The document discusses biohydrogen production from wastewater and provides an overview of key methods and factors. It notes that while biohydrogen is a sustainable fuel, large-scale commercial production faces challenges related to substrate conversion efficiency, product inhibition, and installation costs. The paper reviews various reactor configurations and pretreatment techniques used to optimize production. It finds that integrating dark and photo fermentation shows promise in addressing inhibition issues and improving yields, but widespread commercialization will require further reductions in costs.

1. Biohydrogen production from industrial
wastewater: An overview
Harshidaa S Nair
102119026
4th year chemical engineering

2. Introduction
• About 87 % of world’s energy needs are met by fossil fuels.
• These is world wide concern over the use of fossil fuels and non renewable sources of energy
• World is looking for alternate sources of non polluting and efficient sources of energy
• Hydrogen is a sustainable fuel which generates water and energy, it overcomes the negative effects
from the usage of fossil fuels.
• Hydrogen packs 3 times more energy w/w of petrol or diesel.
• Hydrogen burns in oxygen to form water.
Oxidation of
H2

3. Methods of bio hydrogen Production
• Old technique- Steam Reformation- Fossil fuels are heated
with steam with high pressure to produce hydrogen
• It is an unsustainable and costly process.
• Modern techniques- In recent times microbes are used to
produce biohydrogen
• Algae Photosynthesis
• Dark Fermentation
• Photo Fermentation
Algae Photosynthesis
Microalgae like cyanobacteria and
green algae can produce
biohydrogen after derivation of
their photosynthetic metabolism.
Photo Fermentation
Purple non-sulphur
photosynthetic bacteria grow on
organic acids like lactic acid,
butyric acid, and acetic acid and
generate photosynthetic
biohydrogen using light under
anaerobic conditions
Dark Fermentation
Dark fermentation is the fermentative conversion
of organic substrate to biohydrogen. It is a
complex process manifested by diverse groups of
bacteria, involving a series of biochemical
reactions using three steps similar to anaerobic
conversion.

4. Dark Fermentation
• Anaerobic fermentation in absence of light breaks down the
carbohydrate to form hydrogen and other organic acids.
• The intermediate compounds like volatile fatty acids (VFA) and
alcohols are also produced.
• Bacteria like Clostridia sp., and Enterobacter sp. are used for dark
fermentation
• The technical and economic issues like storage, distribution and
expensive infrastructure are the major barriers for commercialisation.
• Dark fermentation is a type of biological production of hydrogen.
• Dark fermentation is carried out by obligate anaerobes and facultative
anaerobes in the absence of light and oxygen.
• In dark fermentation, bacteria act on the substrate and generate hydrogen.
• The substrate for the dark fermentation is lignocellulosic biomass,
carbohydrate materials like wastewater from industry, sugar-containing
crop residues, and municipal solid waste.

5. Need for Pretreatment in Bio hydrogen Production
Combination of pretreatment methods are
effective to eliminate the methanogens and
increase the efficiency. Its negative effect on
hydrogen yield (HY)is the irreversible
deterioration of all the microbes and mainly the
hydrogen producers.
AB can produce a surrounding layer
around its cell surface which protects it
from severe environmental conditions
whereas MB do not produce this
protective layer.
Example - Combination of heat
shock with acid treatment and
achieved higher hydrogen yield
as compared to single method
Inhibits the hydrogen consuming
bacteria methanogenic bacteria (MB) in
mixed culture thus aids in selection the
group of hydrogen producing bacteria
acidogenic bacteria (AB).
Pretreatmen
t of
Inoculum

6. Types of Pretreatment Techniques
Physical Pretreatment -
Microwave
 Pretreatment is the
irradiation of
electromagnetic waves of
300 MHz to 300 GHz
frequency which produces
heat in liquids and it
disrupts the cell wall of
biomass and increases the
solubility of the medium.
 The rearrangement of
dipoles due to the heat
generated is the reason for
disruption.
 Inactivates the non-
hydrogen producers by
extracting the contaminant
in inoculum which causes
adverse effect to hydrogen
producers.
 When pre-treated with
microwave, the Clostridium
Physical Pretreatment -
Heat shock treatment (HST)
 Thermal treatment helps for
the growth of hydrogen
producing bacteria which
suppresses the activity of
methanogens.
 Treatment kills the non sporing
bacterium- the methanogens
and the non sporulating
hydrogen producing bacteria.
 Parameters of HST –
temperature: 80-104 °C and
exposure time:15-120 mins.
 Microwave treatment shows
higher efficiency than the
thermal treatment in terms of
cell solubilisation when
compared under same
temperature

7. Chemical Pretreatment
 Bromo ethane sulfonic acid (BESA)
destructs methanogens without disturbing
the hydrogen producing bacteria.
 Iodopropane restricts the functioning of
B12 enzyme which carries methyl group.
 Acetylene disturbs the metabolic pathway
of methanogens. Reduces methanogenic
functions.
 Acid treatment was used to kill MB which
survives at narrow pH range and protects
the acidogenic bacteria which can survive
at drastic pH range.
 The pH ranges between 5 and 5.5 provides
an efficient hydrogen production by
suppressing the activity of methanogens.
 The acid treated inoculum shows higher
biohydrogen production, whereas the base
treated inoculum shows higher HY due to
the difference in microbial distribution.
Biological Pretreatment
 Employs bacteria or enzymes to
increase the hydrolysis rate by
breaking the bonded structures in
wastewater.
 In Enzymatic treatment the reaction
takes place under mild conditions
without formation of any toxic
products.
Mechanical Pretreatment
 Ultrasonication propagates in the
form of bubbles which when
collapsed produces highly active
radicals, shear force, high
temperature and pressure.
 The input energy control in inoculum
helps to suppress the methanogens
and protects the hydrogen producing
bacteria.
 Apart from these pretreatment
processes the freezing thawing,
infrared, forced aeration also
provides efficient treatment.
 In forced aeration the
anaerobic sludge will be
inoculated with aerated sludge
to suppress the activity of
methanogens.
 The infrared and freeze thaw
treatment provides efficient
removal of methanogens.
 In freezing and thawing the
extreme changes in
temperature leads to the
intracellular formation of ice
crystals take place and it leads
to the disruption of microbial
cells.

8. Factors affecting Bio Hydrogen Production
pH
 pH limits the growth of bacteria
and shows the total solvent
concentration.
 Lower pH affects the activity of
hydrogenase enzymes and inhibits
the action of hydrogen producers.
 pH of 5 reduces the hydrogen
production due to lesser biomass
growth. Effective production of
hydrogen happens above 5.5 pH.
 pH varies inversely with the HY.
Optimum pH for high production of
hydrogen and lower solvent
production is between 5.5 and 6.5.
Composition of nutrients
 Nitrogen- important source for the growth
and enhances the hydrogen production.
 The inorganic salt like urea doesn't
contribute in the production of hydrogen.
 The stabilization of dark fermentation
process is enhanced by carbon to nitrogen
ratio (C/N ratio) and affects the
productivity and HPR.
 Increase in phosphate content alters the
cellular reducers from production of
hydrogen and thus VFA concentration
increases and decrease the overall
hydrogen production.
 The enzyme functioning can be enhanced
Partial Pressure
 Partial pressure is increased due
to the accumulation of hydrogen
which leads to the inhibition of
forward reaction in reactor
according to Le Chateliers
Principle
 The extraction of generated
hydrogen from the system and
maintaining partial pressure
overcomes the increase in partial
pressure.
 The introduction of nitrogen gas
helps to extract the hydrogen gas
by 68%.

9. Temperature
 Different temperature ranges in anaerobic
fermentation are: ambient (15–30 °C),
mesophilic (30–49 °C), thermophilic (50–64
°C), hyperthermophilic (65–80 °C), extreme
thermophilic (> 80 °C).
 Mesophillic temperature of (30– 49 °C)is
efficient for hydrogen production using mixed
culture. Whereas the energy recovery was
possible only at ambient temperature.From
25- 60 °C fermentative hydrogen production is
effective.
 The increase of temperature from optimum
limit leads to degradation of enzymes involved
in hydrogen production process.
 Thermodynamically, higher temperature
increases entropy of the system and favours
hydrogen production, but economically the
Wastewater as a substrate for
fermentation
 Organic rich wastewater can be
used as a substrate for efficient
biohydrogen production since it
reduces the overall treatment cost.
 Industrial wastewater contains
higher degradable organic matters
which balances the energy applied
and recovered.
 Increased biodiesel production
leads to excessive discharge of
glycerol and it can be effectively
used as substrate for biohydrogen
production.
 The increase in substrate
concentration leads to the lower
Organic loading rate (OLR)
 The total conversion of
carbohydrate is inversely
proportional to OLR in the
reactor.
 OLR affects the performance of
the reactor. The highest HY was
obtained at lower glucose
feeding rate.
 Increase in OLR increases the
microbial diversity and thereby
decreasing its hydrogen yield.
 The hydrogen production can be
enhanced by optimising HRT and
OLR in continuous system -

10. Hydraulic retention time (HRT)
 It is also known as fermentation time. It restricts the
methanogenic process during acidogenic
fermentation.
 HRT can be controlled by dilution rate in continuous
stirred tank
 Shorter HRT lesser the growth of slow growing
microorganisms like methanogens. Washout of
methanogens reduces the cost and size of reactors.
 HRT for hydrogen gas generation from dark
fermentation is usually 0.5-24h. Optimum HRT has to
be determined based on the substrate and bioprocess
used.
Volatile fatty acid concentration
 The increment in VFA concentration leads to
the lysis of the cells through the permeation of
proton into the cell membrane of sporing
bacteria and thus disruption of cells takes
place.
 The substrate degradation efficiency, HPR and
HY decreases with increase in VFA
concentration.
Reactor configuration for biohydrogen production
Bioreactor configuration is the main impacting parameter that affects the biohydrogen
production.
Continuous stirred tank
reactor (CSTR)
Anaerobic Fluidized Bed
Reactor (AFBR)
Membrane bioreactor
(MBR)
Packed bed
reactor (PBR)

11. Continuous stirred tank reactor
(CSTR)
 Widely used for continuous production of
hydrogen and microorganism in which
liquid can mix constantly due to the
constant regime of mixing pattern.
 It facilitates higher contact between the
substrate and the microorganism and thus
obtains higher mass transfer.
 Due its mixing pattern it is unable to hold
large number of microbes responsible for
fermentation and the washout of microbes
takes place at low HRT.
 Cell Immobilisation prevents washout of
microbes.
 Example- In a CSTR where the yeast
industry wastewater was treated the yeast
cells were immobilized in polyvinylidene
fluoride (PVDF) membrane, it reduced the
Membrane bioreactor (MBR)
 It integrates the semi permeable
membrane with the biological process
and it is the combination of micro or
ultra filtration with suspended growth
bioreactor.
 It consolidates the features of
membrane filtration and activated
sludge treatment used for retention of
microorganisms inside the reactor and
the parameters such as HRT and SRT
can be controlled.
 The increase in SRT increases the
retention of biomass and thus
increases the substrate utilization rate
there by decreasing the production of
hydrogen.
 Fouling of membrane is caused by
formation of Extracellular polymeric
Reactor configuration for biohydrogen production

12. Anaerobic Fluidized Bed Reactor (AFBR)
 Excellent stirring capacity, efficient mass transfer
characteristic and low biomass washout compared to
CSTR or PBR.
 It helps in the removal of gas produced, constant mixing
of solid or liquid and reduces the pressure drop.
 Work both at lower hydraulic reaction time and higher
organic loading rate.
 Immobilizing the biomass culture within the reactor
enhances the hydrogen yield.
Packed bed reactor (PBR)
 Used for the conversion of biomass with high organic
content.
 The can work efficiently at lower HRT without any
extraction of biomass, where the beds of the reactor
have a granule or biofilms.
 The substrates are passed through column packed
with biocatalyst. The performances of reactor for
immobilized cells are based on the kind of
immobilization.
 Advantages-simple operation, anaerobic environment
maintained by purging nitrogen and the elevated rates
of reactions were possible through its operation

14. Limitations and challenges of biohydrogen production
• Despite of the remarkable merits the major limitation faced
in biohydrogen production is limited substrate conversion
potential and remaining fractions of substrate (wastewater)
exist in acidogenic effluent during acidification process.
• The prolonged buildup of acidogenic metabolites leads to
decrease in pH. This in turn causes inhibition of hydrogen
producing microbes and reduces hydrogen yield
• The productivity of hydrogen is reduced when more
metabolic by products such as lactic acids, ethanol, and
propionic acid were accumulated as these compounds
surpass the productivity of hydrogen.
• Integration of fermentation process with other approaches is
the viable option to overcome the other metabolites.
• Another major limitation in continuous biohydrogen reactor is
the biomass wash out. In order to overcome this limitation ,
immobilizing the biomass in support material and granulation
in reactorsis the promising choice.
• Another challenge associated is the competition of
methanogens over hydrogen producers.
• Selective enrichment and bioaugmentation are the emerging
strategies to overcome the suppressive growth of hydrogen
producing microbes.

15. Advancements and way forward for commercial application
The development of hydrogen production technologies is not widely accepted
commercially due to its high cost of production and lower yield of hydrogen.
Major challenges- selection of microorganism, optimization of operational
factors, design of reactors, lack of storage for hydrogen, fuel cell
technologies and distribution networks. These needs to be addressed for
the production in commercial scale.
Latest Developments:
 Bioaugmentation- Introduction of cultured
microorganisms required to speed up the
rate of degradation of a contaminant.
 Bio stimulation- addition of various
forms of rate limiting nutrients and
electron acceptors, such as
phosphorus, nitrogen, oxygen, or
carbon (e.g. in the form of molasses) to
increase microbial growth.

16.  Cell Immobilization- Biomass is attached to inert or
supporting material. This material helps the biomass to
remain in the reactor. Thus it increases the yield and
productivity of biohydrogen.
 Genetic engineering -manipulation in genes -
insertion of desirable gene in the microbial
strain to make it as a potent inoculum for
biohydrogen production
 Basic improvement can
be achieved by using
cheaper substrate with
pretreatment, high
yielding strain and cost-
efficient reactors.

17. Process integration
Presently Integration of dark and photo fermentation anaerobically have widely adopted in large scale since the
energy retrieval is high and increases the efficiency of wastewater treatment.
It reduces the inhibition of biohydrogen caused by the accumulation of metabolites during fermentation process.
The process integration can improve the performance and suppresses the issues caused by single process.
Dark fermentation is the efficient concept for biohydrogen production as compared to other methods and the
integration of other system with dark fermentation increases the energy recovery.
The integration of dark and photo fermentation helps to resolve the problems on inhibition of substrate and
also increases the yield of hydrogen.
Organic acids from dark fermentation
are fed directly into the reactors of
photo fermentation. Allows for large
amount of very pure biohydrogen and
whole range of useful by products to
be produced.

18.  The research paper talks about recent
challenges, updates and operational changes
of various biohydrogen producing reactors.
 Limited substrate conversion and low
productivity are the two main challenges
faced.
 The optimizations of operating conditions are
necessary to upgrade the production with
economic viability.
 Integration of dark and photo fermentation
has high scope. It resolves inhibition of
substrate and also increases the yield of
hydrogen
 Various pilot scale studies have been
undergone with different technologies;
however, commercialisation is still a great
challenge due to its higher installation cost.
Reference:-Research Paper used:
https://doi.org/10.1016/j.biteb.2019.100287
https://www.sciencedirect.com/science/article/pi
i/S2589014X1930177X
Conclusion

19. Thank You