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Vol. 19 No. 3 - July 2013

Algae: The Source of Hydrogen Energy

By: S. K. Mandotra1, M. R. Suseela2 , P.W. Ramteke3


With the industrial revolution in the 20th century the use of fossil fuel increased markedly and with the passage of time this requirement kept on increasing. We now consume about 13 terrawatts (1 TW = 1012 W = 3.2 EJ/year) of energy worldwide, and approximately 80% of that comes from burning fossil fuels (Johansson, 2004). Although major part of energy demand comes from the fossil fuel, which lay the foundation for the industrial society, but the use of fossil fuels are increasing day-by-day causing threat to environment and disappearing at an alarming rate. Combustion of fossil fuels is adding about 6 gigatonnes (Gt =109 tons) of carbon per year to the atmosphere (IPCC, 2007). The average facade temperature of the globe has increased more than 10 F since 1900 and the speed of warming has been almost three folds since 1970. This increase in earth’s average temperature is called global warming. Various viral diseases such as ebola, hanta and machupo were appeared due to warmer climates. The marine life is also facing serious problems due to the increase in temperatures. Based on the study on past climate shifts, notes of current situations and computer simulations, many climate researchers assume that because of greenhouse gas discharges, the 21st century might experience temperature rise of about 3 to 80 C, climate pattern shift, melting of ice sheets and rise in sea levels.

It is because of these environmental issues one should consider more environmental friendly solutions to satisfy the current energy consumption. Biomass which has been used for centuries is one of the most attractive alternatives for fossil fuels. Presently various types of biofuels (biodiesel, biogas, bioalcohols etc.) derived from biomass are being used in the energy sector. Currently, biomass contributes about 12% of today’s world energy supply, while in many developing countries it contributes 40-50% energy supply (Demirbas, 2001). Though the burning of these fuels also emits green house gases but in comparison to fossil fuels their emissions are negligible. We need biofuels as a temporary solution until something better is available. Such a temporary solution must last for many years.

The rate at which the solar energy is trapped and converted into various useful energy derived products by the plants cannot be achieved by any other artificial means. Photosynthesis is the key process involved in the plants that synthesises various end products in the form of biomolecules, which can be further converted into the biofuels. Not only the end product but during the whole process there are few side products also which are being used as potential energy sources. The major part of the biofuel production takes place from variety of natural crop products like rapeseed, soybean, mustard, flax, sunflower, canola, palm oil, corn oil, hemp, jatropha and waste vegetable oils etc. But there are some other sources too including animal waste which can be used for biofuel generation. For eg. Mekong Delta people are using ‘tra’ and ‘basa’ fishes to produce the non-toxic, clean biofuel. Another amazing source of biofuel (Hydrogen) is the bacteria that live inside the guts of termites which helps to digest wood and other plant products and produces hydrogen.

In spite of having variety of available options stated above, researchers are continuously exploring cost effective sources of biofuel, which can fulfill the future energy demands. As it is well known that in the future there will be huge shortage of land as well as fresh water. Under these circumstances algae seems to be the most promising candidates for biofuel production, unlike other oil producing crops it do not require land and fresh water to grow, rather it can be grown in the waste water like industrial effluent, sewage and salt water. Most importantly algae are able to grow year round with very less multiplication time (2-3 weeks) while other oil producing crops are generally annual and can be harvested once a year for oil production. Apart from synthesizing good amount of lipids, algae are the only known eukaryotes except blue-green algae (prokaryotes) with both oxygenic photosynthesis and a hydrogen metabolism.

Biological hydrogen production has several advantages over hydrogen production by any other mean. Hydrogen gas from algae due to its high energy content on a mass basis, easy availability and renewable nature is thought to be the ideal future fuel. Hydrogen gas synthesized form algae can be used to generate electricity which can be further utilized in transportation and domestic sectors. Green algae synthesizes hydrogen aerobically as well as anaerobically, during light periods however, the production of hydrogen gas is less in comparison to the dark periods. The reason behind this is the sensitivity of hydrogenase enzyme towards the photosynthetic oxygen. Blue-green algae (cyanobacteria) also synthesizes hydrogen gas in association with nitrogen fixation which occurs in a special structure called heterocyst. There are two major types of hydrogenases enzymes present in the algae: Fe-hydrogenases which is commonly present in the green algae and Ni-Fe-hydrogenases which is present in cyanobacteria.

What are biofuels?

Biofuel is solid, liquid or gaseous fuel derived from any biological carbon source including treated municipal and industrial wastes (Yuan et al., 2008). Also known as agrofuel can be produced directly or indirectly from biomass (organic material) including plant materials and animal waste. Whereas biomass is defined as the renewable energy resource derived from the carbonaceous waste of various human and natural activities including the by-products from the timber industry, agricultural crops, raw material from the forest, major parts of household waste and wood. Biofuels derived from biomass do not release net carbon dioxide (CO2) to the atmosphere; rather they recycle the CO2. The advantages of biomass energy as an alternative energy source is its renewable nature, free from net CO2 emissions and its abundant availability in the form of agricultural residue, city garbage, cattle dung and firewood. Biofuels are divided into three main categories: First-generation biofuels are made largely from edible sugars and starches, second-generation biofuels are made from non-edible plant materials and third-generation biofuels are made from algae and microbes.

Worldwide bioethanol is the most commonly used biofuel. It is an alcohol made by fermenting the sugar components of biomass. Today, it is made mostly from sugar and starch crops. Cellulosic biomass, like trees and grasses, are also used as feedstocks for ethanol production. Ethanol can be used as a fuel for cars in its pure form, but it is usually used as a gasoline additive to increase octane and improve vehicle emissions.

Biodiesel is a mixture of fatty acid alkyl esters made from vegetable oils, animal fats or recycled greases. It can be used as a fuel for vehicles in its pure form, but it is usually used as a petroleum diesel additive to reduce levels of particulate matter, carbon monoxide, hydrocarbons and air toxics from diesel-powered vehicles. In the United States, most biodiesel is made from soybean oil or recycled cooking oils. Animal fats, other vegetable oils, and other recycled oils can also be used to produce biodiesel, depending on their costs and availability. In the future, blends of all kinds of fats and oils may be used to produce biodiesel.

Biogas is produced by the process of anaerobic digestion of organic material by anaerobes. It can be produced either from biodegradable waste materials or by the use of energy crops fed into anaerobic digesters to supplement gas yields. Biological H2 production, known as “green technology”, has received considerable attention in recent years. Biohydrogen can be produced by a various methods, which includes water electrolysis, thermo catalytic reformation of hydrogen-rich organic compounds, and biological processes. H2 gas, which is derived from algal biomass, is expected to become one of the key energy resources for global sustainable development.

Biofuels from algae

Algae are referred to as eukaryotic as well as prokaryotic, chlorophyll containing plant-like organisms that are usually aquatic, photosynthetic and ranges from unicellular to multicellular forms (marine algae). They do not exhibit true roots, stems, leaves, vascular tissue and have simple reproductive structures. Due to their wide availability they are ubiquitous in nature that grow in almost every drop of sunlit water and every gram of fertile soil, on the barks of trees, surfaces of animal shells, rocks and as symbionts in plants.Algae are found in the fossil record dating back to approximately 3 billion years in the Precambrian period and at presentmicroscopic algae are found in almost all water bodies, covering over 75% of the earth surface.

Microalgae mass cultures have been considered for almost fifty years as potential biofuel sources, with the first conceptual engineering analysis presented in the late 1950's. Relative to terrestrial plants, microalgae are more efficient at converting sunlight into chemical energy, and require a smaller footprint and less water for cultivation (Dismukes et al., 2008). Microalgae contain lipids and fatty acids as membrane components, storage products, metabolites and sources of energy. Algal oils posses characteristics similar to those of fish and vegetable oils, and can thus be considered as potential substitutes for the products of fossil oil. Direct lipid extraction from microalgae appears to be more convenient and cost effective than fermenting algae to produce either methane or ethanol.

More recently, emphasis has been on higher value fuels, particularly H2gas, preferably coupled to utilization of CO2 from power plants. H2 gas produced by the algae during the photosynthetic process is considered as the most potent future energy carrier because it is renewable and does not produces green house gas (CO2) during combustion, it also liberated large amount of energy per unit weight and can be easily converted into electricity by the fuel cell. Biological H2 production has several advantages over hydrogen production by photoelectrochemical or thermochemical processes. H2 production by the photosynthetic microorganisms like algae is very cost effective in nature which only requires a transparent closed box with very low energy requirement in comparison to hydrogen production by solar battery-based water splitting. Till now lot of research has been done in the field of hydrogen production from algae. Chlamydomonas reinhardtii, a type of green algae is considered to be the most potential species for hydrogen production. Apart form this various cyanobacterial species have also been reported for their high hydrogen production. Researchers are continuously exploring the new culture techniques by which they can fulfil the future energy demand.

Biological Hydrogen Production:


Hydrogen production occurs during the process of photosynthesis. The process of photosynthesis occurs in the chloroplasts of plant cells. Chloroplast contains the reaction centres, which are generally known as the photosystems. There are two types of photosystems; photosystem-I (PS-I) and photosystem-II (PS-II). During the process of photosynthesis both the photosystems works simultaneously and absorbs light photons to create electron reducing potential. Upon light absorption the PS-II splits water molecule into oxygen gas and the electron released cascades through a series of reactions. The cascade of reactions creates a proton gradient across the thylakoid membrane which houses these reaction centres. The proton gradient drives the ATP synthase protein to generate adenosine triphosphate (ATP), an energy medium used in living organisms. At the same time photon absorbed by PS-I excites electron which is used by the ferredoxin (Fd), a water soluble protein, to reduce NAP+ to NADPH. ATP and NADPH synthesized during the photosynthetic process are then used to reduce the CO2 to built hexoses and other organic material. Plants lack the hydrogenase enzyme, present in green algae and cyanobacteria, which can catalyze the reduction of protons to H2 under specific conditions (Benemann, 1997).

Hydrogen production in green algae

Hydrogen production in green algae is catalyzed by the Fe-hydrogenase enzyme which mediates the donation of high potential-energy electrons to protons. The hydrogenases containing no other metal than Fe are called Fe hydrogenases, also known as "Fe-only" hydrogenases. Two families of Fe-Hydrogenases are recognized: first is cytoplasmic, soluble, monomeric Fe- Hydrogenases, found in strict anaerobes such as Clostridium pasteurianum and Megasphaera elsdenii. They are extremely sensitive to inactivation by O2 and catalyse both H2 evolution and uptake; the second is periplasmic, heterodimeric Fe- Hydrogenases from Desulfovibrio spp., which can be purified aerobically and catalyse mainly H2 oxidation. Cytochrome C3 and cytochrome C6 act as physiological electron donors or acceptors for Fe- Hydrogenases.

Photo-chemical H2 production

H2 production in green algae occurs within the chloroplast and it is light dependent process. The process of photosynthetic electron transport in green algae can operate with a photon conversion efficiency of 85-90% (Ley & Mauzerall., 1982; Greenbaum, 1988). The process of H2 production starts with the absorption of photons (in PS-II) that result in the photo-splitting of water molecules, as a result proton, electron and a molecule of O2 is produced. The electrons which are produced by the H2O oxidation by PS-II are carried by number of electron carriers (plastoquinone, cytochrome b6f complex and plastocyanine) to PS-I. As a result PS-I transfer the electron to ferredoxin, the reduced ferredoxin servs as the physiological electron donor to the Fe-hydrogenases. Finally Fe-hydrogenases accept the electron from ferredoxin and, using available protons, synthesise molecular hydrogen.

Nonphotochemical H2 production

During photochemical process light energy is used for the photolysis of H2O followed by electron generation which are then used by Fe-hydrogenases to produce H2. In non photochemical H2 production the electrons for the generation of H2 comes from the oxidation of cellular endogenous substrate which is explained by the breakdown of starch under anaerobic conditions. The process of glycolysis converts starch into pyruvate, during this process NAD+ oxidises to NADH + H+ followed by the transfer of electron to plastoquinone pool (PQ) mediated by NAD(P)H plastoquinone reductase complex, latte on via PS-I electron transfers to ferredoxin and finally with the transfer of electron to Fe-hydrogenases complex H2 is produced. In an another pathway, during anaerobic conditions pyruvate oxidises to acetyl-CoA by pyruvate-ferredoxin oxidoreductase complex, this complex also converts oxidised form of ferredoxin to its reduced form which then be used to reduce Fe-hydorgenases complex for the generation of H2 (Posewitz et al., 2009).

H2 production via sulphur deprivation

Melis and co-workers in the year 2000 reported that by depriving sulphur in the culture medium of Chlamydomonas reinhardtii, H2 production can be enhanced several folds. The chemistry behind this process lies in the modification of electron transport process in the chloroplast as a result of the partial inactivation of PS-II. Sulphur deprivation plays an entirely different effect on both oxygenic and mitochondrial respiration. On giving sulphur deprivation, after 100 h the photosynthetic activity of C. reinhardtii significantly decreases from ~44 (mmol O2) (mol chlorophyll)-1s-1 to ~2 (mmol O2) (mol chlorophyll)-1s-1. The reason behind the significant decrease in the level of photosynthesis is the decrease in the amount of sulphur containing amino acids; cysteine and methionine, which are needed in the biosynthesis of the proteins that are frequently required for the replacement of D1 kDa reaction centre protein in H2O oxidising PS-II complex, present in the chloroplast. The photosynthetic rate of C. reinhardtii decreases as compared to the rate of mitochondrial respiration during sulphur deprivation. After some time the closed culture of C. reinhardtii become anaerobic as all the oxygen present in the culture is exhausted (Ghirardi et al., 2000).

During partial inactivation of PS-II the electron produced during photolysis of H2O cannot be consumed for the production of molecular oxygen, rather they are accepted by the proton available in the cell and with the help of Fe-hydrogenases enzyme, converted into H2. Though sulphur deprivation produces good amount of H2 gas, but it is necessary to add sulphur to the growth medium, because sulphur is the key ingredient of amino acids (cysteine and methionine) that polymerise to form certain proteins which are useful for the sustainability of the cell. So after some saturation limits of H2 production the cell must be replenished with sulphur which do not let the cell die and again enable the cell to produce molecular hydrogen (Melis et al., 2000).

Hydrogen production in blue-green algae (cyanobacteria)

Cyanobacteria often referred to as "blue-green algae." While most algae is eukaryotic (multi-celled), cyanobacteria is the only exception. They are primitive in occurrence with some fossils dating back almost 4 billion years (Precambrian era), making them among the oldest organisms in the fossil record. Cyanobacteria may be single-celled or colonial. Depending upon the species and environmental conditions, colonies, sheets or even hollow balls may form from filaments. Some filamentous colonies show the ability to differentiate into three different cell types. Vegetative cells are the normal, photosynthetic cells formed under favourable growing conditions. Climate-resistant spores may form when environmental conditions become harsh. A third type of cell, a thick-walled heterocyst, contains the enzyme nitrogenase, vital for nitrogen fixation. Photosynthesis in cyanobacteria uses water as an electron donor and produces oxygen as a by-product. The photosynthesis occurs in membranes called thylakoids, with chlorophyll being employed to absorb the sun's rays.

Hydrogen production occurs in 14 different genera of cyanobacteria under wide range of culture conditions (Lopes Pinto et al., 2002). The process of hydrogen production in cyanobacteria occurs in the special structure called heterocysts. The chief enzymes involved in the formation of hydrogen are hydrogenase and nitrogenase. In comparison to hydrogenases the enzyme nitrogenase is less sensitive to oxygen which is produced during photosynthesis process by the photolysis of H2O. Cyanobacteria has develop a special mechanism to protect nitrogenase form oxygen i.e. the localization of nitrogenase inside the heterocysts where the concentration of oxygen is very low or completely absent that makes the process of H2 production very efficient. H2 gas is produced in the reaction when nitrogenases reduce atmospheric nitrogen into ammonia.

For production of hydrogen there are three enzymes present in the Cyanobacteria: 1) a nitrogenase, evolving H2 during N2 fixation; 2) an uptake hydrogenase, reutilizing this H2; and 3) a bidirectional (reversible) hydrogenase (Hansel & Lindblad, 1998; smith, 1990; Rao & Hall, 1996; Schulz, 1996; Benemann, 1996). The nitrogenase enzyme is made up of two sub units: one is dinitrogenase which is heterotetramer (α2β2), its main role is to break the atom of nitrogen. The second one is dinitrogenase reductase, it is a homodimer and its main role is to mediate the transfer of electron from the external source (ferredoxin of flavodoxin) to the dinitrogenase unit of nitrogenase (Flores & Herrero, 1994; Masepohl et al., 1997; Orme-Johnson, 1992; Dutta et al., 2005).

The second main enzyme present in the Cyanobacteria is hydrogenases. In different cyanobacterial species this enzyme is present in two different forms: uptake hydrogenases and bidirectional (reversible) hydrogenases respectively. Every hydrogenase found in cyanobacteria bind one iron and one nickel atom at its active site. These Ni-Fe-hydrogenases are classified into multiple groups out of which only NAD(P)H-dependent bidirectional hydrogenases can evolve hydrogen using electron from either NADPH or NADH (Vignais et al., 2001; Carrieri et al., 2008). The enzyme uptake hydrogenases catalyse the oxidation of H2. This enzyme is found in the thylakoid membrane of the heterocysts and transfers the electron from H2 to oxygen through the respiratory chain and the reaction is known as oxyhydrogenation. The enzyme uptake hydrogenase is made up of two sub-units, the larger subunit is responsible for the uptaking of H2 and the smaller subunit is responsible for the reduction of oxygen. The cyanobacterial strains which contain uptake hydrogenases cannot synthesise net H2 because the H2 formed is again re oxidized by this enzyme (Dutta et al., 2005). The enzyme bidirectional (reversible) hydrogenase is associated with the cytoplasmic membrane. It is believed to be a common cyanobacterial enzyme, and its presence is not linked to nitrogenase. The enzyme synthesises the H2 via the reaction 2H+ + 2e- H2 (g). As discussed earlier the sources of the electrons are NADPH and NADH (Tamagnini et al., 2002).

Improvement of algal strains for H2 production

Among the hydrogenases present in all the hydrogen producing algal (blue-green & green algae) strains Fe-hydrogenase is the most sensitive to the oxygen gas, this sensitivity is due to the binding of oxygen molecule at the unoccupied coordination site located at the active centre of the enzyme (Hall et al., 1995). Sustained hydrogen production can only be achieved when H2ase enzyme will remain active in all adverse conditions. Various approaches by which hydrogen sensitivity can be achieved include genetic engineering and physiological separation of oxygen and hydrogen production. Through site directed mutagenesis or point mutation at hydA gene which encodes Fe-hydrogenases, oxygen sensitivity may be achieved. This can be done by identifying that particular gene in the DNA sequence which encodes those amino acids where the oxygen irreversibly binds. Substitution of these regions with the help of genetic engineering may result in the oxygen tolerant algal strain (Das et al., 2006). Uptake hydrogenase present in the cyanobacteria is responsible for the oxidation of H2 where as the reversible hydrogenase synthesises good amount of H2. By various genetic engineering techniques the activity of uptake hydrogenase can be down regulated or can be completely eliminated and with the increased activity of reversible hydrogenase the H2 production rate of the cyanobacterial can be enhanced. For example in a mutant strain of Anabaena (AMC 414), the large sub unit of the uptake hydrogenase (hupL) was inactivated which result in the H2 production at the rate which is twice the rate of the parent strain (Zhang et al., 1983). The synthesis of uptake hydrogenase can also be blocked by giving such culture conditions which are deficient in Ni, as Ni is required for the assembly of the holo enzyme and for the catalytic activity of the enzyme (Hall et al., 1995). With advanced molecular techniques now it is possible to over express Fe-hydrogenase encoding hydA gene under strong promoters for enhanced hydrogen production. Genetic manipulation at transcriptional regulatory site results in the constitutive expression of hydA gene which was found to be active under repressed conditions only (Mishra et al., 2004).


1, 2Algology Laboratory, National Botanical Research Institute, Lucknow, India and 3Dept. of Biological Sciences,Sam Higginbottom Institute of Agriculture, Technology & Sciences, Allahabad, India. E-mail: <[email protected]>

This article has been reproduced from the archives of EnviroNews - Newsletter of ISEB India.

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