Biofuels are renewable energy sources derived from biomass, including types such as bioethanol, biodiesel, and biogas, used in various industries. The history of biofuels spans from the production of biodiesel by Rudolf Diesel in the 1890s to the commercial approval of biofuels by the EPA in 1998, with significant production growth thereafter. Methodologies for generating biofuels include anaerobic digestion, fermentation, and photobiological processes, highlighting the potential of waste biomass and microorganisms in sustainable energy solutions.

1. Biofuel

2. Introduction
 Biofuel is a type of renewable energy source derived from
biomass (microbial, plant, or animal materials). Examples: ethanol,
biodiesel, green diesel, biogas, etc.
 Biofuels can be solid (sawdust, briquettes, straw), liquid
(bioethanol, biomethanol, biodiesel), or gaseous (biogas).
 Bioethanol is made from sugarcane, corn, algae, rice straw, and
food waste.
 Biodiesel is made from vegetable oil, algal lipids, animal fats.
 Biomethane can be produced from waste organic material,
sewage, agriculture waste and domestic wastes.

3. History
 In 1890s Rudolf Diesel was the first person who made biodiesel from
vegetable oil.
 In 1970s and 1980s Environmental Protection Agency (EPA) suggested that
fuel should be free from sulphur dioxide, cabon monoxide and nitrogen
oxides.
 In 1998 EPA allowed the production of biofuel on a commercial level which
was the alternative source of petrol.
 In 2010 the production of biofuels reached up to 105 billion liters worldwide.
 In 2011, European countries were the largest that made biodiesel almost
about 53%. The International Energy Agency set a goal to reduce the usage
of petroleum and coal which will be switched on to biofuels by 2050.

4. Classification of Biofuels

5. Common Types of Biofuels

6. Application of Biofuel
 Bioethanol is mostly used as a transportation fuel and has
applications in the cosmetic and alcoholic beverage production
industry.
 Biohydrogen can be used for generating electricity, fertilizer, and
methanol production; in oil refineries for removal of impurities; as a
reducing agent, hydrogenating agent, or rocket engine fuel; and in
cryogenics, pharmaceuticals, etc.
 Biodiesel can be used in railways, aircraft, vehicles, and generators,
and is also useful for cleaning oil spills.
 Biobutanol can be used as solvents, plasticizers, and cosmetics and
has applications in textile industry.

7. Potential Sources of Biofuel

8. Feedstock Conversion Techniques
1. Anaerobic digestion (AD): It is carried out by
microorganisms for the conversion of organic waste to
produce gaseous biofuel, i.e., biogas.
2. Fermentation: It requires pretreatment and saccharification
for the release of simple sugar molecules which are then
fermented to produce liquid biofuel (ethanol).
3. Photobiological: Reactions are being explored to generate
biogas (biohydrogen) via biomass conversion using
phototrophic organisms.

9. Biohydrogen
 Hydrogen is an attractive fuel due to its high energy content of
118.7 kJ/g, which is about four times greater than that of
ethanol and over twice as high as methane.
 Different species of bacteria, cyanobacteria, green algae, and
plant produce hydrogen to dispose electrons generated during
metabolic reactions.

10. Fermentation of Waste Biomass

11. Biological Pathways to Produce Hydrogen

12. Pathways
 Biophotolysis involves splitting water using light energy and not
requiring an exogenous substrate. in the presence of a bacterial
hydrogenase and an appropriate electron carrier, molecular hydrogen
can be generated.
 Photofermentation: This process involves photosynthetic organisms
that use energy from light for the anaerobic conversion of organic
molecules to release hydrogen and carbon dioxide.
 Dark fermentation: Anaerobic fermentative bacteria convert sugar
substrate to release hydrogen along with organic acid byproducts.
Theoretically, it produces 4 moles of H2, 2 moles of acetic acid, and 2
moles of CO2.
 Microbial Electrolysis Cell: Microbes break down organic matters into
hydrogen, carbon dioxide and other products.

13. Dark Fermentation (DF)
 In the DF of glucose as the model substrate, H2-producing bacteria
initially convert glucose to pyruvate through glycolytic pathways
producing adenosine triphosphate (ATP) from adenosine diphosphate
(ADP) and the reduced form of nicotinamide adenine dinucleotide
(NADH).
 Pyruvate is oxidized to acetyl coenzyme A (acetyl-CoA), carbon dioxide
(CO2) and H2 by pyruvate ferredoxin oxidoreductase and hydrogenase.
 Depending on the type of microorganism and environmental conditions,
pyruvate may also be converted to acetyl-CoA and formate which may
be further converted into H2 and CO2.
 Acetate and butyrate are the most common products of DF.

14.  Clostridia are the main hydrogen producers in dark fermentation
systems with mesophilic mixed cultures at a pH of 5.5. E. coli can
boost hydrogen production by redirecting pathways to acetate and
butyrate.
 Clostridium butyricum primarily produces butyric acid during
fermentation, along with acetate and hydrogen.

15. Microorganisms involved in Hydrogen production
Biophotolysis Photofermentation Dark fermentation
Chlamydomonas
reinhardtii
Chlorobi-aceae Clostridia
Synechococcus elongatus Chromatiaceae Methylotrophs
Anabaena variabilis Rhodospeudomonas
palustris
Rumen bacteria
R. capsulata Methanogenic bacteria
R. Sphaeroides Archea
Rhodospirillum rubrum Escherichia coli
Ulva sp. Enterobacter
Chlorella vulgaris Citrobacter
Dunaliella tertioale Alcaligenes
Chloroccum littorale Bacillus

16. Sources
 Second generation biomass – waste biomass.
 Agricultural residues like lignocellulosic biomasses (e.g. rice
straw, wheat straw, and corn stalks), agro-industrial wastes like
those from food processing industries (e.g. olive mill wastewater
and cheese whey), effluents from livestock farms and aquatic
plants.
 Waste generated from biofuel production such as crude glycerol
de-oiled algal cake, or cotton seed cake, etc.

17. Application
 The fuel of the future because of its high energy content,
environmental friendliness of production, and also because it can
give substantial social, economic, and environmental credentials
 Hydrogen can also help address global warming and increase
pollution problems.
 Hydrogen can be used directly in combustion engines because of
its highest energy per unit weight, i.e. 143 GJ per ton among
known gaseous biofuels, or to produce electricity via fuel cell
technologies.

18. Methane

19. History
 In 1868, Bechamp noted that microbes were responsible for
methane formation from decomposing organic matter.
 In the 1890s, Omelianski suggested that microorganisms were
involved in producing acetic acid, butyric acid, and hydrogen
through biofermentation and that microbes likely facilitated the
reaction between carbon dioxide and hydrogen that generates
methane.
 In 1910, it was shown that methane is produced from the reaction
of carbon dioxide and hydrogen, as well as from the
decarboxylation of acetic acid.

20. Introduction
 Methane (CH ) is a colorless and odorless gas, the simplest
₄
alkane, and one of the most abundant greenhouse gases.
 Properties-
 Boiling Point: -161.5°C
 Melting Point: -182.5°C
 Molecular weight: - 16.04 g/mol.
 Methane is a key energy source and the main component of
natural gas, used for electricity, heating, and vehicle fuel as
biogas.

21. Microorganisms
Hydrolytic
bacteria
Acidogenic
bacteria
Acetogenic bacteria Methanogenic
bacteria
Clostridium
sp.
Escherichia Thermotoga lettingae Methanosarcina
barkeri
Bacteroides
sp.
Clostridium Syntrophaceticus
schinkii
Methanosarcina
maze
Cellulomonas
sp.
Lactobacillus Thermacetogeneum
phaeum
Methanosarcina
thermophile
Ramsay Clostridium ultunense
Pullammanappallil

22. Process

23.  Hydrolysis: Anaerobic microorganisms break down organic polymers
(biomass) into simpler substances. Complex materials like carbohydrates,
fats, and proteins are converted into sugars, fatty acids, and amino acids. Any
larger molecules that remain undecomposed will proceed to the next stage.
 Acidogenesis: Fermentative microbes convert sugars, fatty acids, and amino
acids into short-chain volatile fatty acids, producing butyric, lactic, propionic,
and valeric acids.
 Acetogenesis: In this stage, carbon and energy sources react to produce
acetate, using products and energy from earlier stages. This acetate is then
utilized in the final stage of methane production.
 Methanogenesis: The final stage of anaerobic digestion is methanogenesis,
where acetate is converted into methane, carbon dioxide, and hydrogen
sulfide.

24. Biological Methods

25. Biogas Upgrading Systems
 Hydrogenotrophic methanogens uptake substrates such as H2, CO2,
formate, and methanol-
 Methylotrophic methanogens utilize substrates like trimethylamine,
dimethyl sulfate, and methylated ethanolamine-

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