Biofuel Production from Microalgae PDF

Biofuels Production from Microalgae Prof. Khaled N.M. Elyamany,Ph.D. (ParOwn scholarship 2009-2010, USA) (CIMO scholarship 2011-2012), Helsinki, Finland) (DAAD scholarship 2012 – 2020, Germany) (JICA sholarship 2024-2025, Japan...

Biofuels Production from Microalgae Prof. Khaled N.M. Elyamany,Ph.D. (ParOwn scholarship 2009-2010, USA) (CIMO scholarship 2011-2012), Helsinki, Finland) (DAAD scholarship 2012 – 2020, Germany) (JICA sholarship 2024-2025, Japan) 27.07.2024 Fossil Fuels Fossil Fuel: A hydrocarbon fuel, such as petroleum, derived from living matter of a previous geologic time. Coal, Oil, Natural Gas 2  When Fossil Fuels are Exhausted 3 7/27/2024  Why Consider Biofuels? Environmentally friendly fuel Produced from renewable resources Save to use in diesel vehicle Reducing the global warming and GHG effect Biodiesel contains no sulfur no lead or aromatics Ayhan Demirbas, 2009: Political, economic and environmental impacts of biofuels: A review, applied energy 86, 108-117 7/27/2024 4  Microalgae Algae are photosynthetic organisms CO2 + H2O + Light Energy  Biomass Algae very diverse: microscopic to giant kelp Efficient, rapid growth, can double biomass in a day Produce 50% of oxygen but are less than 1% of all plant biomass Few species have been studied for biofuel potential 5 Storing the Sun’s Energy (Photosynthesis) What is needed – Sunlight – CO2 – Nutrients Storage of Energy – Lipids and oils – Carbohydrates http://www.veggievan.org/downloads/articles/Biodiesel%20from%20Algae.pdf  Microalgae species Oil content (% dry weight) Chlamydomonas reinhardtii 25-80 Botryococcus braunii 25-75 Chlorella vulgaris 14-22 Chlorella pyrenoidosa 46.7 Chlorella protothecoides 57.9 Chlorella emersonii 28-32 Crypthecodinium cohnii 20 Cylindrotheca sp. 16-37 Dunaliella primolecta 23 Dunaliella salina 6 Dunaliella tertiolecta 35.6 Isochrysis sp. 25-33 Monallanthus salina >20 Nannochloris sp. 20-35 Nannochloropsis sp. 31-68 Neochloris oleoabundans 35-54 Nitzschia sp. 45-47 Phaeodactylum tricornutum 20-30 Schizochytrium sp. 50-77 Tetraselmis suecica 15-23 Spirulina platensis 4-9 Spirulina maxima 6-7 Scenedemus obliqus 12-14 Scenedesmus dimorphus 16-40 Prymnesium parvum 22-38 Pleurochrysis carterae 30-50 Hormidium sp. 38 7  Microalgae Biotechnology Use of microalgae and cyanobacteria – Oil productivity exceed that of agricultural crops – Grow in aquatic medium – Grow in non-cultivable land Technological problems – Operating costs – Biomass productivity – Process stability in full outdoor conditions – Water footprint Angles et al. 2017; Del Río et al. 2017; Elsayed et al. 2017; Patil et al. 2017; Rodolfi et. al. 2009 ; Řezanka et al. 2017; Chisti 2007 27.07.2024 Biofuels Production from Microalgae Khaled Elsayed 8 Lipid biosynthesis pathway EUKARYOTIC CELL, Apr. 2010, p. 486–501 9 Packaging of TAGs Into Lipid Droplets Riekhof, Sears, and Benning, Euk. Cell, 2005 10  Biodiesel Production CH2— OCOR CH2—OH R1COOCH2 Catalyst | | | CH— OCOR2 +3 HOCH3 CH — OH R2COOCH2 | | +| CH2—OCOR3 CH2— OH R3COOCH2 Triglyceride Methanol Glycerol Methyl esters (Parent oil) (Alcohol) (FAME) Biodiesel Glycerol Chisti, 2007 11  Biodiesel feedstocks Oil content Output Land use Water Production Acid value Biodiesel (% in dry (L oil/h. (M2 footprint cost of Oil Yield (%) Raw weight year) year/kg (M3/ton) US$/L biomass) biodiesel) material Soybeans 18 636 18 4200 0.40-0.60 0.2 90 Rapeseed 41 974 12 4300 0.99 2.0 87 Sunflower 40 1070 11 6800 0.62 0.1 90 Oil palm 36 5366 2 5000 0.68 6.1 95 Castor 48 1307 9 24700 0.92-1.56 4.6 89 Microalgae* 50 97800 0.1 591-3276 3.96 -10.56 8.9 60 CT&F - Ciencia, Tecnología y Futuro - Vol. 5 Num. 2 Jun. 2013 Pag. 85 - 100 12 Strain selection ((No strains have yet demonstrated to actually exhibit improved productivities under full sunlight in outdoor cultures)) ((Low cost microalgae biomass production for fuels will require developing superior strains and low cost harvesting process and locations at favorable sites)) John R. Benemann Biotechnology and bioengineering Vol. 110. No. 9, September, 2013 7/27/2024 13 Egyptian conditions Availability of sunlight Average temperature 27 °C Availability of water resources Average sunlight (12 h/day) Availability of wind power Average wind speed is Availability of non-cultivable land 10.5 m/sec Source: Egyptian Meteorological Center, 2015 Howard Passell et al, 2013: Journal of Environmental Management,129,103-111 Biofuels Production from Microalgae Khaled Elsayed 14 27.07.2024 Algae for biodiesel 15  Hypothesis 1: It is possible to select thermo-tolerant microalgae strains able to grow in high temperature based on process oriented strategy ? 27.07.2024 Hypothesis 1 16  Our new screening strategy criteria Process oriented screening to select robust strains With high growth rate (Optical density, Cells no./L) Can tolerate temperature fluctuations(Egyptian Conditions) High lipid content (Neutral lipid(g)/Cell) 27.07.2024 Introduction 17  Sampling, Isolation and Characterization Sampling Isolation Purification Uni-algal cultures Temperature stress Promised strains Molecular identification Fig. Isolation and growing of the different isolated species on liquid media of 0.05% Wuxal. NaCl 1% (v/wt) at room temperature under normal, natural light for two weeks. 27.07.2024 Biofuels Production from Microalgae Khaled Elsayed 18  1.0 0 days Optical density at 700 0.8 7 days Results of temperature 0.6 0.4 17 days 0.2 stress (40 ºC) 0.0 E- 9 5 2B 3B 5B 6B 45 E- 3 E- 4 5 E- 8 2 a M a1 a1 a1 H3 H4 B K E- E- E- E- E- B B B E- S S E- K K K K K E- K K K K K K K Uni-algal species 6.010 1 0 0 days 7 days Cells number/L 4.010 1 0 17 days 2.010 1 0 0 K 5 K a13 K a14 K 5 K B a9 K H38 K 42 5 K B K B K B K 6B 4 a1 M Fig. : The surviving twelve species 2 3 5 SH K E- E- E- E- E- B B B E- S E- E- E- E- E- E- K showing high growth rate and lipid Uni-algal species production at 40 ⁰C. 2.010 - 1 2 0 days Average lipids (g/cell) 7 days 1.510 - 1 2 17 days 1.010 - 1 2 5.010 - 1 3 0 K Ba9 K 5 5 E- 8 K 42 K a13 K a14 K 5 K B K B K B K B K4 M K H3 1 2 3 5 6 Ba SH E- E- E- E- E- B B E- E- S E- E- E- E- K Uni-algal species 27.07.2024 Biofuels Production from Microalgae Khaled Elsayed 19  Molecular identification of the 12 microalgal resistant species Steps: 1. DNA extraction of (DNeasy Plant Mini Kit) 2. PCR reaction of the extracted DNA 3. Electrophoresis of PCR products 4. Purification of PCR products 5. Send for sequencing at GATC 20 Imaging the formed lipid vesicles as response to temperature stress Samples: Algal samples grown for 10 days at 40°C Instrument: LMS 510 Staining: NR for 20 minutes Samples (with and without NR) Centrifugation at 4000 rpm for 4 minutes 2 µl Confocal Laser Scanning Microscopy (CLSM) Examination Imaging http://www.ljmu.ac.uk/GERI/facilities/90456.htm 21 Visualization of neutral lipid bodies Lipid vesicles clearly distinguished from the rest of the chloroplast It range in size 0.2 to 1.5 µm Fig.: Confocal fluorescence microscopy study of lipid content in different algal species: A – Micractinium sp. YACCYB33, B – Synechocystis sp. PAK12, C – Pedinomonas noctilucae C34, D – Chlorella sorokiniana KLL-G018, E – Synechocystis sp. ElfSCS31, F – Chlorella variabilis NC64A, G – Synechocystis sp. PCC 6803, H –Desmodesmus pannonicus GM4n, I – Desmodesmus intermedius CCAP. 27.07.2024 Biofuels Production from Microalgae Khaled Elsayed 22  Laboratory results Optimization the different suitable conditions for Nile red assay Imaging the formed neutral lipid vesicles as a response to temperature stress Khaled N. M. Elsayed; Tatiana A. Kolesnikova; Anja Noke; Gerd Klöck (2017): Imaging the accumulated intracellular microalgal lipids as a response to temperature stress. 3 Biotech Journal. DOI: 10.1007/s13205-017-0677-x https://www.ncbi.nlm.nih.gov/pubmed/28439814 DOI:10.1007/s13205-017-0677-x 27.07.2024 Biofuels Production from Microalgae Khaled Elsayed 23  Hypothesis 3: It is possible to cultivate the 12 selected promised strains through two cultivation stages (nutrient replete stage and nutrient limited stage) in pilot scale photobioreactors as a first step towards full outdoor conditions 27.07.2024 Biofuels Production from Microalgae Khaled Elsayed 24 Pilot scale-up production General scale-up production involves transitioning the volume from Small laboratory flasks Laboratory scale photobioreactors Pilot scale photobioreactors Industrial scale photobioreactors Two stage cultivation strategy Biomass production (Nitrogen replete condition) Lipid or TAGs accumulation (Nitrogen starvation condition)  Carbon partitioning in algae Atmospheric CO2 CO2 CO2 from industrial exhaust gases CO2 in the form of soluble carbonate Triose phosphate (G3P) Lipids Protein Carbohydrate  Starch Versus TAG Accumulation From Hicks et al., Plant Physiol, 2001 From Hicks et al., Plant Physiol, 2001  Two stage cultivation strategy Two stage cultivation strategy Biomass production (Nitrogen replete condition) Lipid or TAG accumulation (Nitrogen starvation condition) 7/27/2024 28 Effect of nitrogen starvation of NaNO3 on growth rate & lipid content of the12 selected microalgae strains Used media: BG11.NaCl 1% (wt/v) Time: 18 days Measured parameters: OD(700), Cells number/L, Neutral lipids (g/cell) Only four strains survived Normal cells Starved cells (without N source) 27.07.2024 Biofuels Production from Microalgae Khaled Elsayed 29 Experimental design Two different tested stains: 1. Desmodesmus intermedius 2. Synechocystis sp. PCC 6803 Measured parameters: Optical density Cells counting Neutral lipids (Nile red assay) Temperature (°C) light intensity 30 Pilot scale cultivation Tested strains: Desmodesmus intermedius CCAP Synechocystis sp. PCC 6803 Used media: BG11.NaCl 1% (wt/v) Running time: 40 days Bioreactor: 10 Liter working volume Desmodesmus intermedius Desmodesmus pannonicus Synechocystis sp. PCC 6803 27.07.2024 Biofuels Production from Microalgae Khaled Elsayed 31  Neutral lipids extraction calculations Desmodesmus intermedius CCAP 258/36 100 g biomass (50% N) yielded 14 g neutral lipids 14% neutral lipids Synechocystis sp. PCC 6803 100 g biomass (50% N) yielded 13.5 g neutral lipids Fig.: The extraction of microalgal lipids and preparation of biogas substrate a. Extraction of neutral lipid using Hexane b. Evaporating 13.5% neutral lipids the solvent by rotary evaporator at 45 °C at 335 vacuums (mbar) 32  Biodiesel formation Desmodesmus intermedius CCAP 14% neutral lipids 18.2 g FAME Synechocystis sp. PCC 6803 (Biodiesel) 13.5% neutral lipids 9.8 g Glycerol Desmodesmus intermedius CCAP % of produced biodiesel 91% Fig.: The transestrification of Desmodesmus intermedius CCAP neutral lipids with methanol in presence of KOH as a catalyst with Synechocystis sp. PCC 6803 molar ratio percent methanol: microalgae oil 12:1 respectively. % of produced biodiesel 90% 27.07.2024 Biofuels Production from Microalgae Khaled Elsayed 33  Biogas production from microalgae Hannover University Composition Biogas CH₄ (40-75%) CO₂ (20-55%) H₂O (0-10%) H₂S, NH₃ … (0- 1%) Source: FNR: An introduction to biogas. Fachagentur für Nachwachsende Rohstoffe, e.V., 2nd edition , January 2009. 34  Anaerobic Biodegradation of Organic Matter Hydrolysis Acidification Acetic acid formation Methanogenesis Source: FNR: An introduction to biogas. Fachagentur für Nachwachsende Rohstoffe, e.V., 2nd edition , January 35 2009.  Biogas experiment design Inoculum collection Calcination process at 550 ºC Feeding step Provide nitrogen gas to keep anaerobic condition Incubation at 37⁰C Experiment time was 30 days 27.07.2024 Biofuels Production from Microalgae Khaled Elsayed 36 Biogas experimental design Fig.: Set-up of different steps during batch biogas experiment a. Determination of organic matter content, b. Filing each fermentor with inoculums (300 ml sludge), c. connecting each fermentor with sensor, d. running the experiment using DasyLab 7.0 software for data analysis. 37  Biogas production Fig.: The net accumulated methane production of both microalgae strains during the first run (before neutral Desmodesmus 224 mL CH4/gVS lipids extraction). Synechocystis 168 mL CH4/gVS * Corn silage produces 350 ml CH4/gVS after thermal pretreatment step 17% increase in case of Desmodesmus 35.6 % increase in case of Synechocystis Desmodesmus 270 mL CH4/gVS Fig.: The accumulated methane production of both Synechocystis 261 mL CH4/gVS microalgae strains during the second run (after neutral lipids extraction). *FNR:, e.V., 2nd edition , January 2009. *Herrmann et al. (2016): Optimized biogas production, Bioresource Technol 214: 328-337 27.07.2024 Biofuels Production from Microalgae Khaled Elsayed 38  % of CH4 and CO2 in produced biogas CH4 CO2 Amount [%] [%] Synechocystis 70,07 29,93 Desmodesmus 69,61 30,39 Sugar (+ve c) 69,89 30,11 Sludge (-ve) 67 33 * Silage 54.5 45.4 * Maize 55.5 44.3 * Biogas Production from Maize Grains and Maize Silage: Polish J. of Environ. Stud. Vol. 19, No. 2 (2010), 323-329 27.07.2024 Biofuels Production from Microalgae Khaled Elsayed 39  Biogas experiment results Using microalgae cake (waste) as a substrate for biogas production is possible Produced neutral lipids 14% in Desmodesmus and 13.5% in Synechocystis % of produced biodiesel is 91%, 90% in Desmodesmus and Synechocystis Khaled NM Elsayed. Paul Stopp. Corinna Lorey. Dirk Weichgrebe. Ahmed M Abdelrahman. Anja Noke. Gerd Klöck: The potential of microalgae as promised candidates for biogas production. Ready for submission (Energies Journal) 27.07.2024 Biofuels Production from Microalgae Khaled Elsayed 40 Acknowledgements Prof. Dr. Gerd Klöck Prof. Dr. Mathias Winterhalter Dr. Anja Noke Prof. Dr. Roland Benz My colleagues at Hochschule Bremen and Jacobs University Bremen 27.07.2024 Biofuels Production from Microalgae Khaled Elsayed 41  Thank you for your attention Questions ? 27.07.2024 Biofuels Production from Microalgae Khaled Elsayed 42