INTRODUCTION TO PHOTOBIOPRODUCED HYDROGEN:

The main aim of our project is to produce electricity from Chlamydomonas Reinhardtii.
The photobioproduction of hydrogen in water alga systems has been studied as a suitable
way for clean hydrogen generation from renewable solar energy and renewable bio-
sources.

Chlamydomonas reinhardtii is an alga type capable to produce hydrogen in stress

conditions. Several studies reported sustained hydrogen production for about 50 -100 h in
anaerobic and sulfur deprived conditions. After the end of this hydrogenic phase, algae
cultures have to return to an aerobic and rich of nutrients environment.

The phases of hydrogen production and aerobic growth can be alternated and repeated
indefinitely. The mechanism of algae hydrogen production involves the enzyme
hydrogenase and it is essentially a way through which the cell dissipates the electrons
pressure accumulated within, in order to survive in anaerobic conditions when other
mechanisms are not available.

At the moment, the algae conversion efficiencies of light energy into hydrogen energy at
low light intensities are higher than those can be achieved using direct sunlight. A
maximum of 10% of solar energy conversion efficiency to hydrogen chemical energy has
been determined from theoretical extrapolations; however, currently a 1-3% efficiency
has been achieved with outdoor sunlight. There is a change to approach the theoretical or
laboratory efficiencies by a genetic development of algae strains less efficient in photon
capture or developing photo bioreactors with reduced light absorption.

There is evidence that the gas phase over algae culture can be connected to a fuel cell for
detecting and estimating hydrogen amount by equilibrium cell potential measurements
and calibration curves. This evidence guessed the possibility or a direct linkage of algae
and fuel cells to generate electrical power, also justified for the oxygen-and-Co-free gas
mix phase present during the hydrogenic phase. The present study it has been thought in
order to define the main aspects of a direct fuel cell application of photobioproduced
hydrogen, considering the following factors: variation of hydrogen rate production by
Chlamydomonas reinhardtii; forced low of inert gases (Co2 and N2) in order to assess
the effects of dilution of hydrogen on fuel cell output power; the introduction of delays
between hydrogen production and fuel cells stack feed with the purpose to reduce
peak powers due to point; and , in a general way, the perspective of increasing the power
outcome by means of higher algae efficiencies.

A fuel cell works by catalysis, separating the component electrons and protons of the
reactant fuel, and forcing the electrons to travel though a circuit, hence converting them
to electrical power. The catalyst is typically comprised of a platinum group metal or
alloy. Another catalytic process takes the electrons back in, combining them with the
protons and the oxidant to form waste products (typically simple compounds like water
and carbon dioxide).

In the archetypal hydrogen-oxygen proton exchange membrane fuel cell (PEMFC)

design, a proton-conducting polymer membrane, (the electrolyte), separates the anode
and cathode sides. This was called a “solid polymer electrolyte fuel cell” (SPEFC) in the
early 1970s, before the proton exchange mechanism was well-understood. (Notice that
“polymer electrolyte membrane” and “proton exchange membrane” result in the same
acronym.)

On the anode side, hydrogen diffuses to the anode catalyst where it later dissociates into
protons and electrons. These protons often react with oxidants causing them to become
what is commonly referred to as multi-facilitated proton membranes (MFPM). The
protons are conducted through the membrane to the cathode, but the electrons are forced
to travel in an external circuit (supplying power) because the membrane is electrically
insulating. On the cathode catalyst, oxygen molecules react with the electrons (which
have traveled through the external circuit) and protons to form water – in this example,
the only waste product, either liquid or vapor.
In addition to this pure hydrogen type, there are hydrocarbon fuels for fuel cells,
including diesel, methanol (see: direct – methanol fuel cells) and chemical hydrides. The
waste products with these types of fuel are carbon dioxide and water.

In summary, concerns about global warming and environmental pollution due to the use
of fossil fuels, combined with projections of potential fossil fuel shortfall toward the
middle of the 21st century, make it imperative to develop alternative energy sources that
are clean, renewable, and environmentally friendly. The recently developed single-
organism, two-stage photosynthesis and H2 production protocol with green algae is of
interest because significant amounts of H2 gas can be generated for the first time. Further,
this method does not entail the generation of any undesirable, harmful, or polluting
byproducts and it may even offer the advantage of value-added products as a result of the
mass cultivation of green algae. Foremost, the cellular metabolism and basic
biochemistry that support and much fundamental research on the mechanism of H2
production by S deprivation remain to be done.

DETAILED REPORT:

(a) Green algae are grown photosynthetically in the light (normal photosynthesis)
until they reach a density of 3 to 6 million cells ml 0 1 in the culture.
A Chlamydomonas reinhardtii CC 125 wild type 137C
Mt+ original sample is to be grown in media containing: phosphate buffer a modified
Beijerinck’s solution with MgC12.6H20 instead of MgSO4 7H2O, thus obtaining sulfur –
free medium; Hunter’s solution and sodium acetate. Small quantities of BactrimF, is to be
added, containing 20 mg of trimetoprima and 100 mg of sulfametoxazol for algae media
varying from 70 to 100ml. The environmental temperature was in the range of 20-25oC; a
40 W cool white fluorescent lamp at a 20 cm distance was used for algae culture growth.
The First 100 ml sample needed about a month, after the inoculation of alga, to reach and
appreciable diffuse green color, the following sample needed from 1 to 4 days. The
samples are to be covered to avoid raw contamination, but kept in contact with air,
without CO2 bubbling. There was no necessity of an aseptic environment since algae
sample did not manifest any bacterial or other kind of biological contamination during all
the series of experiments.

Heterotrophic Growth of Chlamydomonas reinhardtii on /Acetate in Chemostat

Culture:
Modified Sagar & Granick media 5 referred to as CR M2 medium consisted of (per litre)
0.3g KzHPO4, 03g MgSOa’7H20, 0.053 g CAC12”2 H20, 0.01g FeCl3”6H20, 0.5 g
sodium citrate and 1 ml of trace metal solution comprising (per 100 ml) of 125 mg
H3BO3, 125 mg ZnSO4” 7H20, 38 mg MnSO4”H20, 25mg COC12” 6 H20, 25 mg,
Na2MoO4”2H20 and 8 mg CuSO4”5H20. Nitrogen was supplied as urea (0.5g/litre), since
it was found to be superior to nitrate in heterotrophic batch cultures. Sodium acetate was
used as sole carbon and energy source. An attempt to use the pH-buffered medium, CR-
M11o containing 1.7g/litre acetate without automatic pH control was unsatisfactory, since
large changes in culture pH occurred.

Media and fermenters were sterilized by autoclaving at 121 oC for 20 min. On cooling, the
medium was inoculated with 5% (v/v) of an inoculum grown at 25oC on CR-M1 medium
1o containing 0.85g/litre acetate and 0.2g/litre urea under a cool white fluorescent tube
(1800 lux) on a 16/8 on/off cycle. The fermenter was permitted to grow in batch mode
until cell density was sufficient to permit feed medium to be supplied. Culture was
continuously discharged via an overflow tube to maintain a constant culture volume (1
litre). Steady-state conditions were considered to have been established when cell
concentrations from at least three samples collected after a period of three residence
times, varied by no more than 10%.
The culture temperature was maintained at 35oC by a water bath and automatic pH
control, with sterile 1 M HCI, was used to maintain culture pH a pH 7-2. Sterile air was
supplied to maintain dissolved oxygen concentrations above 50% saturation during
cultivation and the fermenter was maintained in darkness to prevent photosynthetic
growth. A chemostat culture of C. reinhardtii was operated at a variety of dilution rates
using CR-M2 medium with feed acetate concentrations of 0.85 g/litre and 1.7 g/litre.
(b) Sulfur deprivation is imposed upon the cells in the growth medium, either by
carefully limiting sulfur supply in the medium so that it is consumed entirely, or
by permitting cells to concentrate in the growth chamber prior to medium
replacement with one that lacks sulfur nutrients. Cells respond to this S
deprivation by fundamentally altering photosynthesis and cellular metabolism to
survive
(c) S deprivation exerts a distinctly different effect on the cellular activities of
photosynthesis and respiration. The activity of oxygenic photosynthesis declines
quasi-exponentially with a half-time of 15 to 20 h to a value less than 10% of this
original rate. However, the capacity for cellular respiration remains fairly constant
over the S deprivation period. As a consequence, the absolute activity of
photosynthesis crosses below the level of respiration after about 24 h of S
deprivation. Following this cross point between photosynthesis and respiration,
sealed cultures of S-deprived C.reinhardtii quickly consume all dissolved oxygen
and become anaerobic, even though they are maintained under continuous
illumination.
It is often convenient to consider the process of molecular hydrogen production to
be one that contains three phases:

Dark Phase – This phase involves the culture and growth of Chlamydomonas
reinhardtii cells without the presence of light. By growing the cells in dark
conditions we allow the organism to allot resources to the expression an synthesis
of the (FeFe) hydrogenase enzyme, an enzyme crucial to the process of hydrogen
formation.

Light Phase – This phase involves the exposure of the cells to light. This phase
follows the dark phase and is responsible for hydrogen production. The enzymes
that were previously synthesized in the Dark Phase are now put to work and
through a modified form of photosynthesis hydrogen is produced. It should be
considered that the utilized cells have a limited capacity to produce hydrogen.
This means that our cells have a very limited life or a method must be used in
order to rejuvenate the hydrogen production capabilities by the cells.

Regenerative Phase - This phase involves the introduction of sulfur to the media
in which the cells were grown. During this phase no hydrogen is produced
because the exposure of the cells to sulfur–rich conditions allows normal
photosynthesis to occur leading to the production of molecular oxygen. In both
the Dark Phase and Light Phase the cells are grown in media that has been
depleted of hydrogen.

Sulfur-deprived Conditions

Sulfur-deprived media are required for cellular proliferation during the Light
Phase and the Dark Phase. This is because in the absence of sulfur the
mechanism of electron transport within the chloroplasts is modified via partial
inactivation o photosystem II by the organisms. Photosystem II is the unit of the
chloroplast responsible for the transfer of electrons to final electron carrier
oxygen allowing the cell to produce and excrete molecular oxygen, the common
by product of photosynthesis. During its partial inactivation photosystem II is no
longer able to use important enzymes in the transport of electrons to hydrogen and
a buildup of electrons occurs in the chloroplasts. Still, water must be split (source
of electrons) in order to allow energy rich compounds to be produced as a result
of photosynthesis. Under these conditions the cell must find a way to allow the
electrons to drain out (usually through an electron acceptor) as the cell cannot
simply turn off its ability to produce the electrons. Protons become the cell’s
choice for an electron acceptor, which will alleviate the burden of a clogged
electron transport chain .

Another effect of sulfur deprived conditions includes the development of

anaerobic conditions within the cell. This is important because any oxygen
present will bind to the (FeFe) hydrogenase enzymes, which will prevent the cell
from producing molecular hydrogen, Though the effects of sulfur-deprived media
may optimize the yield of molecular hydrogen, the cell is still restricted to a
certain capacity of hydrogen that can be produced. This limitation gives a finite
lifetime on the cell’s ability to produce molecular hydrogen. To restore the cell’s
ability to produce molecular hydrogen, sulfur must be introduced into the media.
The reason for the limitation is that the element, sulfur, is present in two critical
enzymes methonine and cysteine, By removing free inorganic sulfur, from the
media the cell is not able to synthesize anymore of these amino acids necessary
for overall protein synthesis or repair. This leads to protein degradation, which
means the cell is slowly decaying as it produces molecular hydrogen. By feeding
the cell inorganic sulfur atoms, the molecular hydrogen production capacity is
restored and the cells are once again able to produce molecular hydrogen .
A summary of the current state –of-the art in this field is given below:
(a) The absence of sulfur from the growth medium of algae acts as a metabolic
switch, one that selectively and reversibly turns off photosynthetic O2 production.
(b) In the presence of S, green algae do normal photosynthesis (water oxidation,
O2 evolution, and biomass accumulation). In the absence of S and absence of O2,
photosynthesis in C.reinhardtii slips into the H2 production mode.
(c) Reversible application of the switch (presence/absence of S) permits the algae
to alternate between O2 production and H2 production (cycling of the stages), thus
by passing the incompatibility and mutually exclusive nature of the O2 – and H2-
producing reactions.
(d) Interplay between oxygenic photosynthesis, mitochondrial respiration,
catabolism of endogenous substrate, and electron transport via the hydrogenase
pathway is essential for this light-mediated H2 production process.
(e) The release of H2 gas serves to sustain baseline levels of chloroplast and
mitochondrial electron transport activity for the generation of ATP, Which is
needed for the survival of the organism under the protracted sulfur deprivation
stress conditions.
EXTRACTION OF ELECTRICITY IN A FUEL CELL
To deliver the desired amount of energy, the fuel cells can be combined in series and
parallel circuits, where series yield higher voltage, and parallel allows a stronger current
to be drawn. Such a design is called a fuel cell stack. Further, the cell surface area can be
increased, to allow stronger current from each cell.