Biohydrogen is defined as hydrogen
produced biologically, most commonly by algae, bacteria and archaea.
Biohydrogen is a potential biofuel obtainable from both cultivation and from waste
organic materials.
Introduction
Currently, there is a huge demand of the chemical hydrogen.
There is no log of the production volume and use of hydrogen world-wide,
however consumption of hydrogen was estimated to have reached 900 billion
cubic meters in 2011.
Refineries are large-volume producers and consumers of
hydrogen. Today 96% of all hydrogen is derived from fossil
fuels, with 48% from natural gas, 30% from hydrocarbons,
18% from coal and about 4% from electrolysis. Oil-sands processing,
gas-to-liquids and coal gasification projects that are ongoing,
require a huge amount of hydrogen and is expected to boost the requirement
significantly within the next few years. Environmental regulations implemented
in most countries, increase the hydrogen requirement at refineries for gas-line
and diesel desulfurization.
An important future application of hydrogen could be as an
alternative for fossil fuels, once the oil deposits are depleted. This
application is however dependent on the development of storage techniques to
enable proper storage, distribution and combustion of hydrogen. If the cost of
hydrogen production, distribution, and end-user technologies decreases,
hydrogen as a fuel could be entering the market in 2020.
Industrial fermentation of hydrogen, or whole-cell
catalysis, requires a limited amount of energy, since fission of water is
achieved with whole cell catalysis, to lower the activation energy. This allows
hydrogen to be produced from any organic material that can be derived through
whole cell catalysis since this process does not depend on the energy of substrate.
Algaeic biohydrogen
Further information: Biological hydrogen production
(Algae)
In 1939 a German researcher named Hans
Gaffron, while working at the University of Chicago, observed that the
algae he was studying, Chlamydomonas reinhardtii (a
green-algae), would sometimes switch from the production of oxygen to the
production of hydrogen. Gaffron never discovered the cause for this change and
for many years other scientists failed in their attempts at its discovery. In
the late 1990s professor Anastasios Melis a researcher at the University of
California at Berkeley discovered that if the algae culture medium is deprived
of sulfur it will switch from the production of oxygen (normal photosynthesis),
to the production of hydrogen. He found that the enzyme responsible
for this reaction is hydrogenase, but that the hydrogenase lost this function
in the presence of oxygen. Melis found that depleting the amount of sulfur
available to the algae interrupted its internal oxygen flow, allowing the
hydrogenase an environment in which it can react, causing the algae to produce
hydrogen.Chlamydomonas moewusii
is also a good strain for the production of hydrogen. Scientists at the U.S.
Department of Energy’s Argonne National Laboratory are currently trying to find
a way to take the part of the hydrogenase enzyme that creates the hydrogen gas
and introduce it into the photosynthesis process. The result would be a large
amount of hydrogen gas, possibly on par with the amount of oxygen created.
It would take about 25,000 square kilometres to be sufficient
to displace gasoline use in the US. To put this in perspective, this area
represents approximately 10% of the area devoted to growing soya in the US.
The US Department of Energy has targeted a selling price of $2.60 / kg as a
goal for making renewable hydrogen economically viable. 1 kg is
approximately the energy equivalent to a gallon of gasoline. To achieve this,
the efficiency of light-to-hydrogen conversion must reach 10% while current
efficiency is only 1% and selling price is estimated at $13.53 / kg. According
to the DOE cost estimate, for a refueling station to supply 100 cars per day,
it would need 300 kg. With current technology, a 300 kg per day
stand-alone system will require 110,000 m2 of pond area,
0.2 g/l cell
concentration, a truncated antennae mutant and 10 cm pond depth. Areas of
research to increase efficiency include developing oxygen-tolerant FeFe-hydrogenases
and increased hydrogen production rates through improved electron transfer.
Bacterial biohydrogen
Process requirements
If hydrogen by fermentation is to be introduced as an
industry, the fermentation process will be dependent on organic
acids as substrate for photo-fermentation. The organic acids are necessary
for high hydrogen production rates.
The organic acids can be derived from any organic material
source such as sewage
waste waters or agricultural wastes. The most important organic acids are acetic acid
(HAc), butyric
acid (HBc) and propionic acid (HPc). A huge advantage is that
production of hydrogen by fermentation does not require glucose as
substrate.
The fermentation of hydrogen has to be a continuous
fermentation process, in order sustain high production rates, since the amount
of time for the fermentation to enter high production rates are in days.
Fermentation
Several strategies for the production of hydrogen by
fermentation in lab-scale have been found in literature. However no strategies
for industrial-scale productions have been found. In order to define an
industrial-scale production, the information from lab-scale experiments has
been scaled to an industrial-size production on a theoretical basis. In
general, the method of hydrogen fermentation is referred to in three main
categories. The first category is dark-fermentation,
which is fermentation which does not involve light. The second category is
photo-fermentation, which is fermentation which requires light as the source of
energy. The third is combined-fermentation, which refers to the two
fermentations combined.
Dark fermentation
There are several bacteria with a potential for hydrogen
production. The Gram-positive bacteria of the Clostridium
genus, is promising because it has a natural high hydrogen production rate. In
addition, it is fast growing and capable of forming endospores,
which make the bacteria easy to handle in industrial application.
Species of the Clostridium genus allow hydrogen
production in mixed cultures, under mesophilic
or thermophilic
conditions within a pH
range of 5.0 to 6.5. Dark-fermentation with mixed cultures seems promising
since a mixed bacterial environment within the fermenter, allows cooperation of
different species to efficiently degrade and convert organic waste materials
into hydrogen, accompanied by the formation of organic acids. The clostridia
produce H2 via a reversible hydrogenase (H2ase) enzyme (2H + 2e <=> H2)
and this reaction is important in achieving the redox balance of fermentation.
The rate of H2 formation is inhibited as H2 production causes the partial
pressure of H2 (pH2) to increase. This can limit substrate conversion and
growth and the bacteria may respond by switching to a different metabolic
pathway in order to achieve redox balance, energy generation and growth - by
producing solvents instead of hydrogen and organic acids.
Enteric bacteria such as Escherichia coli and Enterobacter
aerogenes are also interesting for biohydrogen fermentation.) Dissecting
the roles of E. coli hydrogenases in biohydrogen production. FEMS Microbiol
Lett 278:48-55.</ref> Unlike the clostridia, the enteric bacteria produce
hydrogen primarily (or exclusively in the case of E. coli) by the
cleavage of formate (HCOOH --> H2 + CO2), which serves to detoxify the
medium by removing formate. Cleavage is not a redox reaction and it has no
consequence on the redox balance of fermentation. This detoxification is
particularly important for E. coli as it cannot protect itself by
forming endospores. Formate cleavage is an irreversible reaction, hence H2
production is insensitive to the partial pressure of hydrogen (pH2) in the
fermenter.
E. coli has been referred to as the workhorse of
molecular microbiology and many workers have investigated metabolic engineering
approaches to improve biohydrogen fermentation in E. coli.
Whereas oxygen kills clostridia, the enteric bacteria are facultative
anaerobes; they grow very quickly when oxygen is available and transition
progressively from aerobic to anaerobic metabolism as oxygen becomes depleted.
Growth rate is much slower during anaerobic fermentation than during aerobic
respiration because fermentation less metabolic energy from the same substrate.
In practical terms, facultative anaerobes are useful because they can be grown
quickly to a very high concentration with oxygen and then used to produce
hydrogen at a high rate when the oxygen supply is stopped.
For fermentation to be sustainable at industrial-scale, it
is necessary to control the bacterial community inside the fermenter.
Feedstocks may contain micro-organisms, which could cause changes in the
microbial community inside the fermenter. The enteric bacteria and most
clostridia are mesophilic; they have an optimum temperature of around
30 degrees C as do many common environmental microorganisms. Therefore,
these fermentations are susceptible to changes in the microbial community
unless the feedstock is sterilised, for example where a hydrothermal
pretreatment is involved, sterilisation is a side-effect. A way to prevent
harmful micro-organisms from gaining control of the bacterial environment
inside the fermenter could be through addition of the desired bacteria.Hyperthermophilic
archaea such as Thermotoga neapolitana can also be used for hydrogen
fermentation. Because they operate at around 70 degrees C, there is little
chance of feedstock contaminants becoming established.
Fermentations produce organic acids are toxic to the
bacteria. High concentrations inhibit the fermentation process and may trigger
changes in metabolism and resistance mechanisms such as sporulation in
different species. This fermentation of hydrogen is accompanied production of
carbon-dioxide which can be separated from hydrogen with a passive separation
process.
The fermentation will convert some of the substrate (e.g.
waste) into biomass
instead of hydrogen. The biomass is, however, a carbohydrate-rich by-product which
can be fed back into the fermenter, to ensure that the process is sustainable.
Fermentation of hydrogen by dark-fermentation is restricted by incomplete
degradation of organic material, into organic acids and this is why we need the
photo-fermentation.
The separation of organic acids from biomass in the outlet
stream can be done with a settler tank in the outlet stream, where the sludge
(biomass) is pumped back into the fermenter to increase the rate of hydrogen
production.
In traditional fermentation systems, the dilution rate must
be carefully controlled as it affects the concentration of bacterial cells and
toxic end-products (organic acids and solvents) inside the fermenter. A more
complex electro-fermentation technique decouples the retention of water
and biomass and overcomes inhibition by organic acids.
Photo-fermentation
Photo-fermentation refers to the method of
fermentation where light is required as the source of energy. This fermentation
relies on photosynthesis to maintain the cellular energy levels.
Fermentation by photosynthesis compared to other fermentations has the
advantage of light as the source of energy instead of sugar. Sugars are usually
available in limited quantities.
All plants, algae and some bacteria are capable of photosynthesis:
utilizing light as the source of metabolic energy. Cyanobacteria
are frequently mentioned capable of hydrogen production by oxygenic
photosynthesis. However the purple non-sulphur (PNS) bacteria (e.g. genus Rhodobacter)
hold significant promise for the production of hydrogen by anoxygenic
photosynthesis and photo-fermentation.
Studies have shown that Rhodobacter sphaeroides is highly
capable of hydrogen production while feeding on organic acids, consuming 98% to
99% of the organic acids during hydrogen production.Organic acids may be
sourced sustanably from the dark fermentation of waste feedstocks. The
resultant system is called combined fermentation (see below).
Photo-fermentative bacteria can use light in the wavelength
range 400-1000 nm (visible and near-infrared) which differs
from algae and cyanobacteria (400-700 nm; visible).
Currently there is limited experience with
photo-fermentation at industrial-scale. The distribution of light within the
industrial scale photo-fermenter has to be designed to minimise self-shading.
Therefore any externally illuminated photobioreactor must have a high ratio of high
surface area to volume. As a result, photobioreactor construction is
materials-intensive and expensive.
A method to ensure proper light distribution and limit
self-shading within the fermenter, could be to distribute the light with an optic fiber
where light is transferred into the fermenter and distributed from within the
fermenter. Photo-fermentation with Rhodobacter sphaeroides require
mesophilic conditions. An advantage of the optical fiber photobioreactor is
that radiant heat-gain can be controlled by dumping excess light and filtering
out wavelengths which cannot be used by the organisms.
Combined fermentation
Combining dark- and photo-fermentation has shown to be the
most efficient method to produce hydrogen through fermentation. The combined
fermentation allows the organic acids produced during dark-fermentation of
waste materials, to be used as substrate in the photo-fermentation process.
Many independent studies show this technique to be effective and practical.
For industrial fermentation of hydrogen to be economical
feasible, by-products of the fermentation process has to be minimized. Combined
fermentation has the unique advantage of allowing reuse of the otherwise
useless chemical, organic acids, through photosynthesis.
Many wastes are suitable for fermentation and this is
equivalent the initial stages of anaerobic digestion, now the most important
biotechnology for energy from waste. One of the main challenges in combined
fermentation is that effluent fermentation contains not only useful oroganic
acids but excess nitrogenous compounds and ammonia, which inhibit nitrogenase
activity by wild-type PNS bacteria.The problem can be solved by genetic
engineering to interrupt down-regulation of nitrogenase in response to nitrogen
excess. However, genetically engineered bacterial strains may pose containment
issues for application. A physical solution to this problem was developed at
The University of Birmingham UK, which involves selective electro-separation of
organic acids from an active fermentation. The energetic cost of
electro-separation of organic acids was found to be acceptable in a combined
fermentation."Electro-fermentation" has the side-effect of a
continuous, high-rate dark hydrogen fermentation.
As the method for hydrogen production, combined fermentation
currently holds significant promise.
Metabolic processes
The metabolic process for hydrogen production are dependent on
the reduction of the metabolite ferredoxin
(except in the enteric bacteria, where an alternative formate pathway
operates).
4H+ + 4 ferredoxin(red) → 4 ferredoxin(ox) + 2 H2
For this process to run, ferredoxin has to be recycled
through oxidation.
The recycling process is dependent on the transfer of electrons from nicotinamide adenine dinucleotide
(NADH) to ferredoxin.
2 ferredoxin(ox) + NADH2 → 2 ferredoxin(red) + 2H+
+ NAD+
The enzymes that catalyse this recycling process are referred
to as hydrogen-forming enzymes and have complex metalloclusters in their active
site and require several maturation proteins to attain their active form. The
hydrogen-forming enzymes are inactivated by molecular oxygen and must be
separated from oxygen, to produce hydrogen.
The three main classes of hydrogen-forming enzymes are
[FeFe]-hydrogenase, [NiFe]-hydrogenase and nitrogenase.These
enzymes behave differently in dark-fermentation with Clostridium and
photo-fermentation with Rhodobacter. The interplay of these enzymes are
the key in hydrogen production by fermentation.
Clostridium
The interplay of the hydrogen-forming enzymes in Clostridium
is unique with little or no involvement of nitrogenase. The hydrogen production
in this bacteria is mostly due to [FeFe]-hydrogenase, which activity is a
hundred times higher than [NiFe]-hydrogenase and a thousand times higher than
nitrogenase. [FeFe]-hydrogenase has a Fe-Fe catalytic core with a variety of
electron donors and acceptors.
The enzyme [NiFe]-hydrogenase in Clostridium,
catalyse a reversible oxidation of hydrogen. [NiFe]-hydrogenase is responsible
for hydrogen uptake, utilizing the electrons from hydrogen for cellular
maintenance.
In Clostridium, glucose is broken down into pyruvate and
nicotinamide adenine dicleotide (NADH). The formed pyruvate is then further
converted to acetyl-CoA and hydrogen by pyruvate ferredoxin oxidoreductase with
the reduction of ferredoxin. Acetyl-CoA is then converted to acetate, butyrate
and propionate.
Acetate fermentation processes are well understood and have
a maximum yield of 4 mol hydrogen pr. mol glucose. The yield of hydrogen
from the conversion of acetyl-CoA to butyrate, has half the yield as the conversion
to acetate. In mixed cultures of Clostridium the reaction is a combined
production of acetate, butyrate and propionate. The organic acids which are the
by-product of fermentation with Clostridium, can be further processed as
substrate for hydrogen production with Rhodobacter.
Rhodobacter
The purple non-sulphur (PNS) bacteria Rhodobacter
sphaeroides is able to produce hydrogen from a wide range of organic
compounds (chiefly organic acids) and light.
The photo-system required for hydrogen production in Rhodobacter
(PS-I), differ from its oxygenic photosystem (PS-II) due to the requirement of
organic acids and the inability to oxidize water. In the absence of
water-splitting photosynthesis is anoxygenic. Therefore, hydrogen production is
sustained without inhibition from generated oxygen.
In PNS bacteria, hydrogen production is due to catalysis by
nitrogenase. Hydrogenases are also present but the production of hydrogen by
[FeFe]-hydrogenase is less than 10 times the hydrogen uptake by
[NiFe]-hydrogenase.
Only under nitrogen-deficient conditions is nitrogenase
activity sufficient to overcome uptake hydrogenase activity, resulting in net
generation of hydrogen.
Rhodobacter hydrogen metabolism
The main photosynthetic membrane complex is PS-I which
accounts for most of the light-harvest. The photosynthetic membrane complex
PS-II produces oxygen, which inhibit hydrogen production and thus low partial
pressures of oxygen most be sustained during fermentation.
The range of photosynthetically active radiation for PNS
bacteria is 400-1000 nm. This includes the visible (VIS) and near-infrared
(NIR)sections of the spectrum and not (despite erroneous writings) ultraviolet.
This range is wider than that of algae and cyanobacteria (400-700 nm;
VIS). The response to light (action spectrum) varies dramatically across the
active range. Around 80% of activity is associated with the NIR. VIS is
absorbed but much less efficiently utilised.
To attain high production rates of hydrogen, the hydrogen
production by nitrogenase has to exceed the hydrogen uptake by hydrogenase. The
substrate is oxidized through the tricarboxylic acids circle and the produced
electrons are transferred to the nitrogenase catalysed reduction of protons to
hydrogen, through the electron transport chain.
LED-fermenter
To build an industrial-size photo-fermenter without using
large areas of land could achieved using a fermenter with light-emitting diodes (LED) as light source.
This design prevents self-shading within the fermenter, require limited energy
to maintain photosynthesis and has very low installation costs. This design
would also allow cheap models to be built for educational purpose
However, it is impossible for any photobioreactor using
artificial lights to generate energy. The maximum light conversion efficiency
into hydrogen is about 10% (by PNS bacteria) and the maximum efficiency of
electricity generation from hydrogen about 80% (by PEM fuel cell) and the
maximum efficiency of light generation from electricity (via LED) is about 80%.
This represents a cycle of diminishing returns. For the purposes of fuel or energy
production sunlight is necessary but artificially lit photobioreactors such as
the LED-fermenter could be useful for the production of other valuable
commodities.
Metabolic engineering
There is a huge potential for improving hydrogen yield by metabolic engineering. The bacteria Clostridium
could be improved for hydrogen production by disabling the uptake hydrogenase,
or disabling the oxygen system. This will make the hydrogen production robust
and increase the hydrogen yield in the dark-fermentation step.
The photo-fermentation step with Rhodobacter, is the step
which is likely to gain the most from metabolic engineering. An option could be
to disable the uptake-hydrogenase or to disable the photosynthetic membrane
system II (PS-II). Another improvement could be to decrease the expression of
pigments, which shields of the photo-system.
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