Happy Biofuel Day
Here is where your presentation begins
https://www.semanticscholar.org/paper/Microscopy-approaches-to-screening-oleaginous-and-Sant-Ferreira/fb1fd68d7fa6831316427564ddad95d314af23d4
Biodiesel
Lecture 2
Energetic Biotechnology
Biodiesel from �Microalgae
Microalgae are increasingly used in biodiesel production due to their high lipid content, fast growth rates, and ability to thrive in various environments. They are considered a promising alternative to traditional oil crops.
Evolution of biodiesel:
https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.970028/full
Comparison of microalgae with other feedstock
As a bioenergy source, microalgae exhibit high photosynthetic efficiency and high yields of biomass and lipid with few environmental restrictions. Microalgae can live on non-arable land, such as beaches, saline and alkali soils, and deserts, and it could also grow in wastewater and seawater.
Schematic of biomass energy production from microalgae:
https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.970028/full
Biodiesel production from lipid in microalgae is characterized by the advantages of high growth rate and lipid yield, tolerance to environmental stress, and low competition for land.
Lipid metabolic pathways in microalgae
In microalgae cell, the synthesis of fatty acid usually occurs in the plastids, and the synthesis of triacylglyceride (TAG) would occur in both of chloroplasts and endoplasmic reticulum. In contrast to phospholipids in biological membranes, TAG does not have structural roles in cells and are used to store energy and carbon. In microalgae cells, two main TAG synthesis pathways exist: the Kennedy pathway and the Monoacylglycerol pathway. In these two pathways, TAG is synthesized through esterification between acetyl-CoA and hydroxyl groups on glycerol. The biosynthesis of fatty acids begins with the carboxylation of acetyl-CoA and acetyl-CoA carboxylase (ACCase)-mediated catalysis to form malonyl-CoA. ACCase is a critical control factor in fatty acid synthesis. In microalgae cells, malonyl-CoA is first transferred to the acyl carrier protein (ACP), and then undergoes a series of acyl chain-elongation reactions. Finally, the C16 or C18 products are catalytically synthesized by multiple subunits of fatty acid synthases.
The elongation of fatty acids is terminated by two types of enzymes. First, the acyl groups in the chloroplast acyltransferase are removed by ACP, and the newly synthesized fatty acid is directly transferred from ACP to glycerol-3-phosphate (G-3-P). Second, acyl-ACP thioesterase hydrolyzes acyl-ACP and releases free fatty acids. Free fatty acids are transferred out of the chloroplast to generate glycerides.
https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.970028/full
Video (5 min):
https://www.youtube.com/watch?v=Vtq1AXrNqD4
Video (20 min):
https://www.youtube.com/watch?v=E4nITFwwtLY
Triacylglyceride (TAG) from algae is used to synthesize biodiesel through transesterification:
https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.970028/full
Production process of microalgal biodiesel:
Representation of direct and indirect transesterification
https://www.sciencedirect.com/science/article/pii/S0016236124006951
Microalgal Cultivation: (a) Open Raceway Pond (b) Tubular photobioreactor (c) Erlenmeyer flask
https://www.sciencedirect.com/science/article/pii/S0016236124006951
Video (5 min):
https://www.youtube.com/watch?v=D0IP4j6xmn0
Video (3 min):
https://www.youtube.com/watch?v=BX7DGFqscag
Video (8 min):
https://www.youtube.com/watch?v=jsuy36SqWLY
Video (6 min):
https://www.youtube.com/watch?v=8NmQeCxgYNI
Video (21 min):
https://www.youtube.com/watch?v=Vdy6F3-Gg1M
Biomass, Lipid, and Biodiesel Productivity using Microalgae under different cultivation modes
https://www.sciencedirect.com/science/article/pii/S0016236124006951
The methods of cultivation consists of three categories(a) in-situ/laboratory scale cultivation carried out in
Wastewater Cultivation: Microalgae can be cultivated in wastewater, which provides essential nutrients like nitrogen and phosphorus. This method not only reduces the cost of cultivation but also helps treat wastewater, contributing to environmental sustainability.
Video (4 min):
https://www.youtube.com/watch?v=2ityWnawjsw
Factors affecting lipid productivity
Microalgae need a variety of nutrients, including carbon, nitrogen, phosphorus, potassium, magnesium, calcium, and trace metals such as iron, manganese, boron, cobalt, copper, and molybdenum for their growth. Lipid production is greatly influenced by nutrient as well as
To obtain the high levels of lipids, the next factors
should be modified:
- light
https://www.sciencedirect.com/science/article/pii/S0016236124006951
Video (5 min):
https://www.youtube.com/watch?v=dJjpXBeZHNQ
Isolation, Inoculation and Cultivation of Microalgae
Video (5 min):
https://www.youtube.com/watch?v=EkDVq4rl30U
Video (2 min):
https://www.youtube.com/watch?v=nIKOfSGnaY8
Isolation, Inoculation and Cultivation of Microalgae
Video (5 min):
https://www.youtube.com/watch?v=lPjYyZoi3HU
Video (2 min):
https://www.youtube.com/watch?v=LpzLlGXrNJU
Main species of microalgae used in biodiesel production
Chlorella: Known for its high lipid content (up to 40% of its dry weight) and fast growth rate. Chlorella is easy to cultivate and can adapt to different environmental conditions, making it a popular choice for biodiesel production.
Plantae (Kingdom)
Viridiplantae (Subkingdom)
Chlorophyta (Phylum (Division))
Chlorophytina (Subphylum (Subdivision))
Trebouxiophyceae (Class)
Chlorellales (Order)
Chlorellaceae (Family)
Chlorella (Genus)
https://www.marinespecies.org/aphia.php?p=taxdetails&id=160576
Video (1 Min):
https://www.youtube.com/watch?v=hIrCG8sRqw0
Main species of microalgae used in biodiesel production
Nannochloropsis: Often used due to its ability to produce a high quantity of lipids (up to 50% of its dry weight) and its resilience in outdoor conditions. It is frequently grown in open ponds and photobioreactors. The algae of the genus Nannochloropsis differ from other related microalgae in that they have chlorophyll a and completely lack chlorophyll b and chlorophyll c.
Chromista (Kingdom)
Harosa (Subkingdom)
Heterokonta (Infrakingdom)
Ochrophyta (Phylum)
Eustigmatophyceae (Class)
Eustigmatales (Order)
Monodopsidaceae (Family)
Nannochloropsis (Genus)
https://www.marinespecies.org/aphia.php?p=taxdetails&id=376045
Video (4 min):
https://www.youtube.com/watch?v=TcV8HoNyk4M
Main species of microalgae used in biodiesel production
Botryococcus braunii: Notable for its exceptionally high lipid production, sometimes exceeding 70% of its dry weight. It produces hydrocarbons that are easily converted into biodiesel, but its slower growth rate poses challenges for large-scale production.
Plantae (Kingdom)
Viridiplantae (Subkingdom)
Chlorophyta (Phylum (Division))
Chlorophytina (Subphylum (Subdivision))
Trebouxiophyceae (Class)
Trebouxiales (Order)
Botryococcaceae (Family)
Botryococcus (Genus)
Botryococcus braunii (Species)
Video (5 min):
https://www.youtube.com/watch?v=7wkBIUwtDi0
Video (3 min):
https://www.youtube.com/watch?v=Gi8TSdDSqoA
https://www.marinespecies.org/aphia.php?p=taxdetails&id=160590
Main species of microalgae used in biodiesel production
Dunaliella salina: Grows well in high-salinity environments and is known for its ability to accumulate lipids under stress conditions. It is also used in biodiesel research
Plantae (Kingdom)
Viridiplantae (Subkingdom)
Chlorophyta (Phylum (Division))
Chlorophytina (Subphylum (Subdivision))
Chlorophyceae (Class)
Chlamydomonadales (Order)
Dunaliellaceae (Family)
Dunaliella (Genus)
Dunaliella salina (Species)
Under optimal growth conditions, Dunaliella typically accumulates 5-20% lipids based on its dry weight.
Under stress conditions (e.g., nitrogen starvation or high salinity), the lipid content can increase significantly, reaching up to 20-50% of its dry weight.
By manipulating cultivation conditions, especially inducing stress, Dunaliella can enhance its lipid accumulation, making it a promising candidate for biodiesel production.
Video (2 min):
https://www.youtube.com/watch?v=a7X_0walwRQ
https://www.marinespecies.org/aphia.php?p=taxdetails&id=160590
Main species of microalgae used in biodiesel production
Scenedesmus is known for its ability to accumulate significant amounts of lipids, especially under stress conditions such as nitrogen or phosphorus starvation. Lipid content can range from 20-40% under normal conditions and up to 50-55% of the dry weight when stressed.
Plantae (Kingdom)
Viridiplantae (Subkingdom)
Chlorophyta (Phylum (Division))
Chlorophytina (Subphylum (Subdivision))
Chlorophyceae (Class)
Sphaeropleales (Order)
Scenedesmaceae (Family)
Scenedesmoideae (Subfamily)
Scenedesmus (Genus)
Video (1 Min):
https://www.youtube.com/watch?v=4VhcVNyZw-4
Video (7 min):
https://www.youtube.com/watch?v=RWs68CeQyBo
https://www.marinespecies.org/aphia.php?p=taxdetails&id=160590
Monoraphidium is a genus of green algae (Chlorophyta) recognized for its potential in biodiesel production. Its small, single-celled structure and adaptability make it a promising candidate for sustainable biofuel development. Monoraphidium can accumulate lipids, ranging from 20-30% of its dry weight under normal conditions and up to 40-55% under nutrient stress conditions (e.g., nitrogen or phosphorus limitation).
Main species of microalgae used in biodiesel production
Plantae (Kingdom)
Viridiplantae (Subkingdom)
Chlorophyta (Phylum (Division))
Chlorophytina (Subphylum (Subdivision))
Chlorophyceae (Class)
Sphaeropleales (Order)
Selenastraceae (Family)
Monoraphidium (Genus)
https://www.marinespecies.org/aphia.php?p=taxdetails&id=160590
Video (7 min):
https://www.youtube.com/watch?v=P81ig3_CTKE
Main species of microalgae used in biodiesel production
Tetraselmis is a genus of green microalgae (Chlorophyta) commonly used in biodiesel production due to its high lipid content, rapid growth rate, and adaptability to various cultivation conditions. They have a cell wall made of glycoprotein instead of cellulose, making them less rigid and easier to process compared to some other microalgae.
Under standard conditions, Tetraselmis species accumulate 20-30% of their dry weight as lipids, and this can increase up to 40-50% when exposed to nutrient stress, such as nitrogen limitation.
Plantae (Kingdom)
Viridiplantae (Subkingdom)
Chlorophyta (Phylum (Division))
Chlorophytina (Subphylum (Subdivision))
Chlorodendrophyceae (Class)
Chlorodendrales (Order)
Chlorodendraceae (Family)
Tetraselmis (Genus)
Video (2 min):
https://www.youtube.com/watch?v=3Vew3G9IRUE
https://www.marinespecies.org/aphia.php?p=taxdetails&id=175584
Main species of microalgae used in biodiesel production
Phaeodactylum is a genus of diatoms. Known for its ability to accumulate lipids and grow rapidly in various conditions, Phaeodactylum, particularly the species Phaeodactylum tricornutum, is a promising candidate for sustainable biofuel development.
Phaeodactylum tricornutum can accumulate lipids ranging from 20-30% of its dry weight under normal conditions, and up to 40-55% when subjected to nutrient stress, such as nitrogen or phosphorus limitation.
Phaeodactylum cells are diatoms, meaning they have a silica-based cell wall called a frustule. The cells are elongated and can exist in different morphs (e.g., fusiform, triradiate), depending on environmental conditions.
Chromista (Kingdom)
Heterokontophyta (Phylum)
Bacillariophytina (Subphylum)
Bacillariophyceae (Class)
Bacillariophycidae (Subclass)
Bacillariophycanae (Superorder)
Naviculales (Order)
Phaeodactylineae (Suborder)
Phaeodactylaceae (Family)
Phaeodactylum (Genus)
Phaeodactylum tricornutum (Species)
https://www.marinespecies.org/aphia.php?p=taxdetails&id=175584
https://www.semanticscholar.org/paper/Identification-of-the-mechanism-of-mixotrophy-in-Villanova/f16901bf374a241b934b8cc955e24232226dfb8e
Video (2 min):
https://www.youtube.com/shorts/EB2WBRk1AyU?app=desktop
Main species of microalgae used in biodiesel production
Spirulina maxima is a cyanobacterium (often referred to as blue-green algae) known for its high protein content, rapid growth rate, and potential use in biodiesel production. While it is primarily cultivated as a superfood and dietary supplement, Spirulina has shown promise as a feedstock for biofuel due to its ability to produce lipids under certain conditions. Under standard conditions, Spirulina maxima generally contains 5-10% of its dry weight as lipids, which is lower than some other microalgae. However, under nutrient stress or altered environmental conditions, its lipid content can increase to 20-25% of its dry weight. It is a promising candidate for biodiesel production due to its resilience, adaptability to harsh environments, potential for wastewater cultivation and vivid growth.
Kingdom: Bacteria
Phylum: Cyanobacteria
Class: Cyanophyceae
Subclass: Oscillatoriophycidae
Order: Spirulinales
Family: Spirulinaceae
Species: Spirulina maxima
Video (2 min):
https://www.youtube.com/watch?v=vdXm7p9c4mE
Video (9 min):
https://www.youtube.com/watch?v=UfG8Cbpw2Po
Use light microscopy and transmission electron microscopy as tools for lipid imaging and quantification for the screening of yeast and microalgae as potential feedstock for biodiesel production.
https://www.semanticscholar.org/paper/Microscopy-approaches-to-screening-oleaginous-and-Sant-Ferreira/fb1fd68d7fa6831316427564ddad95d314af23d4
Biomass pretreatment
Pretreatment is a crucial step for the extraction of valuable compounds from microalgae. There are different pretreatment methods including, mechanical (high pressure homogenization, bead milling, ultrasonication, microwaves, hydrodynamic cavitation and pulsed electric field), thermal (steam explosion and freeze thaw cycles), chemical methods (Acidic alkaline, oxidative, and organo-solvents), biological methods (enzyme, fungi and bacteria), which are comprehensively compared. Chemical pretreatments using alkaline and acidic solutions are regarded as the most common microalgae pretreatment method, as it is straightforward, and the results are reproducible. Even though treatments by concentrated acid and base are greatly effective on cellulose hydrolysis, there are some undesirable impacts, such as toxicity, the necessity of costly non-metallic equipment, and corrosiveness.
The hydrolysis of a microalgal cell wall is the main issue in biofuel production. A cost–benefit method with a positive energy balance for cell wall hydrolysis
should be optimized.
Biological pretreatment is an environmentally friendly technique that has yielded promising results in microalgae because of the absence of lignin in their structure. Pretreatment with bacteria is one of the promising approaches of biological pretreatment. Generally, bacteria can produce enzymes required for the digestion of different biomasses; therefore, they do not encounter further problems associated with enzyme deactivation. Finding new bacterial isolates for biological pretreatment development is an essential step toward attaining an efficient and economic pretreatment method.
Pretreatment with fungi requires long incubation time (weeks to months), whereas, bacterial and enzymatic pretreatments can be completed by only a few hours to days.
Bacterial pretreatment is conducted through different approaches. The most common method is the inoculation of a specific amount of hydrolytic bacteria in microalgal biomass. In this process, bacteria are added to a culture at the stationary phase, and their morphological characteristics and color are evaluated to assess the hydrolytic process. Moreover, the released reducing sugar is measured to calculate the enzymatic activity of bacteria.
In another approach, bacteria coexist with microalgae from the beginning of the cultivation; as such, when the culture reaches the stationary phase, nutrients enhancing the growth of bacteria are added; as a result, bacterial community significantly increases by more than 100 times in cell numbers in some instances, leading to its hydrolytic activity.
The next bacteria could be used: Bacillus, Pseudomonas, Clostridium,
Flavobacterium, Alteromonas, Aeromonas, Aquimarina, Thalassobius,
Pseudobutyrivibrio etc.
https://www.microbiologyresearch.org/content/journal/ijsem/10.1099/ijs.0.02345-0
End of Lecture 2