1 st Semester. 2 nd Semester. 4 th Semester. 5 th Semester. Algal Ingredient. Biomass (Algae growth) Mixed culture. Biodiesel Production

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1 Biomass (Algae growth) Biodiesel Algal Ingredient 1 st Semester Biodiesel Production Growth Conditions Water Chemistry ph ph, DO Reactor Design & Culture 2 nd Semester Algae Sp. CSTR, Plug flow High rate Pond Biochemical Analysis 4 th Semester Chlorophyll Protein Oil Extraction Transesterification COD, TN, TP Mixed culture Pure culture 5 th Semester Lipid TODAY TSS, FSS,VSS Marine Algae 3 rd Semester Oil 17/04/2009 Fresh water algae Heterotrophic growth Carbohydrate Literature Results 1

2 Determination of Algae oil Instructors : Dr. Li Ling LIN Dr. Jack Jie-Dar Cheng Dr. Paris Honglay Chen Dr. Der-Guey LIN Advisor : Prof. Paris Honglay Chen, PhD MPH PE Speaker : Ramaraj Rameshprabu Course 9109 : 3 rd Year Seminar 17/04/2009 Sustainable Resources & Sustainable Engineering Research group 2

3 CONTENTS 1. Introduction 2. Literature Review 3. Methodology 4. Results & Discussions 5. Conclusion 17/04/2009 3

4 1. INTRODUCTION Algal oil : Biodiesel No standard method for algae oil Understand determination processes Research Purpose : Set up reliable procedure for Algae Oil determination. 17/04/2009 4

5 2. LITERATURE REVIEW No Method Process Evaluation Reference 1 Expeller/Press 2 Solvent oil extraction Without Soxhlet apparatus 3 Solvent oil extraction With Soxhlet apparatus 4 Acceptable, Reliable Supercritical Fluid /CO 2 extraction 5 Enzymatic extraction 5-2 Osmotic shock 2.6. DETERMINATION METHODS ( Table) Not complete process "pressed" out with an oil press. use solvent to extract the oil Solvent + Soxhlet apparatus supercritical fluid /CO 2 extraction uses enzymes to degrade the cell walls release cellular components Rapesee, Sunflower Rice Bran Oil1 % less compare with Soxhlet Time consuming Hexane is the best algae oil extraction Expensive, high time consuming Time consuming Very expensive Xylanase, Pectinase, Cellulase Cell wall breakdown (microbes) JAOCS, Vol. 83, no. 5 (2006) JAOCS Vol. 71, No.1(Nov 1994) Bioreso Tech, Vol 97, Issue 6, April 2006, AOAC, AOCS Acta Alimentaria, Vol. 35 (2), pp (2006) Biotech Letters,Vol 8, Number 7 / July, 1986 Biochem 42 (2003), pp Ultrasonic-assisted extraction cells walls to break and release oil Algae (cell wall breakdown) J Ind Microbiol Biotechnol (2008) 6 Fast paralysis Temp 500 C +heating rate of 600 Cs 1 57 % oil Algae Journal of Biotechnology 110 (2004)

6 3. METHODOLOGY 3.1. Algal Culture conditions 3.2. Instrumentation 3.3. Experiment process 10/01/2009 6

7 3. Methodology Sample (Rice bran preparation). Extraction (2 g of rice bran was mixed with 20 ml hexane and mixed with a vortex stirrer for 1, 2, 5 and 10 min. The oil hexane miscella was centrifuged and recovered ) in a drying oven ) Evaporation ( To evaporate the hexane Estimation (the percentage oil was measured gravimetrically after evaporating the hexane in a drying oven. Oil 17/04/2009 7

8 Method selection Find out the problems 1. Volume effect Study (1), (2)? 2. Shake Time effect Study (1), (2), (3)? 3. Sonification Study (1), (2)? Experimental results Set up the standard procedure Confirm Tests 17/04/2009 Set up the standard procedure in this study 8

9 10/01/ Hexane Volume Study (1) & (2) Sonification drying oven ). Study (1) & (2) 3. Methodology Sample (10 ml of algae) Extraction Evaporation (To evaporate the hexane in a Estimation (the percentage oil was measured gravimetrically after evaporating the hexane in a drying oven. ) Oil Hexane solvent Extraction method Shake Time effect Study (1), (2), (3) Confirm Test 6

10 4. Results & Discussions 4.1. Solvent volume effect 4.2. Shake time effect 4.3. Sonification effect 4.4. Set up the standard procedure 4.4. Confirm tests 17/04/

11 4. Results & Discussions Solvent volume effect Sampling date: 7 Jan Sample Source: Effluent of reactor 2 Sample size: 3 ml & 10 ml Solvent: Hexane Heavy shake time: 1 min 30 sec 30 sec Hexane volume (ml) Preweight (g) Weight (g) Increase the volume of Hexane Weight Preweight (g) Mean (g) Oil (g/l) 3 ml ml Improved sensitivity /04/

12 4. Results & Discussions Solvent volume effect Sampling date: 7 Jan Sample Source: Effluent of reactor 2 Sample size: 10 ml Solvent: Hexane Heavy shake time: 1 min 30 sec 30 sec Hexane volume (ml) 3 ml Preweight Increase Weight the (g) (g) volume of Hexane Weight Preweight (g) Mean (g) Oil (g/l) ml Improved sensitivity Small conclusion: 10 ml Hexane is good 17/04/

13 4. Results & Discussions Shake time effect Sampling date: 7 Jan Sample Source: Effluent of reactor 2 Sample size: 10 ml Solvent volume: Hexane 10 ml Shake time (min) Preweight (g) 2 min 5 min 10 min Weight (g) Weight Preweight (g) Increase the Shake time Improved sensitivity Mean (g) Oil (g/l) /04/

14 4. Results & Discussions Shake time effect Sampling date: 13 Jan Sample Source: Effluent of reactor 2 Sample size: 10 ml Solvent volume: Hexane 10 ml Shake time (min) Preweight (g) 10 min 15 min 20 min Weight (g) Weight Preweight (g) Increase the Shake time Improved sensitivity Mean (g) Oil (g/l) /04/

15 15 Sampling date: 13 Jan Sample Source: Effluent of reactor 2 Sample size: 10 ml Solvent volume: Hexane 10 ml Shake time (min) Preweig ht (g) Weight (g) Weight Preweight (g) Mean (g) Oil (g/l) 10 min 20 min 30 min 4. Results & Discussions Shake time effect Small conclusion: 20 min Shake time is good

16 4. Results & Discussions Sonification effect Sample treatment Mean (g) Oil (g/l) w/o Sonifer Sonifier 1 min Sonifier 3 min Sonifier 5 min Improved sensitivity Increase the Sonification time 17/04/

17 4. Results & Discussions Sonification effect Sampling date: 6 Feb Sample Source: Effluent of reactor 2 Sample size: 10 ml Solvent volume: Hexane 10 ml Sample treatment Mean (g) Oil (g/l) w/o Sonifer Sonifier 1 min Sonifier 3 min Sonifier 5 min Small conclusion: 10 min Sonifier 6 min Sonification time is reliable Sonifier 7 min Sonifier 8 min Sonifier 9 min Sonifier 10 min /04/

18 4. Results & Discussions 4.4. Set up the standard procedure 1. wash algae one time min of Sonification 3. Sample cool down to room temperature 4. Suck up 10 ml sample to test tube. 5. Centrifuge 2 min and discard the supernatant. 6. Add 10 ml of hexane in to the tube. 7. Sake 20 min 8. Let test-tubes stand for 24 hr. 9. Weight another set of test-tubes (pre-weight) 10.Suck up 8 ml hexane extract into dry test-tubes. 11.dry the test-tubes by oven 12.Weight the test-tubes 13.Formula: oil (g/l) =[Weight - Preweight (g)]/0.008 (L). 17/04/

19 4. Results & Discussions 4.4. Set up the standard procedure Sample (wash algae one time 10 min of Sonification, Sample cool down to room temperature, Suck up 10 ml sample to test tube, Centrifuge 2 min and discard the supernatant). Extraction (Add 10 ml of hexane in to the tube, Sake 20 min, Let test-tubes stand for 24 hr, Weight another set of testtubes (pre-weight), Suck up 8 ml hexane extract into dry test-tubes, ) drying oven ) Evaporation ( To evaporate the hexane in a Estimation (the percentage oil was measured gravimetrically after evaporating the hexane in a drying oven. 17/04/2009 Oil 19

20 4. Results & Discussions 4.4. Confirm test???????????? 17/04/

21 5. Conclusion 10 ml Hexane volume is good 20 min Shake time is good 10 min Sonification time is reliable 17/04/

22 Reference AOAC, (1990 & 2003). Official methods of analysis. (16th ed. ed.), Association of Official Analytical Chemists, Washington,DC. Handbook of Microalgal Culture. Biotechnology and Applied Phycology, Blackwell Science, Oxford pp /01/

23 Reference (cont.) Robert A. Andersen, (2005) Algal culture Techniques, Physiological Society of America Elsevier Academic Press, California USA. Otto Pulz, (2004) Valuable products from biotechnology of microalgae, Appl Microbial Biotechnology 65: J.M.Berg, J.L. Tymoczko, and L. Stryer, Biochemistry. 5th ed. 2002, New York: W.H. Freeman. 23/01/

24 Reference (cont.) Susan R. Mikkelsen Eduardo Corto n (2004), Bioanayltical Chemistry John Wiley & Sons, Inc., Hoboken, New Jersey. pp 11 Frederick M. Ausubel et al (2003, ) Current Protocols in Molecular Biology, John Wiley & Sons, Inc. pp /01/

25 Thank you for your attentions 17/04/2009 Sustainable Resources & Sustainable Engineering Research Group 25

26 3.1. Algal culture No. Parameter Conditions/Occurrences 1 Cultures Mixed microalgae cultures 2 Medium Filtered River Water [0.45µm] 3 Feeding Batch Feed System 4 Photobioreactor CSTR system 5 Light Intensity [Mean] [µmol-1m-2 per µa] 6 Temperature C 27.5 C [Mean] 7 ph COD 4.90 (mg/l) 9 TN 6.72 (mg/l) 10 DO 3.98 (mg/l) 11 TP 0.62 (mg/l) 12 FSS 0.05 (g/l) 13 TSS 0.11 (g/l) 13 VSS 0.06 (g/l) 15 Detention Time 10 Days 10/01/ Experiment Setup SRSE Lab, Dept of SWC;NCHU 26

27 3.1.1.Current Available Species Chlorella Oscillatoria Oedogonium Anabaena Coccochloris Phtoconis 10/01/

28 3.2. Instrumentation Sonifier Branson Sonifier Model 450 Centrifuge Hettich EBA 3S 10/01/

29 4. Results & Discussions Sonification effect Sampling date: 13 Jan Sample Source: Effluent of reactor 2 Sample size: 10 Solvent volume: Hexane 10 ml Heavy shake: 20 min, then stand about 24 hrs Sample treatment w/o Sonifer Sonifier 1 min Sonifier 3 min Sonifier 5 min Preweight (g) Weight (g) Weight Preweight (g) Mean (g) Oil (g/l) * After heavy shake was done, test tube was stood for around 24 hrs, then other steps were finished. * In order to reduce hexane loss and simplify procedure, we suck hexane 8 ml directly from upper layer of test tube to dry and weigh. 17/04/2009 So, [oil] (g/l) = Mean (g)/ (L)

30 4. Results & Discussions Sonification effect Sample treatment Preweig ht (g) Weight (g) Sampling date: 6 Feb Sample Source: Effluent of reactor 2 Sample size: 10 ml Solvent volume: Hexane 10 ml Weight Preweight (g) Mean (g) Oil (g/l) w/o Sonifer Sonifier 1 min Sonifier 3 min Sonifier 5 min /04/

31 4. Results & Discussions Sonification effect * After heavy shake was done, test tube was stood for around 24 hrs, then other steps were finished. * In order to reduce hexane loss and simplify procedure, we suck hexane 8 ml directly from upper layer of test tube to dry and weigh. So, [oil] (g/l) = Mean (g)/0.008 (L). 17/04/

32 Increase the volume of H 2 SO 4 Didn t improve sensitivity 17/04/