SCREENING OF PHOTOSYNTHETIC BACTERIA
FOR HIGH YIELD HYDROGEN PRODUCTION
Jyumpei Kobayashi, Tomoe Komoriya and Hideki Kohno
Department of Applied Molecular Chemistry
Graduate School of Industrial Technology
Nihon University
Narashino, Chiba, JAPAN
[email protected]
ABSTRACT
Environmental bacteria were screened and examined for hydrogen production. Seventy six
strains of photosynthetic bacteria were screened. In those strains, 4 strains indicated higher
hydrogen production rate (ml/mg h) than that of Rhodobacter sphaeroides RV which has been
used for hydrogen production. Moreover, 5 strains which indicated high hydrogen production
rate were examined further conditions that were carried out using various organic acids as
substrates for hydrogen production. I-2A-K strain indicated the highest average hydrogen
production rate. Strains which had the highest substrate conversion efficiency (%) are I-1A-J and
I-2A-H strains. However, in a practical hydrogen production, it is speculated that compositions
of substrates are diverse and complex. So, in order to select the proper bacteria for conditions
that will be carried out, further examination must be needed.
INTRODUCTION
Hydrogen attracts great attention as clean and efficient energy carrier in stead of fossil fuel. It
has also needed significantly as potential energy carrier, due to rapid advances in fuel cell
technologies (Edwards et al., 2007). However, most of hydrogen is currently generated by
reforming of natural gas or light oil with steam at high temperature. Biological hydrogen
production stands out in environmentally harmless and safety processes compared with these
methods. Moreover, biological methods have the advantage which has both energy generation
and disposal of organic wastes. But it also has problem that biological methods are difficult to
produce a large amount of hydrogen. Photosynthetic bacteria are favorable candidates for
biological hydrogen production due to their high conversion efficiency and versatility in the
substrates they can utilize. In previous studies, various photosynthetic bacteria and conditions
have examined. But theoretical yield has not been reached. In order to improve further hydrogen
production efficiency, we screened and examined photosynthetic bacteria which can take place of
RV with high hydrogen production rate in this study.
EXPERIMENTAL METHODS
Bacteria
Bacteria were isolated from lakes and rice paddy fields in Tokyo and Chiba area in Japan. They
were cultivated in an enrichment culture medium, which consisted of basal medium (Nakada et
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al., 1993), sodium acetate (7.3mM) and yeast extract (1%). Cultured bacteria were formed
colony and isolated for agar plate medium of aSy medium, which consisted of basal medium,
sodium succinated (36mM) yeast extract (1%) and ammonium sulfate (9.5mM) under aerobic
and dark conditions (Mao et al., 1986). And they were cultivated in aSy medium for pre-culture.
The pH of these media was adjusted to be 6.8. These media were sterilized before being used for
culture. The enrichment culture was cultivated anaerobically in presence of nitrogen gas under
illuminated (3000lux) using halogen lamps for ten days. The agar plate medium was cultivated
aerobic and dark conditions for two weeks. Pre-culture was cultivated anaerobically under
illuminated (10,000lux) for three days.
Hydrogen production
The hydrogen production was carried out by using of the GL medium which consisted of a basal
medium which contain ten folds concentration of PO4 buffer, organic acid (75mmol/l) and
sodium L-glutamate (2mmol/l). The pH of the medium was adjusted to 7.0. The cells were
centrifuged (9,000rpm 15min) and re-suspended by a basal medium. The re-suspended solution,
a basal medium and an agar solution witch consisted of a basal medium and agar powders (4%)
were added and mixed in a Roux bottle (200cm3). Then the mixed solution was adjusted to be
30ml, OD1.5 and 2% of the agar for measuring absorbance of the re-suspended solution.
Thereafter, the mixed solution was cooled and hardened in the bottle which was lay down as in
by Figure 1. Figure 2 shows an overall hydrogen production instrument. The hydrogen
production was carried out at 30ºC under a light intensity of 10,000lux using halogen lamps for a
week (168h). The produced hydrogen was collected in a measuring cylinder that was installed up
side down and filled with sodium hydroxide solution to trap CO2 gas which has possibility to
generate.
Evaluation of hydrogen production rate
In order to evaluate the hydrogen production in different kinds of bacteria, we used the hydrogen
production rate. This rate was consisted of the properties of the hydrogen production (ml), dry
weight of bacteria (mg) and time (h). The dry weight used for the hydrogen production was
calculated by measuring the relationship between the absorbance and the dry weight.
The substrate conversion efficiency (%) was also needed to evaluate the bacteria. In order to
determine the substrate conversion efficiency, the substrate concentration (mmol/l) was measured
using HPLC and the theoretical yield was used. Five kinds of substrate were used for the
hydrogen production in this study. The theoretical yield was calculated using follow equations.
C3H6O3 + 3H2O → 6H2 + 3CO2
C2H4O2 + 2H2O → 4H2 + 2CO2
C4H8O2 + 6H2O → 10H2 + 4CO2
C4H6O4 + 4H2O → 7H2 + 4CO2
C4H6O5 + 3H2O → 6H2 + 4CO2
(1)
(2)
(3)
(4)
(5)
Equation (1) indicates the theoretical yield of lactate. Equation (2) is on acetate, (3) is on
butyrate, (4) is on succinate, (5) is on malate.
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(A)
(B)
Figure 1. Basic experimental methods. (A) Use for pre-cultures and hydrogen. (B)
Immobilization of bacteria with agar in Roux bottle.
Medium
H2
Bacteria
Light
Halogen lamp
Water bath
Water bath (NaOH aq)
Figure 2. The instrument of the hydrogen production and collection. Roux bottle was stopped
up by silicon rubber penetrated by an injection needle to thread produced hydrogen. The
hydrogen passed through TYGON tube connected an injection needle at tip.
Dry weight of bacteria
To determine the dry weight, bacteria were centrifuged and re-suspended by pure water and the
re-suspended solution was measured its absorbance. The membrane filter which was rinsed by
pure water and dried in advance was measured its weight. The re-suspended solution was
filtrated by aspirating using the membrane filter. The used membrane filter was dried and
measured its weight. The dry weight was calculated by subtracting the weight of filter from the
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weight of used filter. The calibration curbs were generated by the relationship between the
absorbance and the dry weight.
RESULTS AND DISSCUSSIONS
Isolation and examination of hydrogen production
Seventy six strains of bacteria were obtained and examined the hydrogen production using
D,L-lactate as substrate. Table 1 shows the hydrogen production (ml), dry weight at 1.5 of OD
(mg), and hydrogen production rate (ml/mg h) of the five strains that indicate high hydrogen
production rate (ml/mg h) and RV. The time (h) was 168 in any cases. In Table 1, four strains of
bacteria show the higher rate than that of RV. I-2A-H only has the higher hydrogen production
(ml) than RV and the others indicate lower values. Five strains except for RV indicate the lower
dry weights than RV. These cause the higher hydrogen production rates of five strains than RV.
Hydrogen production using different substrate in five strains
Five strains and RV were examined the hydrogen production using acetate, butyrate, D,L-malate
and succinate besides D,L-lactate. Table 2 shows these hydrogen production rate (ml/mg h) and
sbstrate conversion efficiency (%). For acetate, the hydrogen production rates are not higher than
the case of D,L-lactate. But the substrate conversion efficiencies are higher than D,L-lactate. It is
caused by the theoretical yield of acetate that is lower than D,L-lactate. Acetate is usually used
for synthesis of poly hydroxyl butyrate (PHB). Therefore it can be thought that a part of acetate
for the hydrogen production might be used to synthesize PHB. For that reason, it is generally
thought that acetate is not suitable for the hydrogen production. But all strains indicate high
substrate conversion efficiency. Especially, H-1A-I indicates the highest hydrogen production
rate of all. It is seemed that this strain has the lower ability of the PHB synthesis than the others.
From these results, acetate has possibility of favorable candidates for the hydrogen production.
On butyrate, it produced no hydrogen in every strain. It indicates that all of six strains don’t have
the enzymes which can utilize butyrate. Succinate is an intermediate substance of TCA cycle.
Therefore, results of succinate indicate efficiency of TCA cycle each strains have. L-malate was
also intermediate substance of TCA cycle. On the other hand, D-malate was metabolized through
a different path way (Kokua et al., 2002). If the bacteria have the higher substrate conversion
efficiency in succinate than in D,L-malate, it indicates that the conversion efficiency of D-malate
is lower than L-malate. Efficiency of D-malate in RV, H-1A-I and H-1A-J is lower than that of
L-malate. I-2A-K and I-2A-H has the higher efficiency of D-malate than of L-malate. I-1A-R has
almost same efficiency at both D-malate and L-malate.
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Table 1. Hydrogen production, dry weight and hydrogen production rate of 6 strain including RV
strain
Hydrogen
Dry weight
Hydrogen production
production (ml)
(mg)
rate (ml/mg h)
RV
663
15.091
262
I-2A-K
568
12.623
268
H-1A-I
600
13.453
265
H-1A-J
515
13.136
233
I-1A-R
635
13.320
284
I-2A-H
676
13.994
289
Table 2. Hydrogen production rate and substrate conversion efficiency of 6 strain including RV
strain in D,L-lactate, acetate, butyrate, succinate, D,L-malate, and average
D,L-lactate
Acetate
Butyrate
Succinate
D,L-malate
Average
(Hydrogen production rate (ml/mg h) / substrate conversion efficiency (%))
RV
262/47
199/54
0/0
192/54
157/47
162/41
I-2A-K
268/49
194/50
0/0
188/40
203/49
171/38
H-1A-I
265/51
215/57
0/0
195/58
74/21
150/37
H-1A-J
233/34
263/75
0/0
199/56
116/34
162/40
I-1A-R
284/43
237/62
0/0
182/46
148/45
170/39
I-2A-H
289/42
196/56
0/0
174/55
180/59
168/40
CONCLUSIONS
We screened bacteria and examined the hydrogen production. Moreover, we investigated the
hydrogen production about bacteria which has high hydrogen production rate. But there is room
for an examination which bacteria are suitable for hydrogen production. If we simply want to
obtain a large amount of hydrogen, I-2A-K which has the highest average hydrogen production
rate can be suitable. But if we want to place importance for the disposal of wastes, RV can be
superior. There is a method that photosynthetic bacteria and lactic fermentative bacteria are
co-cultured using saccharides as substrate (Asada et al., 2006). If this method is used, H-1A-I or
I-2A-H can be proper for hydrogen production due to their high hydrogen production rate or
substrate conversion efficiency on D,L-lactate. But in any cases, the most suitable bacteria may
change in thought of what substances constitute the substrates which will be used practical.
There are substrates photosynthetic bacteria can utilize besides organic acids used in this study.
In order to select the proper bacteria for conditions that will be carried out, further examination
must be needed.
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