Hydrogen is emerging as a key alternative fuel due to its renewable nature and lack of greenhouse gas emissions during combustion. Biological methods, particularly using microalgae and cyanobacteria, offer efficient hydrogen production, utilizing processes like biophotolysis and fermentation, although environmental factors such as pH and temperature significantly affect yields. Despite challenges in optimizing these processes, the combined use of photosynthetic and anaerobic bacteria presents a promising approach for sustainable hydrogen production.

1. Dr.K.R.Padma
Assistant Professor
Sri Padmavati Mahila
University

2. Importance of Hydrogen as an Alternative Fuel
• Increased levels of CO2 from fossil
fuels cause an increase in the
Greenhouse Effect
• One of the detrimental effects of the
Greenhouse Effect is Global Warming
• Combustion of Hydrogen produces
water, which is not detrimental to the
environment

4.  Hydrogen gas is seen as a future energy carrier by virtue of the fact that it is renewable, does
not evolve the "greenhouse gas" CO2 in combustion, liberates large amounts of energy per
unit weight in combustion, and is easily converted to electricity by fuel cells.
 Biological hydrogen production has several advantages over hydrogen production by
photoelectrochemical or thermochemical processes.
 Biological hydrogen production by photosynthetic microorganisms for example, requires the
use of a simple solar reactor such as a transparent closed box, with low energy requirements.
 Electrochemical hydrogen production via solar battery-based water splitting on the hand,
requires the use of solar batteries with high energy requirements.

18. Biophotolysis of water by microalgae and
cyanobacteria
 Microalgae are primitive microscopic plants living in aqueous environments.
 Cyanobacteria, formerly known as blue-green algae
 Microalgae and Cyanobacteria along with higher plants, are capable of oxygenic
photosynthesis according to the following reaction:
CO2 + H2O  6 [CH2O] + O2.
organic compounds
 Photosynthesis consists of two processes:
 light energy conversion to biochemical energy by a photochemical reaction,
 and CO2 reductin to organic compounds such as sugar phosphates, through the use of
this biochemical energy by Calvin-cycle enzymes.
 Under certain conditions, however, instead of reducing CO2,
 a few groups of microalgae and Cyanobacteria consume biochemical energy to produce
molecular hydrogen
 (Hydrogenase and nitrogenase enzymes are both capable of hydrogen production.

20. Microbial Hydrogen Production
• Types of microbial hydrogen production
• Fermentative
• Photosynthetic (aerobic/anaerobic)
• Most interest in hydrogen production research in US during the Energy
Crisis of the 1970s
• Interest in hydrogen production again in 1990s due to the awareness of
Global Warming, etc.

21.  Biophotolysis is a water-splitting process occurring in biological systems.
 Molecular O2 and H2 are produced, with light as the energy source.
 Biophotolysis proceeds in two distinctive ways—directly and indirectly (Brentner et al., 2010).
 Direct biophotolysis has been best studied in the microalgae Chlamydomonas reinhardtii. It relies
on photosystems (both PSI and PSII) and hydrogenase (Fig. 1).
 Absorption of light in the form of photons by PSII (680 nm) and/or PSI (700 nm) generates a strong
oxidant that can oxidize water into protons, electrons/reducing equivalents, and O2.
 The electrons reduce protons to form H2, according to Equation (1) (Brentner et al., 2010).

27. Theoretically, the energy conversion
efficiency of hydrogenase is higher
than that of nitrogenase. However, it
catalyses a reversible reaction and absorbs
hydrogen in the presence of O2.
Hydrogenase-dependent hydrogen
production therefore requires frequent
anaerobic operations, making it difficult
for large-scale hydrogen production.

29. Anaerobic bacteria metabolize sugars to produce
hydrogen gas and organic acids, but are incapable of
further breaking down the organic acids formed.
the combined use of photosynthetic and anaerobic
bacteria for the conversion of organic acids to
hydrogen. Theoretically, one mole of glucose can be
converted to 12 moles of hydrogen
(In figure)through the use of photosynthetic bacteria
capable of capturing light energy in such a combined
system.
From a practical point of view, organic wastes
frequently contain sugar or sugar polymers. It is not
however easy to obtain organic wastes containing
organic acids as the main components.
The combined use of photosynthetic and anaerobic
bacteria should potentially increase the likelihood of
their application in photobiological hydrogen
production.

30.  Anaerobic digestion process includes hydrolysis/acidogenesis and
methanogenesis.
 As shown in Figure 1, hydrolysis and acidogenesis produce hydrogen gas and
organic acids, which can be further used to produce methane in methanogenesis.
 The hydrogen production step requires 1-2 days hydraulic retention time (HRT)
 and methane production step requires longer HRT (12-20 days).
 If hydrogen gas is not harvested and further used for methane production, it is
called one-stage fermentation process.
 Otherwise it is called two-stage fermentation process

31. • In an anaerobic fermentation process, the hydrogen synthesis pathways are
severely affected by environmental factors, such as pH, temperature and HRT.
• It has widely been accepted in bio-hydrogen research that pH is one of the key
factors affecting the hydrogen production.
• Hydrogen synthesis pathways are sensitive to pH and are subject to end-products
(Craven 1988). Dark hydrogen fermentation reactions can be operated at a
temperature range from mesophilic (25-40o C) to hyperthermophilic (>80o C).
• Up to now, most of dark fermentation experiments are conducted at 35-37o C,
and the possible advantages of operating out of mesophilic range are not
completely clear.
• HRT is also an important parameter for dark fermentation process.
• In continuously stirred tank reactor (CSTR) system, short HRTs were used to
wash out the slow growing methanogens and select for the acid producing
bacteria
• while too short HRT could lead to bad hydrolysis of organic wastes.

33. • Nitrogenase is an enzyme found only in prokaryotes and is concerned with nitrogen fixation
• Nitrogen fixation is a high energy consuming process since it requires several ATP molecules,
and hydrogen is produced as a by-product.
• The nitrogen fixation is carried out by two separate proteins, namely dinitrogenase reductase
(Fe-protein) and dinitrogenase (MoFe-protein).
• The Fe-protein mediates the transfer of electrons from reduced ferredoxin or flavodoxin to
dinitrogenase and brings about conformational changes to the nitrogenase.
• It is an active process, since it requires ATP hydrolysis. Dinitrogenase catalyses both nitrogen
fixation and hydrogenase generation. The nitrogen-fixing cyanobacterium, Anabaena
cylindrica, simultaneously produces hydrogen and oxygen in a nitrogen-free (i.e. argon)
atmosphere.

37. • The nitrogenase enzyme responsible for nitrogen fixation is present in prokaryotes such
as cyanobacteria, and is absent in eukaryotic microalgae.
• The nitrogenase enzyme mediates the reduction of molecular nitrogen into ammonia and
protons into molecular hydrogen with the consumption of reducing power and ATP.
• The efficiency of conversion of light energy to hydrogen by nitrogenase is low (< 1 %)
because of high energy demand.
• The nitrogen-fixing cyanobacteria also possess hydrogenase, which is an uptake
hydrogenase that consumes and reuses hydrogen gas, so that net hydrogen production will
be low.

38. • Nitrogenase is oxygen-sensitive, so the cyanobacteria have evolved mechanisms to
overcome this difficulty.
• The nitrogenase is localized in the heterocysts of filamentous cyanobacteria. The
heterocystous nitrogen-fixing strains like Anabaena are capable of providing an oxygen-
free environment inside the heterocyst so that the oxygen-sensitive nitrogenase can reduce
molecular nitrogen into ammonia and protons into molecular hydrogen.
• The products of oxygenic photosynthesis carried out in vegetative cells are transferred into
heterocysts and decomposed to provide the nitrogenase with reducing power. The
generation of hydrogen can be improved by limiting the supply of molecular nitrogen

39. Advantage:
For large-scale, labour-saving and economical hydrogen
production, nitrogenase is preferred to hydrogenase,
despite its comparatively low theoretical energy
conversion efficiency, because it has the advantage of
catalysing unidirectional production of hydrogen, thus
eliminating the need for a daily anaerobic production-
harvesting cycle.