Empirical study in growth and lipid content with the microalgae Scenedesmus quadricauda, Chlorella vulgaris and Nannochloropsis oculata.

Transcription

1 Empirical study in growth and lipid content with the microalgae Scenedesmus quadricauda, Chlorella vulgaris and Nannochloropsis oculata. Maja Lindblad, Hugo Wikström. 11 February 2014, Härnösand. Algkraft

2 ABSTRACT This is a practical study with an intention to compare three different algae species in growth and lipid content. The algae species are Chlorella vulgaris, Nannochloropsis oculata and Scenedesmus quadricauda. The purpose of the study is to see how Scenedesmus quadricauda performs in comparison to the well-known species Chlorella vulgaris and Nannochloropsis oculata. This study will give a hint to how these species function, which can be useful for the long-term goal to make large scale algae cultivations for biodiesel production in a northern climate. The test has been going on for 28 days, where the growth of the algae has been measured by daily weighing. The three species have had four PET bottles each to grow in, so on the whole there have been twelve cultivations to have under control. Nutrients have been added on a daily basis, though in very small quantities. Extra nutrients were added several times, but mostly to see how the cultivations would respond to it. The results of this test show that Scenedesmus quadricauda grows just as good as the other two species. Since the hypothesis was that Scenedesmus quadricauda would grow far much better, the results are though somewhat surprising. In this study Scenedesmus quadricauda nevertheless is the species with the highest lipid content. The version of Scenedesmus quadricauda that was used in this test has probably been able to adapt to favorable conditions, which has made it slower to take advantage of light and nutrients compared to the wild original version of Scenedesmus quadricauda.

3 TABLE OF CONTENTS 1. INTRODUCTION Background Purpose METHOD 2.1 Equipment Substances and organisms Nutriment Algae Execution Basic conditions for each algae cultivation Method of measurement Determination of lipid content RESULTS Growth Happenings during the study Lipid content DISCUSSION Sources of error Improvements CONCLUSIONS...11 REFERENCES...12 APPENDICES...13 Appendix 1 Web links for utilized equipment...13 Appendix 2 Content of nutrient solution...14

4 1. INTRODUCTION 1.1 Background Microalgae are small organisms that consist of one or more cells. Because of their simple structure they grow fast and have a very effective photosynthesis. The interest for algae as an alternative to fossil fuels started already in the 1970's, but it is not until recently that research of the possibilities with algae really has begun. The advantages with microalgae are for instance their rapid growth and that they can live in very high concentrations of CO2, which they also bind to very effectively. Furthermore algae can be cultivated very cost effectively since they neither need as much water as crops on land nor arable land. (Yanqun et al, 2008) This means that one can effectively make use of non-arable land as for example gravel pits or garbage dumps. For the production of biodiesel microalgae are seen as the only alternative that has potential to replace fossil fuels. Since the society of today are threatened by more expensive fossil fuels and also a global warming caused by the fossil emissions the public advantages are great with microalgae. (Chisti, 2007) EU has even put up a distinct target that 10% of the European fuels shall be renewable till year 2020, whereof only 6% of that bioenergy can be made from food crops. (Patnaude & Werber ) In the situation of today, microalgae are, to say the least, something very relevant. When making biodiesel from algae it is desirable to have algae with as high lipid content as possible. That is because it is from those lipids that oil is made, which later is used to make the very biodiesel. Besides the high lipid content it is also desirable for the microalgae to have a quick growth, at least when cultivating algae commercially. The more the better, in other words. Each alga has its own specific qualities, so for biodiesel production one has to choose a species with the most appropriate qualities. (Chisti, 2007) For making biodiesel for example Chlorella vulgaris, Dunaliella tertiolecta or Nannochloropsis oculata are appropriate algae species. Though, this does not exclude that other species might be more appropriate in other circumstances. (Sieg, 2012: 4252) In relation to this is for instance a local algae culture found in Härnösand with a mix of different algae, though predominantly Scenedesmus quadricauda. When it was sent to Swedish University of Agricultural sciences, SLU, for determination of species it also came out that this algae culture has a lipid content of about 21%. (Gentili, ) This shows that local algae species also can have great potential. 1.2 Purpose This is a study where three different algae species are compared in growth and lipid content. Those species are Chlorella vulgaris SCCAP K-1006, Nannochloropsis oculata SCCAP K-1281 and a local mixed culture, which up to 95% consists of Scenedesmus quadricauda. (Gentili, ) The reason to why these species is used in this study is because both Chlorella vulgaris and Nannochloropsis oculata are well-known algae, which gives a good hint for what Scenedesmus quadricauda can do. Moreover Scenedesmus quadricauda has shown qualities such as high lipid content, which is very beneficial in production of biodiesel. (Zhao et al, 2012) The purpose of this study is to see how Scenedesmus quadricauda stands in comparison to acknowledged algae species such as Chlorella vulgaris and Nannochloropsis oculata. From the results of this study the long-term goal is to make a large scale algae cultivation with the species that proves to have the best growth and lipid content and that also is adapted to the northern climate. In the test all the algae species are cultivated in the same conditions, however Scenedesmus quadricauda is expected to have much faster growth than the others. This comes from a hypothesis that Scenedesmus quadricauda has been able to adapt to the cold and barren northern climate throughout the evolution. Therefore this species should take advantage of most of the light and nutriment it gets by, which under controlled conditions such as in this test should result in a much higher growth compared to the non-northern algae species.

5 2. METHOD 2.1 Equipment Solely for this study a special lab stand has been built, see Figure 1. The reason to why the stand was built is to give all the algae cultivations as similar growing conditions as possible, which for instance is made by having a light source in the middle to have the same radial distance to every algae cultivation-bottle. The lab stand has holes for 12 PET-bottles, which has made it possible to have four parallel cultivations with three algae species each. The PET-bottles were used because a similar material will be Figure 1. The lab stand used in the big scale cultivation, which is meant to be a result of this test. To utilize the lab stand in the best way these things has also been used: A dozen 1,5-liter Loka-bottles, originally with content of various flavors. Common aquarium-hose. Plastic ventilator, 12 pieces, Ø 4/6 by Zolux. Aquarium bubbler, 12 pieces, 2,5 cm x 1,5 cm. Air compressor, power 45 W, voltage/frequency 220V/50Hz, output 70 l/min, pressure >0,025 MPa, model ACO-318 by HAILEA.* Halogen lamp, 2 pieces., power 28W, light flux 370 lm, voltage 240 V, clear colour, E27 socket by Grunda. Furthermore these things have been used in the test: Aluminium holders, weight 0,7 g by Toppits. Kitchen scale, range 1g-5000g, accuracy 1g. Fungi dehydrator, model 6772 by OBH Nordica.* Weighing-machine, model XR 205SM-DR, series 320XR, range 0,01g -205g, accuracy 0,01/0,1 mg by Precisa instruments Ltd, Schweiz.* Centrifuge, model 800D, capacity 6x20 ml, maximal speed 4000 r/min, maximal RCF 1790xg.* For equipment marked with *, see Appendix 1 for web links with more detailed information. 2.2 Substances and organisms Nutrients The nutrients that have been added to the algae cultivations was a mix of 300 ml Substral Gardening nutriment (i.e. Substral Trädgårdsnäring)* and 5 ml each of 10 different vitamin and mineral stocks, see Appendix 2 for more information about these stocks. Substral Gardening nutriment contains:

6 Inorganic NPK-nutrient solution with altogether 6,0% nitrogen, whereof 3,2% are nitrate nitrogen and 2,8% are ammonium nitrogen, 2,0% water-soluble phosphate and 4,0% watersoluble potassium. Water-soluble micronutrients with: 0,01% boron, 0,002% copper as EDTA-chelate, 0,03% iron as DTPA-chelate, 0,01% manganese as EDTA-chelate, 0,002% zinc as EDTA-chelate and 0,001% molybdenum. For substances marked with *, see Appendix 1 for web links with more detailed information Algae The three algae species used in this study were Scenedesmus quadricauda, that to 95% consists of a local mixed culture found in 17 year old bio beds in Härnösand, Chlorella vulgaris and Nannochloropsis oculata. The two later species were both ordered from SCCAP in Denmark and have the numbers K-1006 respectively K Execution Basic conditions for each algae cultivation In this test totally 12 algae cultivations made from three different algae species were under survey. Because one test only needs one cultivation per species it is therefore possible to see this test as four simultaneous tests. The reason for having many parallel tests was to spread the errors of measurement that possibly could occur. Moreover one never knows what can happen, perhaps a cultivation suddenly collapses, which makes it good to have some other cultivations as back-up. Biomass from each of those 12 cultivations came from three reference cultivations, one reference cultivation per algae species. Samples, with a weight of 47 grams, from these reference cultivations were taken to be dried and weighed, so the proportion of dry weighted algae could be calculated per weight unit of cultivation medium. For Scenedesmus quadricauda the quota between dry weighted algae and sample weight was approximately 15%. Chlorella vulgaris and Nannochloropsis oculata had a dry weight percentage of about 30% and 24%. Because the aim was to have the same total amount of biomass in the beginning of each cultivation one could calculate with these proportions and thereby derive which volume of each alga species that should be taken from the reference cultivations. It was based on an assumption that the final volume for the whole cultivation would be about 1200 ml. After that 60 ml of nutrients were added to each cultivation, which made the nutrient concentration approximately 300 ppm. Then, the twelve cultivations had light and flow of air in 16 coherent hours each day in four weeks Method of measurement Everything that was added or taken away from the cultivation bottles was weighted. For measuring the growth, a sample of about 40 g was taken away from each cultivation on a daily basis. In the beginning of the study the daily weighing method was tested in different ways. In the first weighing the 40 g samples were centrifuged, so that the biomass would sink to the bottom and 2/3 of the remaining water could be poured off. What was left were put into aluminium holders and then put into the dryer for vaporizing the last droplets of water. In the following sample-takings the 40 g were poured directly into the aluminium holders, since it was a more time saving method. Before any samples were taken from the cultivations a nutrient solution was added to a certain level in the cultivation bottle. That level was marked in the beginning of the study, so that each sample would be taken from the same volume throughout the study. After the addition of the nutrient solution the cultivation bottles were shaken, just so the added fluid would solve as good as possible. The nutrient solution had an approximate ion concentration of 100 ppm, this with the ion concentration of water subtracted.

7 Samples taken from the twelve algae cultivations were put in to a fungi dehydrator were they dried for about 20 hours. After that the aluminium holders were weighted once again, though with dried biomass the second time. The weight difference, that is the weight of the dry matter, was at first round 5 mg whilst in the last days of the study the difference became 40 mg Determination of lipid content The determination of the lipid content was made after the end of the experiment. The first half of the cultivation bottles were put upside-down in a plastic cup each, just to make the biomass sink into the cups instead of the cultivations bottles. The same thing was made with the remaining bottles about one-and a-half month later. In Figure 2 one can see how it looked like. The bottles stood upside-down like that for a week. What had sedimented into the cups was then centrifuged in 10 minutes at spins, to separate the biomass from the water even more. After that the biomass from the six bottles was put into test tubes together with methanol and stood like that for about a month. Each species made up a test tube á 20 ml respectively. Thereafter some benzine was added into the methanol and biomass mixture. That was then placed for 24 hours on a shaking table, with the spinning frequency 250 RPM. The reason for adding benzine is that it binds to the lipids, which makes it possible to separate them from the rest of the algal cell. Placing the mixture on a shaking table also helped the benzine solving the lipids even more. The three shaken mixtures were thereafter filtrated through a Büchner funnel with vacuum suction. The biomass was thereby separated from the methanol-, benzine- and lipid-mixture, which was placed in a fume cupboard for 24 hours to evaporate all the benzine and methanol. The biomass and the remaining lipids were weighted. The results were then obtained by dividing the weight of the lipids with the total weight of the algae, which is the sum of the lipids and the biomass. When the lipid content was to be decided for the other half of the cultivations some misfortunes occurred, such as a part of the sample with Chlorella vulgaris being dropped and also some holders with the separated methanolbenzine- and lipid-mixture being tipped over in the fume cupboard. Thereby only the results from the first half of the cultivations can be seen as reliable. Figure 2. Extracting the biomass.

8 3. RESULTS 3.1 Growth Measurements of the 28 day long study has been summarized into a growth curve, see Figure 3. The weight of the dry substance from each cultivation has been divided with its earlier volume. This volume has been all the samples that on a daily basis were taken from the cultivation bottles, the dry substance has been all the samples dried biomass. The growth for each and one of the twelve algae cultivations has been summarized to a mean for each algae species. In Figure 3 each species are denominated with its initial letter, the letter M indicates that it is the mean. The big leaps in the graph have to do with extra nutrient solution being added those specific days, see subheading 3.2 Happenings during the study for more information. The weight of the nutrients in these nutrient boosts is thereby the reason for the graph going so straight up. What can be stated from the graph is though that Scenedesmus quadricauda grows as good as the two other algae species. Figure 3. Growth curve for the algae species Chlorella vulgaris, Nannochloropsis oculata and Scenedesmus quadricauda. 3.2 Happenings during the study In a 28-days-long study it is quite natural that unexpected things happen. Down below comes a compilation of the most relevant changes taking place during the study.

9 Day 3 Ventilators for regulating the flow of air in to the cultivation bottles were installed on the lab stand. Day 10 Nannochloropsis oculata starts to flock along the edge of the cultivation bottle and continues to do so till the end of the study. Nutrient boost is added. That means that 5 ml of a mixture of 15 ml pure orange nutrient and approximately 50 ml ordinary tap water is added to every cultivation. Day 15 Nutrient boost is added. That means that 2 ml of a mixture of 15 ml pure orange nutrient and approximately 55 ml ordinary tap water is added to every cultivation. Day 22 Nutrient boost is added. 3 ml of same mixture as in Day 15 is used. Day 25 Scenedesmus quadricauda and Chlorella vulgaris starts to flock on the bottom of the bottles and continues to do so till the end of the study. Something else coming up during the study was that metal ions in the tap water also were counted in the first measurement of the ion concentration in the weak nutrient solution. This solution was added to the cultivations each day. Thereby the ion concentration of the water had to be determined, so that it could be subtracted from the total ion concentration in the nutrient solution. What was then discovered was that the ion concentration of the tap water varied, probably due to how long the water had been flushed and what temperature it in itself had. At last the measurements showed that the ion concentration of the tap water varied between 63 ppm and 91 ppm. What then can be established is therefore that the daily adding of nutrients was not as much under control as what could be desired. 3.3 Lipid content The result of all weighing has been compiled in to a table, see Table 1. For a graphical representation of the results see the graph in Figure 4. The lipid content is the highest for Scenedesmus quadricauda, it is about 20%. Nannochloropsis oculata has the lowest lipid content of about 14%. As mentioned some misfortunes occurred when the lipid content was to be determined, thereby only the result of one half of the cultivations are reliable. These values are the ones represented bellow. Table 1. Algae species C.v. N.o. S.q. Weight of lipids Weight of biomass Lipid content 17.5% 14.4% 20.3%

10 Figure 4. Graphical illustration for the lipid content of the algae species. 4. DISCUSSION The purpose of this study has been to compare the local algal species Scenedesmus quadricauda from Härnösand with the more well-known species Chlorella vulgaris and Nannochloropsis oculata. The hypothesis was that Scenedesmus quadricauda would grow much faster than the other two species. They have been compared in both growth and lipid content and the test has resulted in a graph where it is possible to see that Scenedesmus quadricauda has a just as good growth as the others. The result thereby shows what a competent algal species Scenedesmus quadricauda is, though it does not completely correspond to the hypothesis. 4.1 Sources of error There are many different sources of error in this test to take into consideration. For instance several parts concerning the growth of the algae species have not been under complete control, such as the wavelengths of the light or the temperature which the algae cultivations have been exposed to. Furthermore the flow of air in to every cultivation bottle has only been examined by it looking good, not in any more precise ways. The test has also not been prosecuted in a very sterile environment, thereby there might be a risk for the samples being contaminated. Though, the more laboratory-like environment might not work as good for the originally wild Scenedesmus quadricauda compared to the two other species, which have been extracted specifically from a laboratory. For the test as a whole these sources of error only have a modest importance. A more relevant source of error is the method of measuring the growth of the cultivations. For the daily weighing an ordinary kitchen scale was utilized. It had no better accuracy than for 1 gram, which has given the measurements a margin of error of about 2%. However, all the measurements in this study are included in this margin of error, which still makes it possible to do internal comparisons. Additionally, it is actually inaccurate to say that the volume of the algae samples was measured, since it was their weight that was measured. The difference between the weight and the volume might nevertheless be negligibly small. The question is though how well the measurements of this study can be compared to measurements from other external experiments. Yet another important source of error has to do with the nutrient salts in the nutrient solution that was added. Since the algae did not absorb these salts they were also weighted as a part of the dried biomass. This is possible to see in the large leaps in the graph in Figure 3, showing the growth of the cultivations. The leaps come just the day after the days when nutrient boosts were added.

11 There are also some sources of error concerning the measurement of the lipid content. In this case it has also to do with several steps in the method not being completely under control, as the amount of added methanol and benzine and also if it was proportionally distributed for each biomass. The most important source of error is however the long time between the end of the test and the final result of the lipid content determination. During that time the samples might have become old and thereby not as reliable. Beside this a significant source of error in this study might be an altered genome for Scenedesmus quadricauda. The alga used in the study was taken from an already established cultivation, where the alga has had possibilities to adapt to favorable conditions such as a continuous nutrient flow and a good temperature. A suspicion is therefore that during the long time that the cultivation has been going on numerous generations of Scenedesmus quadricauda have been able to adapt to these conditions. This in turn should lead to a version of Scenedesmus quadricauda being less responsive than the original wild version. Due to changed living conditions this multi culture also might have altered its species composition, which similarly could have an effect on the behavior of Scenedesmus quadricauda. 4.2 Improvements Taking all the sources of error into consideration there are definitely many aspects that can be improved when doing a new study. It is of highest importance to have better equipment for measurement, such as a weighing scale with bigger accuracy. Furthermore one should be more precise and consequent when adding nutrients, solely for having more control over the situation. Another major deficiency in this study was the nutrient solution, with an ion concentration of approximately 100 ppm, that was added every day to keep the level of liquid relatively constant. Since the ion concentration was measured with an ordinary and slightly unreliable TDS measuring device there was no control over the exact amount of added nutrients. For a new study the best would be to only add water to keep the liquid level and then add nutrients at certain, predetermined intervals such as every seventh day. It would make the experiment more systemized. Furthermore it would also be better to utilize deionized water, partly due to having better supervision of what ions are being represented in the algae cultivations. Also being able to completely disregard aspects such as copper ions coming from water pipes, affecting the algal growth conditions negatively, is a good reason for utilizing deionized water. Yet another thing that would improve the analysis of the algae cultivations is usage of a spectrophotometer. It would make it possible to measure the chlorophyll concentration and by that determine the algal growth much more precisely. Also the determination of the lipid content should occur directly after the end of the study, or maybe even during the study, just to see how the lipid content changes. In relation to that one should also know how much methanol and benzine is being added into the samples of each algae species, mainly for being able to make a fair comparison between them. For an experiment such as this also a more sterile environment could be desired. This is however solely a practical study with the aim to apply the result in real life, although in a larger scale. Because of this everything cannot really be under complete control. For instance it is hard to obtain a sterile environment outside a laboratory, and on a large scale there will always be a risk for polluting elements and contamination. If one wants to keep as close as possible to the conditions of reality, one should not need to have more sterile conditions than in this study. In short it is though

12 clear that it would not be such a bad idea to redo this study, mainly for coming closer to the real truth. 5. CONCLUSIONS In this study the three algae species Nannochloropsis oculata, Chlorella vulgaris and a mixed culture that to 95% consists of Scenedesmus quadricauda have been tested in growth and lipid content. The hypothesis was that Scenedesmus quadricauda would have a much faster and superior growth compared to the other two species. This was however not the case. What the results show is that Scenedesmus quadricauda grows just as good as the two other very competent algae species. The lipid content is nevertheless the highest for Scenedesmus quadricauda, it is 20% to be exact. Due to several sources of error it is rather reasonable to redo the experiment to obtain even more trustworthy results. Probably the genome or the composition of the mixed culture version of Scenedesmus quadricauda used in the study is different from its corresponding wild version. This can affect both its ability to grow and the lipid content. To maintain the same productivity with this algal species its fast adaptation to beneficial conditions is something to take into consideration at large-scale cultivations. In a longer time perspective this can for example make it necessary to take in a new sample of wild algae with high capacity for survival in the beginning of each cultivation season.

13 REFERENCES Gentili, Francesco. Researcher, Department of Wildlife, Fish and Environmental studies, Swedish University of Agricultural Sciences, SLU. Contact by mail: Patnaude & Werber Accessed: The Wall Street Journal. Art Patnaude & Cassie Werber. Biofuels Face Uncertainty in Europe- Move Aims to Ease Fears of Rising Food Prices. Sieg, David Algae biofuels at home- How to save money on gas and heating using algae. Information Specialists, Corp. Yanqun, L. Horsman, Man. Wu, N. Lan, C.Q. Dubois-Calero, N., Biofuels from Microalgae. Biotechnol. Prog, 24: Yusuf, Chisti, Biodiesel from microalgae. Institute of Technology and Engineering, Massey University. Palmerston North. Biotechnology Advances, 25: Zhao, G., Yu, J., Jiang, F., Zhang, X., Tan, T., The effect of different trophic modes on lipid accumulation of Scenedesmus quadricauda. Beijing University of Chemical Technology, Beijing. Bioresource Technology, 114:

14 APPENDICES Appendix 1 Web links for utilized equipment Air compressor, Fungi dehydrator, ProductID=PROD823 Weighing-machine, Centrifuge, Substral Gardening dung (i.e. Substral Trädgårds- & Kökslandsgödsel),

15 Appendix 2 Content of nutrient solution The nutrient solution had a content of 300 ml Substral and 5 ml of each of these mineral and vitamin stocks below.