Selection, breeding and engineering of microalgae for bioenergy and biofuel production

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1 Selection, breeding and engineering of microalgae for bioenergy and biofuel production 2 nd seminar, Lisa Krämer Introduction In these days, the maintenance of energy and fuel supply is of urgent importance. Today s society is highly dependent on energy and fuel supply since it is important for all activities of our daily lives. Actual situation As it is shown in figure 1, fuel is mainly produced from mineral oil, which is a fossil energy source. Figure 1. Comparison of fuel and energy production processes in the present and the future; 0zur%2520Kraftstoffherstellung.jpg.thumb.jpg The amount of fossil energy sources like mineral oil, petroleum gas and coal on earth is limited and will be exhausted in the near future, if the consumption is not reduced. In figure 2, predictions for rates of global fossil fuel depletion using different assumptions are given.

2 Figure 2. Predicted rates of global fuel depletion calculated on the basis of proved (1P) and Ultimately Recoveralbe Reserves (URR), assuming 1.5 and 3 % economic growth; Stephens E. et al (2010) Future prospects of micoralgal biofuel production systems. Trends in Plant Science, Vol. 15, No. 10 It can be seen that the higher the economic growth, the faster the global fossil fuels are depleted. If the ultimately recoverable reserves are taken into account, the supply can be maintained until the year 2110 longest. Furthermore, the burning of fossil fuel negatively affects the environment. As it can be seen in figure 3, the carbon dioxide emissions still are increasing. Figure 3. CO2 emission rates from ; Although the carbon dioxide emission rates in Europe, USA and Russia reached their peak, especially the emission rate of china is exploding. Carbon dioxide is one of the so called greenhouse gases and

3 the increased level of CO2 in the atmosphere can be correlated to global warming. The increase in temperature is shown in figure 4. Figure 4. Development of global temperatures of the years 1860 until 2010; The strong increase in global temperature from 1950 on can be correlated to the increase in greenhouse gas emissions due to increasing industry and infrastructure. Since the fuel and energy supply has to be maintained but also the negative changes in environment have to be stopped, alternative strategies for fuel and energy production urgently need to be found. In this review the authors discuss the use of microalgae for the production of biofuel and bioenergy. In figure 1 is shown, that this could be an alternative strategy in the future. Microalgae Microalgae are the oldest eukaryotes and belong to the protists. These algae are mostly unicellular and 1 10 µm in size. Microalgae are photoautotrophic organism, which means that they are able to produce biomass from carbon dioxide, water and light. The big advantage of microalgae for society is that they can be grown on non-arable land, which means that they don t compete for space with crops and food plants. Also microalgae use saline and waste water, therefore not consume fresh water which is very precious and limited. Photosynthesis Microalgae are able to perform photosynthesis which means that they use carbon dioxide in the atmosphere to generate biomass. The molecular processes are shown in figure 5.

4 Figure 5. Molecular processes in photosynthesis; Stephenson P. G. et al (2011) Improving photosynthesis for algal biofuels: toward a green revolution. Trends in Biotechnology, Vol. 29, No. 12 The photosynthesis process can be divided into the light- dependent and light-independent reaction and takes place in the thylakoid membrane of green plants, (micro-)algae and cyanobacteria. In the light-dependent reactions, light energy is captured by the antenna complex, which contains pigments like chlorophyll and carotenoids that are able to absorb violet-blue and red light. The light energy induces the water proteolysis in the photosystems I and II, which is given by the following formula: H 2 O H + + ½ O 2 During water proteolysis, electrons and protons are released. The electrons are carried via an electron transport chain and finally reduce NADP+, which is needed for carbohydrate synthesis later on. The proton gradient induces the enzyme ATP synthase which is able to produce ATP when protons follow the gradient across the membrane. Three molecules of ATP and two molecules of NADPH are needed to fix one carbon atom in the following light-independent reactions, the calvin cycle, where carbon dioxide is fixed into carbohydrate. This reaction is catalyzed by the enzyme ribulose-1,5-biphosphate carboxylase oxygenase (RUBISCO). Carbohydrate is then used for all cellular functions including respiration, biomass generation and lipid synthesis. Photosynthesis is a very inefficient process; the maximum efficiency was determined to be 8-10 %. This inefficiency is also represented by the purple arrows in figure 5. Some efficiency limiting factors are limited photosynthetically active radiation, excitation energy transfer (fluorescence, heat release), photorespiration, light saturation, etc. Biofuel production In comparison to terrestrial plants, microalgae can be processed completely and no waste is generated. This is also shown in figure 6.

5 Figure 6. Production and processing of microalgae; Microalgae generate biomass from carbon dioxide, water, sun light and nutrients. The algae are broken down, the oil is used for the production of biodiesel and glycerol and the biomass contains carbohydrates that can be converted to alcohol but also protein which makes algae biomass an interesting option for animal feed. The carbon dioxide, which was fixed in biomass, is released when biomass is degraded. The carbon cycle is closed, because the carbon dioxide again is fixed by microalgae growth. Selection Microalgae are the oldest eukaryotic organisms and evolved 1.5 to 2 billion years ago (figure 7). Figure 7. Evolution of the earth:

6 Microalgae as eukaryotes perform sexual and asexual replication, which accelerates evolution and thereby adaption to different environments. They have developed a huge genetic diversity, which allows them to grow in a wide range of conditions regarding ph, temperature, water quality, light conditions and nutrient composition. Today, microalgae are found in every sunlit aquatic environment. It is thought that there are more than algal species in total and many of them have in biotechnological sense very interesting properties. By investigating the algal strain-specific publications since 1991 as it was done in figure 8, it can be seen that only a small number of algal strains are object of the current research. Figure 8. Algal publications a) ordered for strain-specificity b) ordered for biodiesel, hydrogen and lipids; Larkum W.D., Ross I.L., Kruse O. and Hankamer B. (2012) Selection, breeding and engineering of microalgae for bioenergy and biofuel production. Trends in Biotechnology, Vol. 30, No. 4. The majority of publications is about lipid formation and yields in microalgae. The amount of publications that concern biodiesel is strongly increasing and passed the number of publications concerning hydrogen. Reasons for the use of only a small number of microalgal strains in research is that these few strains are well documented and some genetic toolsets are available. Also the biomass productivity is limited and the known strains already produce very well which means that there is no urgent need of new strains. Because of their huge diversity, it is very likely that there are strains in environment that have new positive properties like accumulation of desired storage compounds and high value products, resistances in salt, ph or temperature or easy handling. Therefore, the screening for new strains with novel properties is worthwhile. For the collection and screening of new microalgal strains, there are different methods applied which are given in figure 9.

7 Figure 9. Selection criteria for microalgal strains; Larkum W.D., Ross I.L., Kruse O. and Hankamer B. (2012) Selection, breeding and engineering of microalgae for bioenergy and biofuel production. Trends in Biotechnology, Vol. 30, No. 4. Water samples can be taken from every aquatic environment and different strains can be separated by different techniques like extraction from crude water, dilution, antibiotic selection or cell sorting. The newly isolated strains are investigated regarding production of special metabolites by response surface modeling, metabolite profiling or principal component analysis when growth is optimal. To prevent the new strains from genetic drift, they are cryopreserved. Breeding Advantages of microalgae In comparison to terrestrial plant, microalgae offer a number of advantages for breeding. Their life cycles are very short and take hours or a few days. Therefore, harvesting is possible much more often and is seasonally independent. Since microalgae are mostly single cells, they need less space than crops and small scale for experiments is applicable. Furthermore they are able to use saline and waste water and can be cultured on non-arable land. Many strains incorporate high yield of triacylglycerols which can be easily converted into biodiesel. But not only the lipids are of interest but also the biomass can be used for animal feed or carbohydrate extraction.

8 Breeding systems The breeding systems can be divided into open and closed systems. Open systems Open systems are further divided into natural waters like lakes, lagoons or ponds and artificial ponds or containers. These systems offer advantages like low costs and easy construction and operation. But their simplicity also brings limitations like evaporative losses and loss of carbon dioxide to the atmosphere. Also the light utilization of the cells is poor, large areas are required, contamination occur very easily and the stirring is inefficient what result in low productivity. One very common example of an open system is the raceway pond which is shown in figure 10. Figure 10. Raceway ponds; The principle of this type of culture system is given in figure 11. Figure 11. Working principle of a raceway ponds; The system works continuously. Water and nutrients are constantly fed and water with algae is constantly removed. The flow is maintained by a motorized paddle.

9 Closed systems Closed systems are consistent with open systems but the ponds or raceways are covered by a greenhouse as it can be seen in figure 12. Figure 12. Closed systems for microalgae cultivation; These additional expenses implicate quite a number of advantages. Since these systems are closed and not in contact to the environment, also genetically modified species can be grown. The contaminations are prevented and since it is possible to heat these systems, the production can be achieved the whole year round. Because of the coverage, the loss of carbon dioxide to the atmosphere is lowered, resulting in higher growth rates. Photobioreactors Photobioreactors are the most advanced culture systems for microalgae. The principle is given in figure 13. Figure 13. Principle of a photobioreactor; Algae, water, carbon dioxide and nutrients are mixed in the feeding vessel and injected into the actual photobioreactor, where the light is applied. After the algae have generated biomass in the bioreactor, the microalgae are harvested and the cells are separated from the water which is

10 recovered. To gain the algae oil, the algae slurry is pressed and the biomass is separated from the oil. Photobioreactors offer a fully controlled environment regarding temperature, ph, gas composition, light exposure, mixing, etc. Evaporation is prevented as well as contaminations. These systems are space saving and highly productive. Major disadvantages are the complicated technique behind those reactors as well as high capital and production costs. Engineering Goals Microalgae offer many different targets for the application of engineering. Future strains used for biofuel production should accumulate high yields of triacylglycerols which are needed for biodiesel synthesis. Since the efficiency of photosynthesis is low, these structures like the antenna complex and the photosystems but also the on-going reactions are an interesting goal for engineering. Since microalgae are eukaryotic and offer short life cycles, also the production of high value products and co-products could be realized in microalgae. Positive growth properties like high cell densities or robustness in physiological conditions also are very interesting targets for engineering. Requirements For the application of targeted genetic engineering, genome sequences should be known. But also a genetic toolset for the conduction of cloning and transformations is necessary. The model microalga is still Chlamydomonas reinhardtii, where the genome sequence is known and an extensive toolset and a random insertion library is available. Generally, there are two possibilities for engineering of production strains. The first one would be the isolation of highly efficient non-genetically modified strains that can be further improved by random mutagenesis by chemical treatment or UV light. This method offers the advantage that non-gmos can be cultured in open systems. The second one would be to construct new highly efficient strains via targeted metabolic or genetic engineering. For this approach, genome sequences need to be known and the strains have to be characterized. Since the analytical but also the in silico methods get better and faster, this approach will become more successful in the future. Conclusions Up to now, there is no commercial plant producing and processing microalgal biomass into biofuels, because the cultivation but also the processing is very energy intense and therefore costly. But microalgae selection and engineering but also breeding systems offer many targets for optimizations.

11 Since there is extensive research done in this field, in the near future, there will be economic production and processing of microalgae into biofuel and bioenergy. Literature Larkum W.D., Ross I.L., Kruse O. and Hankamer B. (2012) Selection, breeding and engineering of microalgae for bioenergy and biofuel production. Trends in Biotechnology, Vol. 30, No. 4. Man Kee Lam and Keat Teong Lee (2012) Microalgae biofuels: A critical review of issues, problems and the way forward. Biotechnology Advances 30, Stephenson P. G. et al (2011) Improving photosynthesis for algal biofuels: toward a green revolution. Trends in Biotechnology, Vol. 29, No. 12. Trentacoste E.M. et al (2013) Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth. Applied biological sciences. Grobbelaar J.U. (2010) Microalgal biomass production: challenges and realities. Photosynth Res (106), Larkum W.D. (2010) Limitation and prospects of natural photosynthesis for bioenergy production. Current opinion in Biotechnology (21), Stephens E. et al (2010) Future prospects of micoralgal biofuel production systems. Trends in Plant Science, Vol. 15, No. 10