteria can enhance and stabi-

lize hydrogen production process. There are only a few

reports about the immobilization of photo-fermentation

bacteria for improving the bio-hydrogen process [14,15].

However, these reports have not focused on the optimi-

zation of immobilized condition.

Therefore, in this study photo-fermentation bacteria

were immobilized on agar gel granule to explore their

hydrogen production capacity. The some key immobi-

lized parameters, for example, agar granule diameter,

inoculum age, agar concentration, pH, biomass in agar

and light intensity, were systematic optimized for im-

proving photo-hydrogen production by batch culture.

2. Method

2.1. Bacterial Strain and Medium

Photo-fermentative bacterium Rhodopseudomonas fae-

Optimization of Photo-Hydrogen Production by Immobilized Rhodopseudomonas Faecalis RLD-53

calis strain RLD-53, the previously isolated from fresh

water pond, used in this study [16]. The medium for

growth and hydrogen production was same with previous

description by Liu et al. [17]. Acetate of 50 mmol/l was

used as sole carbon source in medium for hydrogen pro-

duction. The strain RLD-53 was pre-cultured at 35℃ for

24 h under light intensity of 2000 lux with incandescent

lamps (60 W) and argon was used to maintain anaerobic

condition.

2.2. Hydrogen Production Procedure

The experiment was carried out in 100 ml serum bottles,

which were sealed by rubber plugs and filled with argon

to maintain anaerobic conditions. 80 ml medium for hy-

drogen production was put into reaction bottles. The bot-

tles with liquid medium were sterilized at 121℃ for 15

min. Operation parameters were as follows: inoculant

age of 24 h, OD660 of 1.68, inoculant volume of 10%

(v/v), the bottles were shaken on the constant tempera-

ture incubation oscillator at 120 rpm, culture temperature

of 35℃, The light intensity of outside surface of the bot-

tles was maintained at 4000 lux by a incandescent lamps

of 60 W.

2.3. Preparation of Immobilized Cell

The cells of photo-fermentation bacteria were harvested

by centrifugation at 6000 rpm for 10 min. Agar was dis-

solved in 20 ml of sterile water at 0.4% (w/v) and incu-

bated at 100℃, and then it was used for the immobiliza-

tion of bacterial cells. Agar solution was cooled to 40-50

℃ and mixed with 20 ml prepared suspension of centri-

fuged R. faecalis RLD-53 in a beaker. The above mixture

was immediately inhaled into a syringe of 50 ml by hand

and until agar gel was formed, and then agar gel of con-

taining bacterial cells was injected into the medium

through a syringe. The dry weight of the bacterial cells in

the each agar gel granule with the average diameter of

about 2.5 mm was approximately 0.113 mg.

2.4. Analytical Method

Hydrogen analysis in evolved gas was performed using a

gas chromatograph (GC) (Model SC-II, Shanghai Analy-

sis Instrument Factory) equipped with a thermal conduc-

tivity detector and a 2-m stainless column packed with 5

Å molecular sieves. The operational temperatures at the

injection port, the column oven and detector were 100,

60 and 105 °C, respectively. Argon was used as carrier

gas at a flow rate of 70 ml/min. The volatile fatty acids in

supernatant of culture broth were determined using a

second GC (Model GC122, Shanghai Analysis Instru-

ment Factory) equipped with a flame ionization detector

and a 30 m×0.25 mm×0.25 mm fused-silica capillary

column. The liquor samples were first centrifuged at

12,000 rpm for 5 min, and then acidified with hydrochlo-

ric acid and filtered through a 0.2-μm membrane before

free acids were analyzed. Nitrogen was used as carrier

gas.

The light intensity (lux) was measured by using a

digital luxmeter (TES1330A, Junkai Co.). Cell concen-

tration was determined by an Amersham Pharmacia Bio-

tech ultraspec 34300 UV/Vis spectrophotometer.

3. Results and Discussion

3.1. The Effect of Agar Gel Granule Size

Agar granule size was an important factor affecting hy-

drogen production by photo-fermentation. Agar granule

size of 0.5 mm, 1 mm, 1.5 mm, 2.5 mm made by differ-

ent sizes of syringe. Acetate of 50 mmol/l, glutamate of

10 mmol/l, reaction system of 80 ml, incubation tem-

perature of 35 and light intensity of 4℃ 000 lux were

used.

Hydrogen production increased gradually with the in-

crease of agar granule size from 0.5 - 2.5 mm (Figure 1).

Agar granule size at 0.5, 1.0, 1.5 and 2.5 mm, cumulative

volume of hydrogen was 170, 210, 240 and 270 ml/re-

actor, respectively, and the control was 218 ml/reactor.

This result indicated that hydrogen yield was higher

compared to other size when agar granule size was at 1.5

and 2.5 mm. Agar granule size at 2.5 mm, hydrogen

yield reached maximum value of 3.15 mol H2/mol ace-

tate, conversion efficiency of substrate was 78.75% and

hydrogen content was between 75% - 85%. In addition,

immobilized bacterial cells can lengthen the time of hy-

drogen production. Hydrogen production of

non-immobilized cell stopped at 192 h, but hydrogen

production of immobilized cell stopped at 264 h. The

utilization efficiency of substrate of immobilized cell

04896144 192 240 288

100

150

200

250

300

Hydrogen production (ml)

Time (h)

0.5 mm

1.0 mm

1.5 mm

2.5 mm

the control

Figure 1. The effect of gel granule size on photo-H2 Produc-

tion.

Optimization of Photo-Hydrogen Production by Immobilized Rhodopseudomonas Faecalis RLD-53 3

was higher compared to non-immobilized cell. Within 48

h of culture, a great quantity of substrate was consumed

for cell growth and the utilization efficiency of substrate

was 0.04 g acetate/l/h. However, the utilization effi-

ciency of substrate of immobilized cell was 0.018 g ace-

tate/l/h, more acetate was for hydrogen production (Fig-

ure 2). These seem to imply that the bacteria were im-

mobilized in agar gel can limit substrate for itself growth

and increase hydrogen production, and lengthen the time

of hydrogen production. So, agar granule size of 2.5 mm

was used follow experiment for further research.

3.2. The Effect of Inoculant Age

In this test, the inoculant age of 12, 24, 36, 48, 60, 72, 84

and 96 h was employed to explore their hydrogen pro-

duction capacity.

The difference in hydrogen production under various

inoculant ages is very obviously (Figure 3). Higher yield

and production rate of hydrogen was obtained when in-

oculant age was 24 h and 72 h, and cumulative volume of

hydrogen was 246 ml/reactor and 233 ml/reactor, respec-

tively. Inoculant age was at 12, 48 and 84 h, the yield and

production rate of hydrogen was similarly and it was

about 200 ml/reactor. Inoculant age at 36 and 96 h, cu-

mulative volume of hydrogen was about 160 ml/re-

actor. However, inoculant age at 60 h, hydrogen yield

only was about 100 ml/reactor. The hydrogen production

by the bacteria cause the results of differences may be

related to physiological state and enzymes activity of

bacteria. We think that a long time of bacteria in the agar,

nitrogenase activity was restored or enhanced leading to

the hydrogen yield in high level. Inoculant age has a di-

rect impact on the cell's physiological state and the

chemical components of culture. Our result was consis-

tent with Felten et al.’s report, which showed that the

Figure 2. The consumption of acetate under different agar

granule size in immobilized photo-hydrogen production.

inoculant age of the immobilized bacteria is the key fac-

tor affecting hydrogen production, R. rubrum of inocu-

lant age of 70 h were immobilized with the highest hy-

drogen production activity [18]. Therefore, inoculant age

of about 24 h and 72 h for photo-hydrogen production is

appropriate.

3.3. The Effect of Agar Concentration

Photo-hydrogen production significantly influenced by

agar concentration, which directly determined the ab-

sorbance of photo-fermentative bacteria, utilization effi-

ciency and transfer rates of substrate.

Agar concentration was in the range of 1% - 4%, agar

gel granule size of 2.5 mm and inoculant age of 24 h

were used in this test. When agar concentration was 1.5%

and 2%, cumulative volume of hydrogen was 254.98

ml/reactor and 249.67 ml/reactor, hydrogen production

capacity was higher than that of other agar concentration

(Figure 4). The performance of hydrogen production

was similar to the control under agar concentration of 1%,

3% and 4%. Agar concentration over 3%, penetration of

light into inside bacterial cell decreased and the growth

rate will be decreased with increasing the agar concentra-

tion, thereby affecting the utilization of its substrate and

the internal mass transfer resistance increased. This result

was similar to Seol. et al.’s research, which indicated that

the substrate and products are easily transferred through

the bead when agar concentration in proper experimental

ranges [19]. In addition, the accumulation of cell me-

tabolite in the agar granule caused cell toxicity and re-

pressed infiltration capacity of substrate, will also affect

the growth and hydrogen production of photo-fermenta-

tion bacteria.

3.4. The Effect of Bacterial Concentration in

Agar Gel

The bacterial concentration in agar influenced utilization

and mass transfer efficiency of substrate. Biomass in agar

was 2, 4, 6, 8 and 10 mg/ml, respectively. Agar gel gran-

ule size, inoculant age, agar concentration, acetate con-

centration and reaction system were 3 mm, 24 h, 2%, 50

mmol/l and 80 ml, respectively.

Result indicated the biomass range of 2 - 4 mg/ml in

agar may help to improve performance of hydrogen pro-

duction and hydrogen yield decreased when biomass was

over 6 mg/ml; high biomass in gel granule for enhancing

the hydrogen production is not obvious (Figure 5). Rea-

son may be due to the substrate into the gel granules was

constant and excessive photo-fermentation bacteria were

employed in hydrogen production process leading to the

consumption of a lot of substrate to maintain the energy

require for their growth, thus reducing the use of sub-

strate for hydrogen production and immobilized cells

Optimization of Photo-Hydrogen Production by Immobilized Rhodopseudomonas Faecalis RLD-53

Figure 3. The effect of inoculant age on photo-H2 produc-

tion.

04896144 192 240

100

150

200

250

Hydrogen production (ml)

Time (h)

1.5%

Figure 4. The effect of agar concentration on photo-H2

production.

activity was prevented. So, the biomass of a certain con-

centration range can promote hydrogen production ca-

pacity of immobilized photo-fermentation bacteria. How-

ever, the biomass in gel granule is too high and substrate

to maintain bacterial physical requirement exceeded,

thereby the hydrogen production capacity reduced. The

suitable cell concentration of 1 mg/ml not only achieved

the highest hydrogen yield but also more important supe-

rior nitrogenase activity [18].

3.5. PH Tolerant Capacity

To determine acids tolerance of immobilized cell, in this

test, the pH of reaction system was adjusted to 4.0, 5.0,

5.5, 6.0 and 6.5, respectively. Agar granule diameter of

2.5 mm, inoculum age of 24 h, agar concentration of 2%,

biomass of 4 mg/ml in agar and acetate of 50 m mol/l

Figure 5. The effect of biomass concentration in agar gel

granule.

were constant in 80 ml system.

A decrease in pH resulting in decrease in the yield and

production rate of hydrogen (Figure 6). In all pH tests,

compared to initial pH, final pH increased slightly due to

the consumption of acetate (Figure 7). At low pH 4.0,

bacteria can not grow and hydrogen also not produced.

Little hydrogen was generated and cumulative hydrogen

volume only was 50 ml at pH 5.0. The lag time of hy-

drogen production was about 72 h and hydrogen produc-

tion rate was 7.3 ml H2/l/h. At pH 5.5, the lag time of

hydrogen production decreased to about 48 h and hydro-

gen production yield and rate started to increase. Cumu-

lative hydrogen volume and maximum hydrogen produc-

tion rate reached 149 ml and 17.7 ml H2/l/h, respectively.

At pH 6.0 and 6.5, the trend of hydrogen production was

similarly within 144 h. After 144 h, hydrogen production

rate increased slightly under pH 6.5. Finally, maximum

Figure 6. Acid-tolerance capacity of strain RLD-53 during

H2 Production.

Optimization of Photo-Hydrogen Production by Immobilized Rhodopseudomonas Faecalis RLD-53 5

04896144 192 240

4.0

4.5

5.0

5.5

6.0

6.5

7.0

Initial pH

Time

(

)

pH4.0 pH5.0 pH5.5

pH6.0 pH6.5

Figure 7. The change of pH under different initial pH dur-

ing entire H2 production.

hydrogen yield of 250 ml and rate of 23.4 ml H2/l/h was

obtained at pH 6.5. Suitable range of pH is at 6.5 - 7.5 for

non-immobilized photo-fermentation bacteria [16,20].

Thereby, above results implied that pH are an important

factors for sustained and efficient hydrogen production in

immobilized strain RLD-53 and the immobilized fer-

mentation bacteria have certain acids-tolerance capacity

with high hydrogen yield. Immobilized photo-fermenta-

tive bacteria can tolerate lower pH of a certain extent,

even at pH 5.0 hydrogen also was produced.

3.6. Requirement of Light Intensity

The growth and hydrogen production of photo-fermenta-

tion bacteria need to apply energy by light condition. So,

light intensity also was an important limiting factor for

photo-hydrogen production. The optimum light intensity

of non-immobilized strain RLD-53 for hydrogen produc-

tion was at 3000-5000 lux [16], and the bacteria were

immobilized on agar, which can prevent the infiltration

of light and light absorption of bacteria. Therefore, the

investigation of light intensity of immobilized bacteria is

necessary.

Effect of different light intensities on the hydrogen

production is depicted in Figure 8. It has been observed

that increased light intensity resulted in an increase in the

total volume of hydrogen and also hydrogen production

rate. The lower light intensity accompanied a long lag

time of hydrogen production. Light intensity was at 1 000

lux, lag time of hydrogen production is 48 h and the low-

est yield of hydrogen of 178 ml-H2/ reactor was obtained.

When the light intensity was at 7 000 lux and 9000 lux,

the hydrogen production capacity was closed, the cumu-

lative volume of hydrogen gas were 246 ml-H2/reactor

and 255 ml-H2/reactor, respectively, the maximum hy-

drogen production rate reached 24 ml- H2/l/h. It can be

Figure 8. The effect of light intensity on photo-H2 produc-

tion.

observed that the hydrogen production under highest

light intensity reached a higher yield. Usually, high light

intensity can inhibit hydrogen production by non-immo-

bilized photo-fermentation bacteria [16, 21]. However,

these results indicated that the range of optimal light in-

tensity for hydrogen production by immobilized phoro-

fermentation bacteria was between 7 000 - 9 000 lux.

This also suggested that the phoro-fer-mentation bacteria

immobilized in agar to prevent the light penetration into

inside bacterial cells and light absorption of the bacterial

photosynthetic system, further influence generation of

electronic and synthesis of ATP, leading to bacterial

growth, nitrogenase activity and hydrogen production

were inhibited. Therefore, light intensity needs to in-

crease to obtain enough supply of light energy for the

growth and producing hydrogen of photo-fermentation

bacteria.

4. Conclusions

Results obtained in this study clearly exhibited the immo-

bilized photo-fermentation bacteria could obviously pro-

mote hydrogen production, the conversion efficiency of

substrate and lengthen time of hydrogen production.

More importantly, it demonstrated that the granule dia-

meter, inoculant age, agar concentration, biomass in agar

and light intensity are key factors affecting photo-

fermentation hydrogen production, and when they are 2.5

mm, 24 or 72 h, 2%, 4 mg/ml and 7000-9000 lux, the

maximum hydrogen yield reached 3.15 mol-H2/molacetate.

The immobilized photo fermentation bacteria not only

can enhance hydrogen production but can increase ac-

ids-tolerance capacity, even at pH 5.0 hydrogen also was

produced, and thus hopefully immobilized photo-fer-

mentation bacteria can be applied in the combination of

dark and photo-fermentation for hydrogen production

with high yield.

Optimization of Photo-Hydrogen Production by Immobilized Rhodopseudomonas Faecalis RLD-53

5. Acknowledgements

This research was supported by the financial support

from the National Nature Science Foundation of China

(No. 30870037, 50821002 and 50638020), State Key

Laboratory of Urban Water Resource and Environment

(HIT) (Grant No.QAK200806). The authors would like

to thank the Key Laboratory of water/soil toxic pollutants

control and bioremediation of Guangdong Higher Educa-

tion Institutes, Jinan University for supporting this study.

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