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.
- 1 Introduction
- 2 Algal biohydrogen
- 3 Bacterial biohydrogen
- 4 See also
- 5 References
- 6 External links
Currently, there is a huge demand for 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.
In 1939 a German researcher named Hans Gaffron, while working at the University of Chicago, observed that the alga he was studying, Chlamydomonas reinhardtii (a green alga), 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 algal 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.
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.
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.
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. 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 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 sustainably 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.
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.
- 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.
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 dinucleotide (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.
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.
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.
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.
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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