3rd-Generation Biofuels: Bacteria and Algae as Sustainable Producers and Converters

Abstract

Biofuels have been commercialized, predominantly bioethanol, biodiesel, and biogas. Mostly, they are based on edible feedstock such as corn, maize, or soybean (so-called 1st-generation (1G) biofuels). The arising competition over arable land with food crops has caused significant debate, as well as the net contribution to climate change mitigation, where it was found that sometimes 1G biofuels perform even worse than petroleum-based fuels, due to land use change, fertilizer usage, and process yields, for instance. Biofuel research has hence targeted lignocellulosic feedstock, which exists in abundance. Due to the stability of these biopolymers, cost-effective 2G biofuels are now only at the verge of commercialization. Processes to break up the biomass into fuels are thermochemical and biochemical, using enzymes. 3G biofuels have been envisioned, where microorganisms are deployed. For instance, since algae can form up to 200 times more biomass per area than terrestrial biomass, they hold great promise for future biofuel production on marginal land or in the ocean. In this chapter, 2G and particularly 3G biofuel concepts, where bacteria and algae are used to obtain biofuels, are discussed. Standard industrial processes, like ethanol fermentation through microorganisms for regular 1G biofuels, are not covered here. Alternative biofuels from bacteria and algae, such as biomethanol or biohydrogen, are also addressed.

Full text

3
rd
-Generation Biofuels: Bacteria and Algae as Sustainable Producers and
Converters
Maximilian Lackner*
Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria
Abstract
Biofuels have been commercialized, predominantly bioethanol, biodiesel, and biogas. Mostly, they are
based on edible feedstock such as corn, maize, or soybean (so-called 1
st
-generation (1G) biofuels).
The arising competition over arable land with food crops has caused significant debate, as well as the
net contribution to climate change mitigation, where it was found that sometimes 1G biofuels perform
even worse than petroleum-based fuels, due to land use change, fertilizer usage, and process yields, for
instance. Biofuel research has hence targeted lignocellulosic feedstock, which exists in abundance. Due to
the stability of these biopolymers, cost-effective 2G biofuels are now only at the verge of commerciali-
zation. Processes to break up the biomass into fuels are thermochemical and biochemical, using enzymes.
3G biofuels have been envisioned, where microorganisms are deployed. For instance, since algae can form
up to 200 times more biomass per area than terrestrial biomass, they hold great promise for future biofuel
production on marginal land or in the ocean. In this chapter, 2G and particularly 3G biofuel concepts,
where bacteria and algae are used to obtain biofuels, are discussed. Standard industrial processes, like
ethanol fermentation through microorganisms for regular 1G biofuels, are not covered here. Alternative
biofuels from bacteria and algae, such as biomethanol or biohydrogen, are also addressed.
Keywords
Biofuels; 1
st
,2
nd
,3
rd
,4
th
generation; Biodiesel; Bioethanol; Biogas; Biohydrogen; Synthetic biology;
Metabolic engineering; Cyanobacteria; Microalgae
Introduction
Bioenergy is energy of biological origin, derived from biomass, and biofuels are fuels produced from
biomass or renewable energy sources (RESs). They can contribute to sustainable transportation and
electricity production. Sustainability is defined as creating and maintaining “the conditions under which
humans and nature can exist in productive harmony, that permit fulfilling the social, economic and other
requirements of present and future generations”(EPA 2015).
Main biofuel feedstocks, which are all produced by sun energy, are the following:
•Wood (forest management residues and fuel timber)
•Crops (annual and perennial ones, such as rapeseed and Miscanthus)
•Wastes (e.g., straw and animal manure)
•Others (e.g., algae)
*Email: maximilian.lackner@tuwien.ac.at
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Biofuels are a means of climate change mitigation, since less net CO
2eq
is emitted than from fossil fuels.
CO
2eq
(CO
2
equivalent) is the amount of CO
2
that corresponds to the same amount of radiative forcing
caused by an emitted greenhouse gas (GHG) such as CH
4
or N
2
O; it facilitates comparison.
Theoretically, biofuels close the carbon cycle (see Fig. 1below).
Other positive aspects are, e.g., less dependency on (foreign) crude oil, local and rural value generation,
and more stable fuel prices. Policy measures to support biofuel proliferation include biofuel blending
obligations and fuel standards, duty exemptions, feedstock subsidies, and R&D and investment support.
Essentially, all fossil-based fuels can be replaced by biofuels, as Fig. 2shows for the example of
petroleum-derived fuels.
Biofuels are solid, liquid, and gaseous fuels derived from renewable resources, suitable for energy
production by combustion processes for light, heat and electricity generation, and propulsion. Combus-
tion of fuels, which are predominantly fossil (e.g., coal, crude oil, natural gas), yields approx. 80 % of
global primary energy production (Lackner et al. 2013). Traditional biomass burning, e.g., of wood, has
Fig. 1 A possible carbon cycle for synthetic fuel production from biomass (Source: Inderwildi and King 2009)
Fig. 2 Substitutability of biofuels with common petroleum-derived fuels (Source: http://unctad.org/en/docs/ditcted200710_
en.pdf. Accessed 4 May 2015)
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been carried out for a long time (Miller 2013). Cheap coal and later crude oil have been increasingly used
as of 1750, with the onset of the industrial revolution. Advantages of liquid fuels such as petrol (gasoline)
and diesel are their high specific energy content (energy density), combined with ease of storage and
handling (see Fig. 3).
Natural gas can be burnt with low emissions. With depleting conventional fossil fuel reserves,
exploiting unconventional reserves has become profitable, e.g., of shale gas and tar sands. All of them
contribute to climate change, as GHGs, predominantly CO
2
and CH
4
, are being emitted into the
atmosphere.
Generations of Biofuels
Based on their feedstock, biofuels can be grouped into families: 1st-generation, 2nd-generation, and
3rd-generation (or advanced) biofuels (see Fig. 4).
As Fig. 4shows, there are many options for biofuels in terms of feedstocks and products. In Fig. 2,
HVO stands for hydrotreated vegetable oil, also named “renewable diesel fuel,”as opposed to “biodie-
sel,”which is reserved for the fatty acid methyl esters (FAME) (Aatola et al. 2008). The process of HTU
(hydrothermal upgrading) is described in Biofuels production via HTU and via pyrolysis; DMF
(dimethylfuran) is a promising alternative biofuel (Tian et al. 2011), as is DME (dimethyl ether) (Wang
et al. 2011).
1st-generation biofuels use agricultural crops. The most prominent examples are biogas, biodiesel, and
bioethanol. They are “drop in replacements,”being compatible with their fossil fuel alternatives.
Biogas (synthetic natural gas (SNG), biomethane) is obtained from anaerobic digestion of sugar and
starch (e.g., from sugar beets, maize, and wheat). It can also be collected from landfills (so-called landfill
gas). Small-scale fermenters are used by farmers, who can be self-sufficient, or produce electricity and
heat (cogeneration) with a gas engine. For details on biogas, see, e.g., Divya et al. (2015) and Deublein
and Steinhauser (2010).
Bioethanol (BioEtOH, BioEt) is also obtained by fermentation of sugars, e.g., from corn, potatoes, or
sugarcane. It can be admixed to conventional gasoline. The fuels E5 and E10 contain 5 % and 10 %
ethanol, respectively. In Brazil and the USA, bioethanol fuel is extensively used. In 2010, US and Brazil’s
usage of corn and sugarcane, respectively, made 90 % of the world’s bioethanol (Philbrook et al. 2013).
Bioethanol can be made from brown algae (Fasahati et al. 2015). A recent book on advances in bioethanol
is by Bajpai (2013).
Fig. 3 Liquid fuels are particularly interesting because of their high energy densities. LH
2
liquid hydrogen, M85 85 %
methanol in gasoline, E85 85 % ethanol in gasoline (Source: Jiang et al. 2010)
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The enlarged biofuels family
Frist generation
Mass production, low technology level
Jatropha
Sunflower
Palm
Cotton
PLANT OIL
BIOGAS
BIOETHANOL
BIODIESEL
BIOMETHANOL
BIOETHANOL
DMF HTU
F-T BIODIESEL
HVO BIOHYDROGEN
BIODME BIOETHANOL
BIODIESEL
JET
FUEL
OILGAE
Rapeseed
Coconut
Advanced Advanced
Near-commercial production, high technology level Test stage production, high technology level, high costs
Castor
Algae
Note:
Sources: UNEP, Assessing Biofuels, 2009; UN-Energy, Sustainable
Bioenergy. Framework for Decision Makers; 2007; EPA, Renewable
Fuels Standard Program Regulatory Impact Analisys, 2010;
Refuel.eu, accessed 03.03.2010; Biofuel Magazine press review,
SAE International, Hydrotreated Vegetable Oil (HVO) as a Renewable
Diesel Fuel, 2008.
1.This figure omits traditional and/or solid biofuels. It only considers
transport biofuels. The full list of crops includes more than 200
sources. Here only the most representative ones are shown.
Maize stover
Wheat stalks
Miscantus
2. Many advanced biofuels can be sourced from almost any type of
biomass. Listed here are the most common or those used in specific
production processes.
Any
Biodiesel
feedstocks
Potato peels
Sugar cane
bagasse
Soybean
Wheat
Manure
Industrial
biodegradable
waste
Residential organic
waste
Sugar beet
Sugarcane
Potato
Cassava
Sorghum
Maize
Sludge
Waste
liquor Wood chips
Beet pulp
Animal fats
Fig. 4 The family of biofuels. HVO hydrotreated vegetable oil; HTU hydrothermal upgrading, DMF dimethylfuran, DME dimethyl ether, FT Fischer–Tropsch (Source:
http://www.grida.no/publications/vg/biofuels/. Accessed 4 May 2015)
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Biodiesel is made by transesterification of plant oils (soybean, rapeseed, coconut, oil palm) with
methanol, yielding FAMEs (fatty acid methyl esters) with glycerol as by-product. Straight vegetable oil
(SVO), also called pure vegetable oils (PVOs) or pure plant oil (PPO), is produced through pressing or
extraction, including refining (filtration), but without chemical modification. It has a too high viscosity for
utilization in standard diesel engines; this is why the plant oils have to be further processed. The necessary
methanol for transesterification can be obtained conventionally or from renewable resources. Depending
on the source, biodiesel is called RME (rapeseed methyl ester), SME (soybean methyl ester), PME (palm
methyl ester), etc. all subsumed under the generic term XME. Waste cooking oil or animal fat can also be
used for biodiesel production. Higher alcohols can be used as a solvent for straight vegetable oil, the
mixture being named BM. BM was mixed with diesel fuel (D) to yield biomix diesel (BMD) (Savvidis
and Sitnik 2010). For a review on biodiesel, see, e.g., Demirbas (2010).
A major disadvantage of 1st-generation biofuels is their lack of sustainability. They are grown on arable
land, where competition with food crops drives up food prices. In 2011, the World Bank and nine other
international agencies produced a report advising governments to phase out biofuel subsidies as the use of
food stock for 1G fuel production was linked to increasing food prices (Price volatility in food and
agricultural markets: policy responses 2007).
Crop growth requires energy-intensive fertilizers, and sometimes, rainforests or other natural pieces of
land are removed to create space for farming (land use change). 1st-generation (1G) biofuels are state-of-
the-art; they are available in pure form or admixed to petroleum-based fuels. They are not considered
sustainable any more. 1G biofuels are out of the scope of this chapter. Lessons learned from 1G biofuels
are discussed in Mohr and Raman (2013).
The Role of Microorganisms
1G and 2G biofuels are made from plants and by/with microorganisms. Animals are not useful, since they
are on a higher trophic state. However, microorganisms can be deployed for biofuel production in two
ways:
1. Conversion of energetic compounds into biofuels (e.g., sugar to ethanol): 2G
2. Production of energetic compounds from sunlight (e.g., by algae): 3G
These two concepts are shown schematically in Fig. 5below.
Leaving thermal methods aside, both 2G and 3G biofuels rely on microorganisms to convert the carbon
feedstock into the desired hydrocarbon biofuels (Ruffing). Microorganisms can produce various biofuels,
Fig. 5 Process steps for (a) 2G (biomass like lignocellulosic feedstock) and (b) 3G (i.e., inorganic carbon feedstock) biofuels
(Source: Ruffing)
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such as alcohols, hydrogen, biodiesel, and biogas, from multiple starting materials (Elshahed 2010). It is
expected that microbially produced biofuels will eventually replace petroleum-based fuels as well as
today’sfirst-generation and second-generation biofuels (Singh et al. 2011). They are called “3rd-
generation biofuels”in this chapter. Also for 2G biofuels, microorganisms (or their enzymes) are used.
2G biofuels focus on lignocellulosic material, which is challenging to break up into fermentable sugars
(Faraco 2013). These 2G technologies utilize the full plant, for instance, dedicated energy crops.
Lignocellulose, which forms the major part of plants, is composed of lignin, hemicellulose, and cellulose.
In order to produce smaller fragment of these molecules, thermal methods or enzymatic methods have
been proposed (Faraco 2013). Pretreatment and subsequent enzymatic hydrolysis are applied to produce
fermentable sugars (saccharification). Fungi were also studied for the pretreatment of lignocellulose (see
Table 1).
In Philbrook et al. (2013), different end products obtained from bacterial treatment of lignocellulose are
presented.
The literature also describes “1.5G”biofuels, which are somewhere between 1G and 2G in terms of
sustainability (see Table 2for an example from China).
As Table 2shows, Kang (2014) sees 1.0G biofuels as grain based and 2.0G biofuels as cellulose based.
1.5G biofuels are non-grain based, but still rely on sugar or starch, e.g., cassava or sweet sorghum in the
case of bioethanol and nonedible oil (e.g., Jatropha) in the case of biodiesel. 3G biofuels are based on
oleaginous material derived from microorganisms (algae, yeasts, bacteria). These can grow heterotrophi-
cally on organic waste/organic feedstock (e.g., sugar) or phototrophically, i.e., using only CO
2
, sunlight,
and nutrients. Photoautotrophic growth is considered the best mode, as sunlight is being used directly,
whereas efficiency losses are encountered in case that energetic C-compounds such as sugars have to be
produced first and are then fed to the microorganisms. Algae could be 200 times more productive per unit
area than a land-based crop (http://www.cbd.int/doc/publications/cbd-ts-65-en.pdf. Accessed
4 May 2015).
Also, “4th-generation biofuels”(4G) are discussed in the literature (L€
u et al. 2011), with varying
definitions. 4G biofuels should be “carbon negative,”e.g., by including CCS (carbon capture and
storage). They are also based on microorganisms and seen as more “advanced”in terms of sustainability
and yield than 2 and 3G biofuels, yet far from commercialization. Also, concepts for 4G biofuels are
vague. 2G and 3G biofuels are where current research is focusing on.
Impact of Biofuels
When replacing fossil fuels by biofuels, one has to consider several dimensions of, e.g., farming,
processing, and use of biofuels. The impact of biofuels is conceptualized in Fig. 6below.
For instance, for crop production, land is needed, and land use change (LUC) can negatively affect the
climate (Panichelli and Gnansounou 2015), as the N
2
O emissions from fertilizer production and use
(Crutzen et al. 2008). Air pollution from biofuel combustion, e.g., SO
x
,NO
x
, and dust, has to be taken into
account as well (Lackner et al. 2013). Note: Apart from combustion, certain biofuels, after purification,
can be used in fuel cells. Another aspect is water consumption (see the concept of virtual water).
A valuable tool is life cycle assessment (LCA) or life cycle impact assessment (LCIA). Figure 7below
compares several biofuels in terms of GWP (greenhouse warming potential), smog formation, and
eutrophication (i.e., excessive fertilizer usage).
It has to be noted that the different biofuels have not yet reached their maximum sustainability potential,
as sometimes the technologies are not mature yet or economies of scale are missing. Nonetheless, a good
indication can be derived, showing, e.g., that methane from manure has a high GWP reduction potential.
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Growth Conditions of Microorganisms
Microorganisms are single-cell or multicell, microscopic living organisms. They include all bacteria and
archaea and almost all protozoa, some fungi, algae, and certain animals, e.g., rotifers. Within this chapter,
we focus on bacteria and algae.
Microorganisms have developed different strategies to obtain energy and carbon. Table 3shows these
growth conditions:
The most common heterotrophic hosts for biofuel production are Escherichia coli (bacteria from the
lower intestine of warm-blooded organisms, fecal indicator bacteria (Koschelnik et al. 2014)) and
Table 1 Fungal strains studied for pretreatment of lignocellulosic biomass
Substrate Species Findings
Duration
(days)
Bamboo Echinodontium taxodii 29 % reduction in lignin 30
Bamboo Coriolus versicolor Enhanced saccharification rate of 37 % 2
Residues
Bamboo Echinodontium taxodii 5.15-fold increase in sugar yields 120
Culms
Bamboo Trametes versicolor 8.75-fold increase in sugar yields 120
Culms
Cornstalk Phanerochaete
chrysosporium
34.3 % reduction in lignin with a maximum enzyme
saccharification of 47.3 %
15
Corn
stover
Ceriporiopsis
subvermispora
Lignin degradation readied 45 % 30
Corn
stover
Irpex lacteus CD2 66.4 % saccharification ratio 25
Corn
stover
Ceriporiopsis
subvermispora
31.59 % lignin degradation with less than 6 % cellulose loss 18
Corn
stover
Cyathus stercoreus 3- to 5-fold improvement in enzymatic digestibility 29
Wheat
straw
Basidiomycetous fungi
Euc-1
4-fold increase in saccharification 46
Wheat
straw
Irpex lacteus 3-fold increase in saccharification 46
Rice
straw
Dichomitus squalens 58 % theoretical glucose yield for remaining glucan 15
Rice
straw
Pleurotus ostreatus 39 % degradation of lignin with 79 % cellulose retention 48
Rice
straw
Phanerochaete
chrysosporium
64.9 % of maximum glucose yield from recovered glucan 15
Cotton Phanerochaete
chrysosporium
33.9 % lignin degradation with 18.4 % carbohydrate availability 14
Stalks
Cotton Phanerochaete
chrysosporium
27.6 % lignin degradation 14
Stalks
Sawdust Grifola frondosa 21 % reduction in lignin and 90 % recovery of cellulose 2
Matrix
Source: (Philbrook et al. 2013)
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Saccharomyces cerevisiae (yeast for baking and brewing) (Gendy and El-Temtamy 2013). Advantages of
these “model organisms”are the following:
•Fast growth rates
•Well-known genetics and regulation
•Availability of advanced molecular tools for genetic engineering
•Established use in the industrial setting (Gendy and El-Temtamy 2013)
Neither E. coli nor S. cerevisiae naturally produce hydrocarbon fuels, so metabolic engineering
techniques (see further below in this chapter) are needed to express their production. For a discussion
of genetically modified algae for biofuel production, see, e.g., Gendy and El-Temtamy (2013).
Table 2 1.5G biofuels are made from nonfood sugar/starch/oil crops
Fuel type
a
Main product Main feedstock Status
1.0G
biofuel
Grain-based ethanol Corn, wheat Industrialized (2004–)
Waste oil-based diesel Waste cooking oil Industrialized (2006–)
1.5G
biofuel
Non-grain, but sugar- or starch-based
ethanol
Cassava, sweet
sorghum
Industrialized (2008–)
Nonedible oil-based biodiesel/bio-jet
fuel
Jatropha Demonstration (2010–)
2.0G
biofuel
Cellulosic ethanol Corn cob
Corn stalk
Demonstration (2010–); three scaled up
(2013)
BtL Agricultural residue Research stage
Source: (Kang 2014)
a
It is not a standard but a conventional way to define biofuel types, 1.5 generation biofuels are produced by using nonfood
sugar/starch/oil crops, to separate from the first-generation food-based fuels
Fig. 6 Pathway to biofuels with inputs and environmental impact (Source: http://www.unep.org/pdf/Assessing_Biofuels-full_
report-Web.pdf. Accessed 4 May 2015)
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Methane manure, optimized
Methane manure + cosubstrate,
optimized
100 % Recycled plant oil ME FR
Ethanol whey Ch
Methanol fixed bed CH
Methanol fluidized bed CH
Ethanol sugar cane BR
Ethanol sugar beets Ch
Ethanol com us
Ethanol rye FRT
Ethanol potatoes CH
Methane sewage sludge
Methane grass biorefinery
Methane bio waste
100 % Soy ME US
100 % Palmoil ME MY
100 % Rape Me CH
Natural gas, EUR03
Diesel, low sulphur EUR03
Petrol, low sulphur EUR03
Reference ( = 100%) is petrol EURO3 in each case. Biofuels are shown in diagram at left ranked by their respective GHG emission reductions.
Production paths from waste materials or residue.
Those with GHG emissions reductions of less than 30%.
Those with GHG emissions reductions of more than 30%.
Fuels that have a total GHG emission reduction of more than 50% as versus petrol.
GWP: global warming potential, SMOG: summer smog potential, EUTR: excessive fertilizer use
100 % Soy ME ER
100 % Rape MR FER
Methane manure + cosubstrate
Methane manure
Ethanol grass CH
Ethanol wood Ch
Ethanol sweet sorghum Ch
Methane wood
100 % Recycled plant oil ME CH
GWP in %
20 40 60 80 100 100 200 200
300 300400 400
500 500
EUTR in %
SMOG in %
100
00
0120
Fig. 7 Comparison of various biofuels in terms of global warming potential (GWP), smog and eutrophication (Source: http://
www.unep.org/pdf/Assessing_Biofuels-full_report-Web.pdf. Accessed 4 May 2015)
Table 3 Microorganisms can draw their energy from sunlight or organic compounds, or both. For details, see, e.g., (Cuellar-
Bermudez et al. in press)
Mode Description Example
Photoautotrophic Light is used as a sole energy source (autotrophic
photosynthesis). CO
2
is converted into energetic
compounds through photosynthetic reactions
Algae, e.g., microalgae, cyanobacteria
Heterotrophic Only organic compounds are used as carbon and energy
source (e.g., glucose, acetate, glycerol)
Escherichia coli and Saccharomyces
cerevisiae (yeast)
Mixotrophic
(bitroph)
Organisms may employ mixotrophy obligately or
facultatively: Energy is derived from different modes.
Combinations are photo- and chemotrophy, litho- and
organotrophy, auto- and heterotrophy
Euglena
Photoheterotrophic These organisms have a metabolism in which light is
needed to use organic compounds as carbon source. The
phenomenon is also known as photoorganotrophic,
photoassimilation, or photometabolism
Purple non-sulfur bacteria, green
non-sulfur bacteria, heliobacteria
Chemotrophic Organisms that oxidize inorganic (chemolithotrophic) or
organic (chemoorganotrophic) compounds as their
principal energy source. Chemotrophs can be either
autotrophic or heterotrophic
Bacteria in “black smokers”that feed on
H
2
S, iron- and manganese-oxidizing
bacteria
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Growth of Microalgae and Cyanobacteria
“Algae”encompass microalgae, cyanobacteria (the so-called blue-green algae), and macroalgae
(or seaweed). Under certain conditions, some microalgae have the potential to accumulate significant
amounts of lipids (more than 50 % of their ash-free cell dry weight) (http://www1.eere.energy.gov/
bioenergy/pdfs/algal_biofuels_roadmap.pdf. Accessed 4 May 2015).
Microalgae (also called microphytes) are microscopic algae. Unicellular in nature, they exist indi-
vidually, in chains and in groups, with sizes between 1 and several 100 mm. In contrast to higher plants,
microalgae lack roots, stems, and leaves. They can be grown photoautotrophically, converting CO
2
into
energetic compounds using sunlight.
Cyanobacteria is a phylum
1
of bacteria that obtain their energy by photosynthesis. Their name stems
from the color of the bacteria, which are often called blue-green algae. Cyanobacteria are no “true”algae,
since cyanobacteria are prokaryotic and algae are eukaryotic cells.
Why Algae?
The biofuel yield varies geographically, with regions that provide optimum growth conditions having
advantageous productivities. Also, the chosen type of crop plays an important role (see Table 4).
Using soybeans, 73 % of the global land area (!) would be needed to cover the global oil demand,
whereas with algae, the space is significantly less, an estimated 0.3–2.7 % (Ullah et al. 2014). Consid-
erations which make microalgae attractive for 3G biofuel production are the following:
1. High productivity per km
2
2. Nonfood-based feedstock resources
3. Use of otherwise nonproductive, nonarable land
4. Utilization of a wide variety of water sources (fresh, brackish, saline, marine, wastewater)
5. Production of both biofuels and valuable coproducts
6. Potential recycling of CO
2
and other nutrient waste streams (http://www1.eere.energy.gov/bioenergy/
pdfs/algal_biofuels_roadmap.pdf. Accessed 4 May 2015.
1
A taxonomic rank below kingdom and above class in biology.
Table 4 Comparison of crop-dependent biodiesel production efficiencies from plant oils. TAGs triacylglycerols
(triglycerides). For the algae, two scenarios are shown
Plant source
Biodiesel
L/ha/year
Area to produce global oil
demand (10
6
ha)
Area required as % of
global land mass
Area as % of arable
land mass
Cotton 325 15,002 100.7 756.9
Soybean 446 10,932 73.4 551.6
Mustard seed 572 8,524 57.2 430.1
Sunflower 952 5,121 34.4 258.4
Rapeseed 1,190 4,097 27.5 206.7
Jatropha 1,892 2,577 17.3 130
Oil palm 5,950 819 5.5 41.3
Algae (10 g
2
day
1
at
30 % TAG)
12,000 406 2.7 20.5
Algae (50 g
2
day
1
at
50 % TAG)
98,500 49 0.3 2.5
Source: (Ullah et al. 2014)
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The US DOE-supported Aquatic Species Program (ASP, 1978–1996) illustrated the potential of algae
as biofuel feedstock.
In the ASP, over 3,000 species of microalgae from a diverse range of environmental habitats were
isolated and screened. The focus of the program was on eukaryotic algae, as they naturally produce
significant amounts of TAG (Ruffing).
Algae fuel, also called oilgae, is made from triglycerides (algal oil) within the algae. It is converted into
biodiesel. The processing technology is essentially the same as that for biodiesel from 2G feedstocks
(http://www.unep.org/pdf/Assessing_Biofuels-full_report-Web.pdf. Accessed 4 May 2015). Algae can
also be fermented anaerobically to yield biogas, which eliminates the need for biomass drying.
Whereas chemotrophic organisms are cultured in fermenters, phototrophic ones have to be exposed to
sunlight to harvest energy. This is achieved in various embodiments, the most common ones being open
ponds (e.g., raceway ponds) and photobioreactors (PBRs). Figure 8shows a figure of typical raceway
ponds and PBRs.
Fermentation tanks and closed photobioreactors need to be fed with CO
2
. Open ponds can take up CO
2
from the atmosphere. CO
2
fertilization will increase the yield, though. As light source, laboratory-scale
photobioreactors often use artificial installations such as LED arrays, whereas large installations need to rely
on sunlight. Energy efficiencies of the reactors shown in Fig. 8above will be discussed in more details later.
In Table 5, the two systems “open raceway pond”and “photobioreactor”are compared.
In Table 6below, the various cultivation approaches for microalgae are shown.
In heterotrophic cultivation, algae are grown without light and are fed a carbon source, such as sugars,
to produce new, higher-value biomass. This approach capitalizes upon mature industrial fermentation
technology (http://www1.eere.energy.gov/bioenergy/pdfs/algal_biofuels_roadmap.pdf. Accessed
4 May 2015).
Production cost is expected to be significantly lower in open ponds; however, PBRs are needed when
pure cultures are to be grown. Open ponds are only suitable for extremophile organisms, such as
halophiles, since else the pond would very fast become populated by other invading microorganisms.
For “grazing”by ciliates, amoeba, rotifers, and other zooplankton taxa, see, e.g., Day et al. (2012).
Fig. 8 Typical reactors to grow microalgae (Source: http://www1.eere.energy.gov/bioenergy/pdfs/algal_biofuels_roadmap.
pdf. Accessed 4 May 2015)
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Table 5 Comparison between microalgae production in open and closed bioreactors
Factor Open systems (raceway ponds) Closed systems (photobioreactors)
Space required High Low
Area/volume ratio Low (5–10 m
1
) High (20–200 m
1
)
Evaporation High No evaporation
Water loss Very high Low
CO
2
loss High Low
Temperature Highly variable Required cooling
Weather dependence High Low
Process control Difficult Easy
Shear Low High
Cleaning None Required
Algal species Restricted Flexible
Biomass quality Variable Reproducible
Population density Low High
Harvesting efficiency Low High
Harvesting cost High Lower
Light utilization efficiency Poor Good
Most costly parameters Mixing Oxygen and temperature control
Contamination control Difficult Easy
Capital investments Low High
Productivity Low 3–5 times more productive
Hydrodynamic stress on algae Very low Low–high
Gas transfer control Low High
Source: (Cuellar-Bermudez et al. in press)
Table 6 Comparative features of microalgal cultivation approaches
Advantages Challenges
Photoautotrophic
cultivation
Closed
photobioreactors
Less loss of water than open ponds
Superior long-term culture maintenance
Higher surface to volume ratio can support
higher volumetric cell densities
Scalability problems
Require temperature maintenance as
they do not have evaporative cooling
May require periodic cleaning due to
biofilm formation
Need maximum light exposure
Open ponds Evaporative cooling maintains
temperature
Lower capital costs
Subject to daily and seasonal changes
in temperature and humidity
Inherently difficult to maintain
monocultures
Need maximum light exposure
Heterotrophic cultivation Easier to maintain optimal conditions for
production and contamination prevention
Opportunity to utilize inexpensive
lignocellulosic sugars for growth
Achieves high biomass concentrations
Cost and availability of suitable
feedstocks such as lignocellulosic
sugars
Competes for feedstocks with other
biofuel technologies
Source: (http://www1.eere.energy.gov/bioenergy/pdfs/algal_biofuels_roadmap.pdf. Accessed 4 May 2015)
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In Table 7below, the productivity of several microalgae species is shown, depending on growth
scenarios.
3G biofuels based on algae and cyanobacteria are not yet commercially available yet. Today’s plants
producing microalgae focus on high-value products such as omega 3 fatty acids. Scale-up (Rogers
et al. 2014) and downstream processing (algae harvesting and dewatering (http://www1.eere.energy.
gov/bioenergy/pdfs/algal_biofuels_roadmap.pdf. Accessed 4 May 2015)) are major challenges that still
need to be overcome.
Figures 9and 10 below show simplified flowcharts on the two basic options of converting algae to
biofuels: Using the entire organism or the extracted oil.
The algae slurry can be anaerobically digested to biogas. Also, thermal methods such as pyrolysis and
gasification are available.
Table 7 Comparison of the growth characteristics and CO
2
fixation performance of microalgae strains under different CO
2
concentrations, temperature, and NO
x
/SO
x
contents. N.S. not specified
Microalgae specie CO
2
(%) Temperature (C) NO
x
/So
x
(mg L
1
)
Biomass
productivity
(mg L
1
d
1
)
CO
2
consumption
rate (mg L
1
d
1
)
Nannochloris sp. 15 25 0/50 350 658
Nannochloropsis sp. 15 25 0/50 300 564
Chlorella sp. 50 35 60/20 950 1,790
Chlorella sp. 20 40 N.S. 700 1,316
Chlorella sp. 50 25 N.S. 386 725
Chlorella sp. 15 25 0/60 1,000 1,880
Chlorella sp. 50 25 N.S. 500 940
Chlorogloeopsis sp. 5 50 N.S. 40 20.45
Chlorococcum
littorale
50 22 N.S. 44 82
Source: (Elshahed 2010)
Fig. 9 Schematic of the potential conversion routes for whole algae into biofuels (Source: http://www1.eere.energy.gov/
bioenergy/pdfs/algal_biofuels_roadmap.pdf. Accessed 4 May 2015)
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The algae oil can be processed to biodiesel through transesterification. For an integrated, cost-
optimized production of algae oil, the coproducts need to be utilized, too, which is depicted in terms of
available options in Fig. 11 below.
Fig. 10 Schematic of the various conversion strategies of algal extracts into biofuels (Source: http://www1.eere.energy.gov/
bioenergy/pdfs/algal_biofuels_roadmap.pdf. Accessed 4 May 2015)
Fig. 11 Overview of the five potential options for the recovery and use of coproducts (Source: http://www1.eere.energy.gov/
bioenergy/pdfs/algal_biofuels_roadmap.pdf. Accessed 4 May 2015)
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Five concepts to recover and use coproducts have been devised:
Option 1: Combustion for maximum energy recovery from the leftovers of lipid extraction and use of the
ash to fertilize and improve soil
Option 2: Production of food and feed supplements by recovering protein from the residues
Option 3: Recovery and utilization of nonfuel lipids and chemicals, e.g., for surfactants and bioplastics
Option 4: Recovery and utilization of carbohydrates in the algae residues and use in fermentation
processes
Option 5: After extraction of (only) fuel lipids: use of the residue as soil fertilizer and conditioner (http://
www1.eere.energy.gov/bioenergy/pdfs/algal_biofuels_roadmap.pdf. Accessed 4 May 2015.
In Kleinová et al. (2012), FAMEs prepared from algae oil TAG (Nannochloropsis and Chlorella
microalgae) were found to meet the requirements of biodiesel standard EN 14 214. However, it was
concluded that FAME from algae oil might show less oxidative stability due to higher level of
unsaturation (Kleinová et al. 2012).
A simplified process flow diagram of how to obtain biodiesel and bioethanol from algae is shown below
in Fig. 12.
Cyanobacteria
Cyanobacteria grow fast, do not need arable land, can use CO
2
, and show genetic tractability; hence, they
have a strong potential as a platform for biofuel production. They have been engineered to produce
various biofuels and biofuel-related compounds, e.g., ethanol and lipids. Challenges for advancing
cyanobacterial fuel production are improving genetic parts, carbon fixation, metabolic flux, nutrient
requirements on a large scale, and photosynthetic efficiency using natural light (see Fig. 13).
Nozzi et al. (2013) writes: “... despite years of research, eukaryotic algae have yet to realize their
industrial potential and synthetic biology techniques for eukaryotic systems remain elusive ...
Cyanobacteria, prokaryotic organisms, combine of the advantages of both eukaryotic algae, as a
photosynthetic microorganism, and E. coli, as a tractable and naturally transformable host.”
Fig. 12 Algae biofuels production approach. Biodiesel and bioethanol can be obtained from microalgal biomass, with glycerol
as by-product (Source: Cuellar-Bermudez et al. in press)
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For more information on cyanobacteria for biofuel production, see, e.g., Nozzi et al. (2013), Parmar
et al. (2011), Lu (2010), Savakis and Hellingwerf (2015), Steinhoff et al. (2014), Leite and Hallenbeck
(2014), and the section on metabolic engineering below.
Assessment of Biofuels
Energy Return Ratios (ERRs)
In order to compare the efficiencies of energy extraction and conversion systems, net energy analysis
(NEA) is carried out, yielding energy return ratios (ERRs) such as the net energy ratio (NER) or energy
return(ed) on investment (EROI) that are used (Brandt and Dale 2011). Key energy metrics are shown in
Table 8below.
NEB (net energy balance) is the difference between the total energy in the biofuel (and its coproducts)
and the total primary energy necessary to produce it. A positive net energy balance is needed for
sustainability. EROI and NER >1 correspond to a positive energy balance. FER and BF
en
are variants,
which refer the energy output to the amount of fossil or renewable energy input, respectively.
Table 9below shows some energy ratios for common 1G and 2G biofuels relative to gasoline.
The comparison of NER of microalgae biomass production in raceway ponds and photobioreactors in a
metastudy (Slade and Bauen 2013) has revealed strong differences (see Fig. 14 below).
NER is defined here as the sum of the energy used for cultivation, harvesting, and drying, divided by the
energy content of the dry biomass. As it can be inferred from Fig. 14, six out of the eight reported raceway
pond concepts have a NER <1, whereas the NER of all PBR was found to be >1. Likewise, this study has
assessed the carbon emissions, expressed in CO
2eq
, for different microalgae growth concepts (see Fig. 15
below).
In this study, the GHG emissions associated with algal biomass production were estimated by
multiplying the external energy inputs (e.g., electricity for pumps, heat energy for drying) by the default
emissions factors described in the EU renewable energy directive (RED) 2009/28/EC (European Union
2009). One can see that, based on today’s growth technologies, the CO
2eq
emissions associated with algal
Fig. 13 Challenges in cyanobacterial chemical production. (1) Improving available biological parts at each level of the central
dogma for engineering artificial pathways in cyanobacteria; (2) improving carbon fixation; (3) improving metabolic yield with
various strategies, A–eliminating competing pathways, B–improving pathway flux, for example, via irreversible steps,
C–improving tolerance to or continuous removal of the target chemical; (4) management of limited resources that may be
stressed upon scale-up; (5) photosynthetic efficiency and bioreactor design. For details on the insert, see the source: (Nozzi
et al. 2013)
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biomass production in raceway ponds are on the same level as emissions from the cultivation and
production of RME (biodiesel). By contrast, production of microalgae in PBR yields emissions that are
greater than those of conventional fossil diesel.
Table 8 Key energy metrics
Name Abbreviation Formal definition
Net energy balance NEB ∑Energyoutput ∑Energyinput
Energy return on investment EROI ∑Energy Output
∑Energy Input
Net energy ratio NER ∑Energy Output
∑Energy Input
Fossil energy ratio FER ∑Energy Output
∑Fossil Energy Input
Energy breeding factor BF
en
∑Energy Output
∑Nonrenewable Energy Input
Source: (Gupta and Tuohy 2013)
Table 9 Energy ratios for gasoline and some first- and second-generation biofuels
Overall energy ratio (OER) Fossil energy ratio (FER) Petroleum energy ratio (PER)
Liquid fuel output Liquid fuel output Liquid fuel output
Liquid fuel Fossil +biomass input Fossil input Petroleum input
Gasoline (USA) 0.81 0.81 0.91
Corn ethanol (USA) 0.57 1.4 5.0
Soy biodiesel (USA) 0.45 3.2 Not available
Cellulosic ethanol (USA) 0.45 5.0 5.0
Sugarcane ethanol (Brazil) 0.30 10 10
Source: (http://unctad.org/en/docs/ditcted200710_en.pdf. Accessed 4 May 2015)
Fig. 14 Net energy ratio for microalgae biomass production: comparison of published values with normalized values
(for direct comparison) (Source: Slade and Bauen 2013)
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Biorefinery Concept
Petrochemical refineries are highly integrated, where the entire crude oil is being used. Cracking and other
processes are used to shift the fractions of final product from fractionated distillation to the usage patterns.
The biorefinery concept builds upon the same idea, where “green crude”(e.g., algae slurry) is processed
into several products, high-value and low-value ones. A schematic concept is depicted below in Fig. 16.
For details on biorefineries, see, e.g., Kamm et al. (2010) and Fang (2013).
Gasoline Alternatives
In 2012, global petroleum consumption was estimated at 89 million barrels per day, and nearly half of it
was for producing gasoline. Our energy demand is projected to increase by more than 50 % over the next
10 years (Arifin et al. 2014). It is estimated that in 2010, the number of 1 billion vehicles was surpassed.
Today, an estimated 1.2 billion vehicles populate the world’s roads, and by 2035, it will be 2 billion
(Voelcker 2014). Most of them run on gasoline. The most common bio-based gasoline alternative is
ethanol. Apart from ethanol, several alternative fuels can be used to replace petroleum-based gasoline and
diesel; they are briefly described below (see also Table 10).
Production of these alternative fuels is possible from petrochemical and renewable resources; processes
can be thermal (e.g., pyrolysis and gasification with subsequent Fischer–Tropsch synthesis: BTL, biomass
to liquid or, alternatively, e.g., RTP, rapid thermal processing) or enzymatic (carried out in fermenters
either with enzymes or with microorganisms).
Bioethanol
Ethanol can be obtained from various carbohydrates. Starch and sugar fermentation are used industrially
(1G biofuels). Ethanol production via fermentation using glycerol as carbon source was carried out
ethanologenic Escherichia coli bacterial (Adnan et al. 2014). This process is interesting, since glycerol is
a by-product of biodiesel production. Bioethanol is widely used as renewable fuel, e.g., in Brazil.
Disadvantages are its lower energy density compared to gasoline and its hygroscopic nature.
Fig. 15 Estimates for CO
2
emissions from algal biomass production in raceway ponds (The default emissions factors used to
estimate carbon dioxide emissions were 83.80 g MJ/l (diesel), 91 g MJ/l (electricity), and 77 g MJ/l (heat). The emissions factor
for the embodied energy infertilizer and for production of PVC lining (in the case of raceway ponds) and PBR was assumed tobe
the same as for heat. Normalized system boundaries allow direct comparison of the original data (Source: Slade and Bauen 2013))
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BioMTBE, BioETBE
The oxygenate additives MTBE (methyl tertiary-butyl ether) and ETBE (ethyl tertiary-butyl ether) can be
added to gasoline in order to increase the octane rating, improve combustion efficiency, and reduce
knocking. Both additives can also be made from renewable resources. Note that MTBE is banned in
several states of the USA due to possible contamination of groundwater (What is MTBE? 2015). The
reason for MTBE’s contamination potential is its water solubility, which makes it more mobile than other
gasoline constituents.
Biomethanol
Methanol is an attractive fuel; it can be burnt or be used in fuel cells. Conversion into higher hydrocarbons
is also feasible (Olah et al. 2006). Using catalysts, methane can be chemically oxidized to methanol
(Fei et al. 2014). It can also be made from glycerol (BioMCN produces biomethanol from by-product
glycerol 2008). Renewably produced methanol can also be used for the production of biodiesel
(transesterification) and dimethyl ether (DME). For the biochemical production of bioalcohols, see also
Minteer (2011).
Biobutanol
Butanol (C
4
H
10
) is comparable to gasoline in its properties (Arifin et al. 2014). It is less corrosive than
ethanol and not hygroscopic. Due to its lower heat of vaporization, butanol-fuelled cars are easier to start
during cold weather than ones running on ethanol.
Butanol can be mixed with gasoline in any ratio, so the existing infrastructure such as pipelines
and storage facilities can be used. Butanol can be obtained petrochemically. Also, it is
accessible through carbohydrate fermentation by Clostridium acetobutylicum in a process known as
acetone–butanol–ethanol (ABE) fermentation with a product ratio of 3:6:1 (Arifin et al. 2014; Gupta and
Tuohy 2013).
Extraction
Biophotolysis
Fermentation Combustion
Gasification
Pyrolysis
Anaerobic Digestion
Pyrolysis
Gasification
Algae
Biomass
Bio-Oil
Methanol Syngas
Algae Oil
Catalytic Synthesis Fermentation
Ethylene Formaldehyde
Acetic Acid
DME:
Dimethyl
ether
Fischer Tropsch
Gasoline Wax Nafta Kerosene Diesel Transesterification
Methylacetate
Methane
Ethanol
Hydrogen
Electricity
Fig. 16 Algae biorefinery concept (Source: Cuellar-Bermudez et al. in press)
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Table 10 Fuel and physicochemical characteristics of petroleum-derived fuels and their potential substitutes
Energy
content
(MJ/L)
Solubility
a
(g/L)
Cetane
number
b
Lubricity
c
(mm
corrected
wear scar) Viscosity
d
cST Density
a
Autoignition
temperature
b
(C)
Boiling
point
a
(C)
Flash
point
a
(C)
Vapor
pressure
a
(mmHg)
Freezing
point
a
(C)
Methanol 16 Miscible 2 1,100 0.6@40 C 0.79 463 65 11 127 98
Ethanol 19.6 Miscible 11 603 1.1 @40 C 0.79 420 78 17 55 114
1-Butanol 29.2 77 17 623 1.7@40 C 0.81 343 117 29 7 90
1-Hexanol 31.7 7.9 23 534 2.9@40 C 0.81 285 158 59 1 45
1-Octanol 33.7 0.59 39 404 4.4@40 C 0.83 270 195 81 0.08 16
1-Decanol 34.6 ~0.04 50 406 6.5@40 C 0.83 255 233 108 <0.1 6
1-Dodecanol 35.3 ~0.004 64 345 9.0@40 C 0.83 275 261 119 <0.1 24
Hydrogenated
bisabolene
e
~37 Immiscible 42 Unknown 2.91 0.82 Unknown 267 111 <0.01 <78
Biodiesel 32.1 Immiscible 60 314 4–6@40 C 0.87
(avg)
177–330 315–350 100–170 <13to5
Petrodiesel
f
40.3 Immiscible 45–50 315 1.8–5.8@40 C 0.84
(avg)
210 150–350 52–96 0.4 12
Petroleum
g
32.1 Immiscible 13–17 711–1,064 0.4–0.8@20 C 0.82
(avg)
246–280 27–225 40 275–475 60
Sources: Akhtar et al. (2015), with
a
Linstrom and Mallard (2015),
b
Harnisch et al. (2013),
c
Weinebeck and Murrenhoff (2013),
d
Viswanath et al. (2007),
e
Peralta-Yahya
et al. (2011),
f
NREL (2009),
g
Louis and Arkoudeas (2012)
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Bio-Propanol
Propanol can be burnt in engines (Gong et al. 2015) and fuel cells (Markiewicz and Bergens 2010). It can
be obtained via metabolically engineered microorganisms from lignocellulose (Deng and Fong 2011),
glucose, or glycerol (Choi et al. 2012).
Biodiesel
Diesel is used in heavy engines (trucks, ships, locomotives, construction machinery). Kerosene, which is
chemically similar to diesel fuel, is deployed for aircraft propulsion in jet turbines. Table 11 below
compares conventional diesel (“summer diesel,”according to the standard EN 590) to three bio-based
diesel substitutes, HVO (hydrotreated vegetable oil), GTL (gas to liquid) fuel, and FAME (fatty acid
methyl ester, or common biodiesel). HVO is sometimes termed “renewable diesel fuel”or “green diesel”
to distinguish it from biodiesel. One can see that the three substitutes are comparable in properties.
Bio-Octanol
Another alternative fuel is 1-octanol (Akhtar et al. 2015). It is similar to diesel fuel (Akhtar et al. 2015).
Traditional industrial production of octanol proceeds by the oligomerization of ethylene using triethyla-
luminium followed by oxidation. This route is known as the Ziegler alcohol synthesis. Production from
renewable sources is feasible via microbial fermentation of organic compounds (Akhtar et al. 2015).
Bio-Jet Fuel
The global aviation industry aims to achieve carbon-neutral growth by 2020 and to cut its CO
2
emissions
by 50 % relative to 2005 levels by 2050 (International Air Transport Association (IATA) 2009).
Renewable jet fuel processes that are currently certified for use in commercial aviation include fuel
produced from a hydroprocessed esters and fatty acids (HEFA) process (also known as hydrotreated
renewable jet fuel) and biomass to liquid (BTL) via a Fischer–Tropsch (FT) process (Winchester
et al. 2013).
For details on renewable aviation fuel, see, e.g., Cremonez et al. (2015).
Table 11 Typical properties of HVO, European EN 590:2004 diesel fuel, GTL, and FAME. HFRR high-frequency
reciprocating rig
HVO EN 590 (summer grade) GTL FAME (from rapeseed oil)
Density at 15 C (kg/m
3
) 775...785 835 770...785 885
Viscosity at 40 C (mm
2
/s) 2.5...3.5 3.5 3.2...4.5 4.5
Cetane number 80...99 53 73...81 51
Distillation range (C) 180...320 180...360 190...330 350...370
Cloud point (C) 5... 25 50...25 5
Heating value, lower (MJ/kq) 44.0 =42.7 43.0 37.5
Heating value, lower (MJ/I) 34.4 35.7 34.0 33.2
Total aromatics (wt-%) 0 30 0 0
Polyaromatics (wt-%)
a
0400
Oxygen content (wt-%) 0 0 0 11
Sulfur content (mg/kg) <10 <10 <10 <10
Lubricity HFRR at 60 C(mm) <460
b
<460
b
<460
b
<460
Storage stability Good Good Good Very challenging
Source: (Aatola et al. 2008)
a
European definition including di- and tri+ -aromatics
b
With lubricity additive
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Gaseous Biofuels
Apart from biogas and synthesis gas (out of the scope of this chapter), biohydrogen is an attractive option.
Biohydrogen
Hydrogen is needed by the chemical industry, e.g., for ammonia production (Haber–Bosch process)
and methanol production (from CO). Today, approx. 95 % of H
2
is obtained from fossil fuels
(steam reforming or partial oxidation of methane, coal gasification), with electrolysis playing a less
important role.
Biohydrogen is H
2
that is produced biologically by algae, bacteria, and archaea. The carbon source can
preferentially be waste. One can distinguish between dark fermentation and photofermentation. There is a
great potential for improving hydrogen yield by metabolic engineering, i.e., the use of GMOs
(genetically modified organisms) (see also below). Algae have been proposed for biohydrogen production
(http://www1.eere.energy.gov/bioenergy/pdfs/algal_biofuels_roadmap.pdf. Accessed 4 May 2015).
An advantage of biohydrogen production is energy efficiency (Kao et al. 2014). For details on
biohydrogen, see, e.g., Demirbas (2011).
Gas to Liquids (GTL)
Biological Conversion of Natural Gas to Liquid Fuel (Bio-GTL)
Methane, which is the main constituent of biogas and natural gas, is the most common gaseous
fuel. Attempts have been made to convert it into a liquid fuel, to meet the huge demand by the
transportation sector. One approach uses certain bacteria, so-called methanotrophs (methanophiles).
These bacteria can be grown aerobically or anaerobically, and they only feed on methane as carbon and
energy source. Methanotrophs were considered for the production of vitamins, antibiotics, single-cell
protein (SCP), and carboxylic acids (Fei et al. 2014). Also an important biopolymer,
poly-b-hydroxybutyrate (PHB), which is a potential replacement for polypropylene (PP), can be produced
by methanotrophs.
Methanotrophic bacteria are a promising concept for liquid fuel production, bypassing oil-rich crops
such as rapeseed, soybean, and oil palms. A research and development roadmap for GTL with
methanotrophs is shown in Fig. 17 below.
Biofuels from Biological Wastewater Treatment (BWWT) Plants
Wastewater from households or industry contains significant amounts of energetic carbon compounds.
Processes in today’s biological wastewater treatment (BWWT) plants are mainly based on the activated
sludge process, where microorganisms oxidize organic molecules to CO
2
. In municipal wastewater, lipids
can represent more than 40 % of the total organic fraction, with the vast majority consisting of TAGs
(triacylglycerols) (Arifin et al. 2014). Specialized oleaginous bacteria could either assimilate lipids
from the wastewater or synthesize them de novo from other carbon sources and store them intracellularly
as neutral lipids, for example, TAGs, wax esters (WEs), or polyhydroxyalkanoates (PHAs)
(Arifin et al. 2014). Such a concept could be realized in a “biorefinery column”as part of a BWWT,
see Fig. 18 below.
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Other Technologies
Microbial Fuel Cells
A microbial fuel cell (MFC) is a device that converts chemical energy directly to electrical energy by the
catalytic reaction of microorganisms (Yu et al. 2012). A configuration that derives energy directly from
Fig. 17 Research and development map of Bio-GTL using methane as a substrate (Source: Fei et al. 2014)
Fig. 18 Conceptual scheme of a “biorefinery column”for biofuel production from wastewater under anaerobic conditions
using specifically enriched lipid accumulating bacterial populations. Abbreviations: FAEE fatty acid ethyl ester, FA M E fatty
acid methyl ester, HAME hydroxyalkanoate methyl ester, PHB polyhydroxybutyrate, PHB pol PHB polymerase, TAG
triacylglycerol, WE wax ester (Source: Muller et al. 2014)
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certain plants is known as a plant microbial fuel cell (PMFC). Potential applications for MFC lie in
biosensors, bioremediation, and wastewater treatment with concurrent electricity production. Electrohy-
drogenesis can be used in microbial fuel cells.
Euglena
Euglena is a genus of single-celled flagellate protists. They live in freshwater and seawater. Euglena show
properties like plants (photosynthesis) but also those of animals (motion and digestion). Euglena were
devised for jet fuel production in Japan (Alternative Jet Fuel 2015).
Archaea
Archaea are one of the three domains (kingdoms) of life, next to bacteria and eukaryota. They are single-
celled microorganisms. These microbes are prokaryotes (like bacteria), meaning that they have no cell
nucleus. Archaea were initially classified as bacteria, receiving the name archaebacterial. By contrast,
eukaryotes are organisms whose cells contain a nucleus. Many unicellular organisms are eukaryotes, such
as protozoa and algae. All multicellular organisms are eukaryotes, including animals, plants, and fungi.
Archaea could be interesting to provide special enzymes for biofuel synthesis, e.g., hyperthermophilic
cellulose (Graham et al. 2011), or thermoacidophilic enzymes (Hess 2008) to break down lignocellulose.
Flue Gas Recycling
Large point sources such as power or cement plants (the latter release 8 % of anthropogenic CO
2
(Cuellar-
Bermudez et al. in press)) end themselves for carbon capture and storage (CCS). For CO
2
capture from
industrial sources, see, e.g., (Kuckshinrichs and Hake 2014; Romano et al. 2013). CO
2
can be captured
from large point sources that burn fossil fuels, biomass can, or both. In carbon capture in processes using
biomass (Bio-CCS), a negative CO
2
balance can be achieved (see Fig. 19 below).
Concepts for the integration of power plants with algae ponds are discussed in Schipper et al. (2013).
For details on bio-CCS, see, e.g., Apel et al. (1994) and Zhao and Su (2014).
Metabolic Engineering
In metabolic engineering, genetic engineering is deployed to modify the metabolism of organisms. It can
involve the optimization of existing biochemical pathways or the introduction of pathway components,
mostly in bacteria, yeast, or plants. The goal is the high-yield production of certain metabolites (http://
www.nature.com/subjects/metabolic-engineering) such as lipids for biofuel use. Anne M. Ruffing (http://
cdn.intechopen.com/pdfs-wm/43693.pdf) writes: “Metabolic engineering is a powerful tool to improve
microbial fuel production, either through engineering the metabolic pathways within the native micro-
organism to encourage high fuel synthesis or though transferring the fuel production pathway into a
model organism for optimization.”
Genetic engineering has come under criticism for foodstuff. One can argue that for biofuel production
crops, the risks are lower. Table 12 provides an exemplary overview of hydrocarbons and fuel precursors
by genetically modified organisms (GMOs).
2G and 3G: Development of microorganisms for cellulose-biofuel consolidated bioprocessings:
metabolic engineers’tricks (Mazzoli 2012).
For a review on microbiological aspects of biofuel production, see Elshahed (2010). For details on
metabolic engineering for biofuel production, see, e.g., Ruffing (2013).
The production of biofuels by in vitro synthetic biosystems was suggested by Zhang (2014). The
concept is to achieve a high product yield, coupled with fast reaction rate, easy product separation, open
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Page 24 of 32

POWER PLANT POWER PLANT + CCS
Electricity:
2,1 TWh/a
0,2 Mt
CO2
0,2 Mt
CO2
2,3 Mt
CO2
2 Mt
CO2
1,7Mt
CO2
NET GHG effect:
0,2 Mt CO2/a
NET GHG effect:
−2 Mt CO2/a
NET GHG effect:
0 Mt CO2/a
Coal fired power plant with CCS
100% coal
Biomass fired power plant with CCS
+ 100% biomass
Biomass fired power plant
100% biomass
Coal fired power plant
100% coal
COAL
6,5 TWh/a
BIOMASS BIOMASS
Electricity:
2,7 TWh/a
Electricity:
2,1 TWh/a
Electricity:
2,6 TWh/a
Biomass
growth
absorbs CO2
from the
atmosphere
Biomass
growth
absorbs CO2
from the
atmosphere
1000 MWth
410 MWe
1000 MWth
400 MWe
1000 MWth
330 MWe
1000 MWth
320 MWe
1,7 Mt CO2/ a reduction
3,9 Mt CO2/ a reduction
1,9 Mt CO2/ a reduction
NET GHG effect
1,9 Mt CO2/a
CoalBiomass
1,9 Mt
CO2
780 kton/a
COAL
6,5 TWh/a
780 kton/a
6,5 TWh/a
1,2 Mton/ a
6,5 TWh/a
1,2 Mton/ a
Fig. 19 Principle of carbon balance when applying Bio-CCS (Arasto et al. 2014)
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Page 25 of 32

process control, broad reaction condition, and tolerance to toxic substrates (Zhang 2014), as opposed to
living “cell factories”of engineered microorganisms.
Enzymes from (extremophile) microorganisms for biofuel processing and production are out of the
scope of this chapter.
Discussion
Biofuels are one option for renewable energy; they cannot be the only one, as Fig. 20 below suggests.
Wind energy needs less space than biomass, and renewable electricity has proven to be very efficient.
Hartmut Michel writes in (http://onlinelibrary.wiley.com/doi/10.1002/anie.201200218/pdf. Accessed
4 May 2015): “Commercially available photovoltaic cells already possess a conversion efficiency for
sunlight of more than 15 %, the electric energy produced can be stored in electric batteries without major
losses. This is about 150 times better than the storage of the energy from sunlight in biofuels. In addition,
80 % of the energy stored in the battery is used for the propulsion of a car by an electric engine, whereas a
combustion engine uses only around 20 % of the energy of the gasoline for driving the wheels. Both facts
together lead to the conclusion that the combination photovoltaic cells/electric battery/electric engine
uses the available land 600 times better than the combination biomass/biofuels/combustion engine.”
Table 12 Hydrocarbon fuels and fuel precursors produced by genetically engineered microorganisms
Hydrocarbon fuel/fuel precursor Concentration range Microbial hosts
Heterotrophic production
FFA 0.5–7 g/L Escherichia coli
0.024–0.2 g/L Saccharomyces cerevisiae
TAG 20–32.6 % dcw, 0.12 g/
L
Chlamydomonas reinhardtii
0.4–0.7 g/L Saccharomyces cerevisiae
FAEE 0.07–1.5 g/L Escherichia coli
N/A Saccharomyces cerevisiae
Fatty alcohols 0.001–1.67 g/L Escherichia coli
Alkanes/alkenes 0.042–0.32 g/L Escherichia coli
Other isoprenoids (lycopene, b-carotene,
amorphadiene)
0.002–1g/L Escherichia coli
0.01 g/L Saccharomyces cerevisiae
Autotrophic production
FFA 0.11–0.20 g/L Synechocystis sp. PCC 6803
0.015–0.06 g/L Synechococcus elongatus PCC 7942
0.051 g/L Synechococcus sp. PCC 7002
TAG 28.5 % dcw Chlamydomonas reinhardtii
FAEE 0.077–0.086 g/L Synechococcus sp. PCC 7002
Fatty alcohols 200 mg/L Synechocystis sp. PCC 6803
Alkanes/alkenes 150 mg/L/OD730 Synechocystis sp. PCC 6803
0.05 g/L Synechococcus sp. PCC 7002
N/A Thermosynechococcus elongatus
BP-1
Isoprene 0.5 mg/L Synechocystis sp. PCC 6803
Source: (Ruffing 2013)
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The author argues that the most sensible utilization of biomass is for the generation of base chemicals
for syntheses purposes. He sees the future for car propulsion in electricity. However, by replacing
conventional fossil fuels with advanced biofuels in the short and medium terms, environmental benefits
can be achieved.
Biofuels have their advantage in high energy densities and compatibility with existing (liquid) fuel
handling systems. Hence, they are a viable option for the transportation sector, being an important
“ingredient”in the mix of renewable technologies.
Conclusions
Due to rising oil prices and depleting fossil fuel reserves, interest in biofuels has increased over the last
decades, with first-generation (1G) biofuels falling behind coal, oil, and gas in several performance
dimensions. Advanced biofuels (2G, 3G) can contribute to sustainable transportation. Their manufactur-
ing involves microorganisms, predominantly bacteria, yeasts, and algae. Such biofuels can replace liquid
and gaseous transportation fuels. As this chapter has outlined, current challenges are the use of lignocel-
lulosic biomass (2nd-generation biofuels), which is available abundantly, by thermal or enzymatic
methods. Hopes are placed on 3rd-generation biofuels, i.e., energetic compounds obtained from micro-
organisms like algae or cyanobacteria from sunlight. These technologies have not yet reached commercial
maturity, and metabolic engineering can support research efforts.
Outlook
Globally, biofuels now provide only 2 % of total transport fuel (http://www.cbd.int/doc/publications/cbd-
ts-65-en.pdf. Accessed 4 May 2015). Most of the approx. 30*10
9
l of biofuels that are used per year relies
on 1G technologies (Ullah et al. 2014).
The IEA (International Energy Agency) predicts that biofuels will constitute approx. 27 % of the world
transport fuel by 2050 (http://www.cbd.int/doc/publications/cbd-ts-65-en.pdf. Accessed 4 May 2015). In
the long run, once electricity storage issues have been solved, vehicles might predominantly be powered
electrically. In the meantime, replacing fossil fuels by renewable ones can help in climate change
Land required to drive 100 kilometres
Square metres
NB: Data assumes the use of fuel-cell
vehicles, with conservative estimates
for long-term cultivation for each crop.
Wind
Hydrogen from lignocellulose
Ethanol from lignocellulose
Methanol from lignocellulose
FT from lignocellulose
Ethanol from sugarbeet
RME from rapeseed
Sources: Hamelinck, C. N. and Faaij, A. P., Outlook for
advanced biofuels, Elsevier, 2005; University of Groningen,
Effective Land Use for Renewable Energy Sources, 2009
Fig. 20 Necessary land needed to generate the energy for a 100 km car trip (Source: http://www.grida.no/publications/vg/
biofuels/. Accessed 4 May 2015)
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Page 27 of 32

mitigation. Further research, particularly in 3G biofuel technology, is needed to bring promising concepts
to commercial maturity.
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