Journal of Environmental Protection, 2010, 1, 426-430
doi:10.4236/jep.2010.14049 Published Online December 2010 (http://www.SciRP.org/journal/jep)
Influence of Metal Ions on Hydrogen Production by
Photosynthetic Bacteria Grown in Escherichia coli
Pre-Fermented Cheese Whey
Fadhil M. Salih1*, Muthana I. Maleek2
1ClearValue Technologies Inc., Sugar Land, U.S.A.; 2Department of Biology, College of Science, Wasit University, Wasit, Iraq.
Received September 15th, 2010; revised October 4th, 2010; accepted October 15th, 2010.
ABSTRACT
The photosynthetic bacteria, Rodospirillum rubrum, produced hydrogen when grown in cheese whey in presence of
light. The production increased three times as much when whey was used after being incubated in presence of Es-
cherichia coli at 37℃ for 5 days, giving a total of 3600 ml of H2 in 10 days. The presence of Fe ions (5 mg/L) enhanced
H2 production of treated whey to about 6000 ml in 10 days. Mo ions (0.3 and 1.6 mg/l) did not affect achieved H2 pro-
duction of treated whey. However, when Fe and Mo ions were present together, the production was comparable with
that of Mo ions alone, i.e. Mo prevented Fe of producing any enhancing effect. The addition of Mn ions (7.68 mg/L) to
treated whey containing Fe (5 mg/L) and Mo ions (8 mg/L) increased H2 production to give about 9500 ml/10 days.
Keywords: Hydrogen Production, Photosynthetic Bacteria, Rodospirillum rubrum, Metal Ions, E. coli, Fermentation
1. Introduction
Hydrogen is an attractive energy carrier because it has
the highest density of energy per weight of any chemical
fuel. It is essentially non-polluting and it is by far the
most abundant element in the universe [1]. Among the
many methods used for hydrogen production are those
depending on biological systems. The light dependent
production of hydrogen by photosynthetic bacteria,
which was first discovered in 1949 with cultures of R.
rubrum [2] and with other photosynthetic bacteria [3]
represents one of the promising systems. It has been
found that in photosynthetic bacteria including R. rubrum
energy dependent hydrogen production occurs for which
the nitrogenase enzyme is responsible and such produc-
tion increased by intracellular accumulation of nitro-
genases [4] and [5]. The activity of the enzyme was
strictly dependent on light and no activity was observed
in the dark [6].
Maximal expression of H2 production capacity was
observed when bacteria were grown photoheterotrophi-
cally on suitable substrates (e.g. organic acids) in pres-
ence of certain amino acids serving as source of nitrogen
[7] and [8]. However, presence of ammonia, even at very
low concentrations repressed synthesis of H2 evolving
system and inhibited H2 production [9] and [10]. Never-
theless, addition of N2 gas at intervals activates H2 pro-
duction [3]. Evolution of H2 increased twice as high
when 5 mg iron/L was present as compared to cultures
containing 0.5 mg/L. This phenomenon was reportedly
related to iron requirement of nitrogenase and ferredoxin
[11]. Molybdenum and iron were also necessary for ni-
trogenase activity through its two proteins (Fe and Mo Fe
proteins) [12]. Other factors that seriously affect the
process of conversion such as inoculum, substrate, reac-
tor type, phosphate, metal ion, temperature and pH are
reported elsewhere [13].
Cheese whey has been used successfully as a growth
supporting medium as for its good contents of nutritive
materials in addition to its high content of lactose (5 to
6%) [14]. Growing R. rubrum in cheese whey in pres-
ence of light produced H2 gas at certain rate [15] and [16].
Similarly, hydrogen was also produced from cheese
whey and other waste materials using anaerobic and pho-
tosynthetic bacteria [17] and [18]. Usually large amount
of cheese whey is released from dairy factories to sewage
every day. Therefore, it was the aim of the present inves-
tigation to make use of this whey in the production of H2
through growing photosynthetic bacteria and to try to
improve such productivity.

Influence of Metal Ions on Hydrogen Production by Photosynthetic Bacteria Grown in Escherichia coli
Pre-Fermented Cheese Whey
427
2. Materials and Methods
R. rubrum S-1 was used. Cells were grown and propa-
gated using RCV medium described by Weaver et al. [12]
except that ammonia was replaced by 7 mM L-serine and
the DL-malic acid by 30 mM lactic acid. For the rest of
the work cheese whey was used as nutrient medium. It
was obtained from a dairy factory immediately before the
release to sewage. Prior to using the whey it was heat
treated at 95℃ for 15 minutes and filtered through chee-
secloth. After heat treatment whey was centrifuged in 50
ml lots at 3000 rpm for 30 minutes in order to get rid of
most of the solid particles. pH was adjusted to 7.0 using
1N NaOH and samples were further autoclaved at 121℃
for 15 minutes.
In order to convert the lactose of the whey into lactic
acid, whey was incubated at 37℃ for 5 days in presence
of E. coli. After treatment whey was centrifuged for 30
minutes and the supernatant was heat treated at 80℃ for
10 minutes to kill E. coli left. Whey pH was adjusted to
7.0.
Screw capped test tubes containing 27 ml RCV me-
dium was inoculated with R. rubrum and incubated at
30℃ overnight in presence of light (60 W, 25 cm dis-
tance). Two subcultures were made successively by 7 ml
inoculums. This method of subculturing was employed
for bacterial adaptation. The content of the third culture
was transferred to 125 ml bottle containing about 100 ml
medium so that the bottle was topped up. The bottle was
closed with rubber stopper leaving a small space above
the medium for gas collection. Needle was inserted
through the stopper so that its distal end does not touch
the liquid. The needle was connected to a rubber tube for
gas collection. Gas collection was made by simple liquid
displacement. The liquid was distilled water containing
40 g NaOH and 200 g NaCl/l. It was convenient for re-
moving CO2 completely from the evoluted gas. The effi-
ciency of CO2 removal was checked using gas chroma-
tography (Shimadzu GC 14A Gas Chromatograph). The
bottle was incubated at 30℃ in a growth box made of
Perspex and illuminated with 2 × 60 W Tungsten lamps.
Growth intensity was measured by using PYE Unicum
SP8-200 UV/VIS spectrophotometer. It was found in the
range of 2.45 at 660 nm and giving a dry weight of
0.00141 g/ml for one-day-old culture. Light intensity was
measured by IL 700 A Research Radiometer, Interna-
tional Light, Newburyport, Ma, USA. It was found to be
20 mW/cm2. H2 concentration was determined using gas
chromatography as well. The gas chromatograph was
equipped with a molecular sieve 5A column (40-60 mesh)
and a thermal conductivity detector.
For the purpose of increasing the efficiency of whey,
varying concentrations of Fe (5, 10 and 20 mg/L), Mo
(0.8, 1.6, 4 and 8 mg/L) and Mn (2.58-5.12 and 7.68
mg/L) were added either individually or in combinations.
3. Results and Discussion
R. rubrum seeded in treated or untreated whey showed
heavy growth in comparison with that grown on RCV
medium (Figure 1). As appeared, cells produced H2 from
both untreated and treated whey. For untreated one the
average rate of H2 production during the first 2 days was
120 ml/L medium/day, and for the first 5 days was 160
ml/L/day followed by slower rate. However, when E. coli
treated whey was used, the rate of H2 production was
about 820 ml/L/day for the first 2 days, and was as high
as 560 ml/L/day in the first 5 days, followed by slower
rate.
For comparison purposes H2 produced by cells grown
in RCV medium was also included in Figure 1. The rates
of production for the first 2 days were 300 and 450
ml/L/day respectively. Production continued at its high
rate up to the eighth day where it almost stopped. The
total amounts of H2 produced during the whole experi-
mental period (10 days) were 960, 3040 and 3600 ml for
untreated whey, RCV medium and treated whey, respec-
tively.
The low productivity of untreated whey can be attrib-
uted to the unavailability of readily useable carbohy-
drates (the major part of carbohydrate was lactose, which
simply, cannot be directly used by this bacteria) [15] and
[16]. However, the evoluted H2 may well be due to the
presence of small amount of lactic acid formed during
cheese curding [14]. This seems unreal because when
lactate and glutamate were added to the untreated whey
(Figure 2) H2 production was not improved. In fact low-
er evolution was seen. However, when whey contained
50% RCV medium H2 production was enhanced to an
extent as high as that of the E. coli treated whey. These
findings imply that the improvement of H2 production in
treated whey could possibly not be due to lactic acid
formation only, but to other product(s) formed as a result
of E. coli growth in the whey.
When ferric chloride at 5, 10 and 20 mg/L was added
to E. coli treated whey, H2 production was largely af-
fected (Figure 3). At 10 mg/L the total amounts of H2
evoluted in 10 days was unchanged but the rate of evolu-
tion was altered. H2 production at 5 mg/L was largely
enhanced giving a total of 6000 ml/L/10 days. The rates
of production at the first 2 and 5 days were 1600 and 935
ml/L/day, respectively. This amount of increase is about

Influence of Metal Ions on Hydrogen Production by Photosynthetic Bacteria Grown in Escherichia coli
Pre-Fermented Cheese Whey
428
Figure 1. H2 production from 125 ml cultures of R. rubrum
grown in untreated whey, ▲; E. coli treated whey, ●; and
RCV medium, ○.
Figure 2. H2 production from 125 ml untreated whey cul-
tures to which RCV medium (○), lactate (●) or lactate +
Glutamate (▲) was added.
Figure 3. Ferric ion effect on H2 production from 125 ml
untreated whey cultures containing 5 mg/L (○), 10 mg/L (●)
or 20 mg/L. Dashed line represents treated whey alone.
double the amount produced by the treated whey alone.
However when 20 mg/L ferric chloride was used, marked
production in H2 evolution was seen giving a total H2 of
1200 ml/L in 10 days period. The increased production
of H2 due to the presence of 5 mg Fe/L can be taken in
support of a previous work [11], in which this phenome-
non was related to iron requirement for nitrogenase and
ferredoxin. However, reduction in H2 production at high-
er ferric chloride concentrations may be due to cellular
intoxication that was easily detected by the poor growth
seen in cultures containing these concentrations.
Figure 4 shows the changes in H2 production from
treated whey containing sodium molybdate at 0.3 and 1.6
mg/L. It is apparent that the presence of molybdate en-
hanced both the rate and the total H2 production. The
average daily production at the first 2 days for 0.3 and
1.6 mg/L were 1050 and 1200 ml/L, respectively. The
rates for the first 5 days for the two molybdate concen-
trations were 625 and 680 ml/L/day, and the total
amounts produced were 3920 and 4200 ml, respectively.
Changes in H2 production from treated whey contain-
ing iron and molybdate together (Figure 5) were not sig-
nificantly different from those seen when molybdate
alone was present (Figure 4). In other words molybdate
abolished the enhanced H2 production exerted by ferric
chloride (5 mg/l). No possible explanation can now be
offered for this discrepancy.

Influence of Metal Ions on Hydrogen Production by Photosynthetic Bacteria Grown in Escherichia coli
Pre-Fermented Cheese Whey
429
Figure 4. Effects of adding Mo ions to E. coli treated whey
on H2 production. Treated whey alone (○), 0.8 mg Mo/L (▲)
or 1.6 mg Mo/L (●).
Figure 5. Effects of adding Mo ions to treated whey con-
taining 5 mg Fe/L + 1.6 Mo/L (○), 4 mg Mo/L (▲) or 8 mg
Mo/L (●).
Large increase in the rate and total amount of H2 pro-
duced was seen when manganese sulphate was added to
treated whey containing 5 mg iron/L and 8 mg Mo/L
(Figure 6). At 2.65 mg Mn/L the rate of H2 production at
Figure 6. Effects of adding Mn ions to treated whey con-
taining 5 mg Fe/L + 8 mg Mo/L on hydrogen production.
2.58 mg Mn/L (○), 5.12 mg Mn/L (▲) or 7.68 mg Mn/L (●).
the first 2 days was 900 ml/L/day and at the first 5 days
was 655 ml/L/day, giving a total 10 days production of
4000 ml. For Mn concentration of 5.12 mg/L the rate at
the first 2 days was 1000 ml/L/day and at the first 5 days
was 760 ml/L/day, with a total production in 10 days of
5400 ml. Much higher rates and total production were
obtained when 7.68 mg Mn/L was used; rate at first 2
days was 1335 ml/L/day and the total 10 days production
was 9500 ml. It is apparent that curves representing the
effect of the presence of the three ions (Figure 6) indi-
cate that high Mn concentration (7.68 mg/L) increased
H2 production by keeping up the initial high production
rate to continue for a period longer than that occurred at
lower Mn concentration. In fact, after 3 days the rate of
H2 production for the two low Mn concentrations was
sharply reduced while that for highest concentration con-
tinued at its high rate for about 7 days.
4. Conclusions
Hydrogen production by photosynthetic bacteria was
enhanced three folds by fermenting the whey with E. coli
prior to incubation with photosynthetic bacteria. Such
productivity was further augmented by the addition of
metal ions such as Mo, Fe and Mn or combinations the-
reof. The highest H2 production was achieved when Mn
(7.68 mg/L) was added to treated whey containing Fe
and Mo ions to give about 8500 ml/10 days. The extent

Influence of Metal Ions on Hydrogen Production by Photosynthetic Bacteria Grown in Escherichia coli
Pre-Fermented Cheese Whey
430
of H2 productivity was metal ions concentration depend-
ent.
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