Bioenergy systems for the future 17 bioenergy production from second and third generation feedstocks

Bioenergy production from
second- and third-generation
feedstocks

17

F. Dalena*, A. Senatore*, A. Tursi*, A. Basile†
*University of Calabria, Rende, Italy, †Institute on Membrane Technology (ITM-CNR),
Rende, Italy

Abbreviations
ABE
ADP
ATP
BGL
CBH
CoA
ED
EDG
EMP
FAME
Fd
GHG
GRAS
HPR
IEA
LAC
LCB
LE
LiP
MnP

MSS
PNS
PPP
PSI
PSII
SHF
SSF
TAG
VFA
WEO
WO
WtE

acetone butanol ethanol
adenosine diphosphate
adenosine triphosphate
ß-glucosidases
cellobiohydrolases
coenzyme A
Entner-Doudoroff
endo-1,4-ß-glucanases
Embden-Meyerhof-Parnas pathway
fatty acid methyl ester
ferredoxin
green house gas
generally recognized as safe
hydrogen production rate
International Energy Agency
laccase
lignocellulosic biomass

ligninolytic enzymes
lignin peroxidase
manganese peroxidase
mushroom spent straw
photosynthetic nonsulfur
pentose phosphate pathway
photosystem I
photosystem II
separately hydrolysis fermentation
simultaneous saccharification and fermentation
triacylglyceride
volatile fatty acids
World Energy Outlook
wet oxidation
waste to energy

Bioenergy Systems for the Future. />© 2017 Elsevier Ltd. All rights reserved.


560

17.1

Bioenergy Systems for the Future

Introduction

In the last few years, industrial research efforts have focused on low-cost large-scale
processing for lignocellulosic feedstocks originating mainly from agricultural residues and municipal wastes or, generically, lignocellulosic biomass for bioenergy
production. Different raw materials (feedstocks) have been employed for processing

first into simple sugars and then into bioenergy (Dalena and Basile, 2014). These
feedstocks (principally crops such as sugar beet and sugarcane, corn, canola, and
appropriate land crops) used for the production of first-generation biofuels have
addressed the global markets to make more biofuels obtained from agriculture
and forestry and inventories and from nonfood crops (second generation). However,
the exploitation of these materials is in conflict with a balanced diet as competing
directly with the food; therefore, they are not so promising even if they have permission to maintain consumption water and the destruction of forests for intensive
cultivation of plants for the second-generation production cycle (Talebnia et al.,
2010; Demirbas et al., 2011).
Unfortunately, the first and the second generations have a material impact on the
use and maintenance of soils and on the expenditure of large amounts of energy that
reduce the economic advantage.
So, the primary concern of researchers interested in biofuels is to find the ideal
production cycle through the use of microorganisms. With this goal, the research is
developing biofuels obtained by a third-generation feedstock in order to minimize
greenhouse gas (GHG) emissions and disposal problems. For example, the consumption of CO2 by algae (present in the whole ecosystem) for growing allows
a removal of this substance to the air, improving the environment. Microalgae have
a high growth ratio, duplicate the population in 24 h, and can grow in salt water or
wastewater.
Studies dating back to 2011 (Kim et al., 2011; Lee and Lazarus, 2011) suggest that
in order to cut CO2 emissions, the demand for bioenergy will increase significantly by
2050. The International Energy Agency (IEA) has also suggested that the use of bioenergy is expected to triple by 2050 to about 135 EJ/yr (IEA, 2010); screenings of
potential bioenergy range from 100 to 300 EJ by 2050.
Production of energy from biomasses, that is, in the form of biodiesel or
biomethane, is one way to reduce both consumption of crude oil and environmental
pollution, because it can be mixed with the already present fuels due to its high octane
number that impedes self-ignition in the gasoline engine (Demirbas et al., 2011).
Waste-to-energy (WtE) technologies convert solid waste into various forms that
can be used to supply energy (Demirbas and Balat, 2010). Energy can be derived from
waste that has been treated and pressed into solid fuel and from waste that has been

incinerated. In fact, WtE can be used to produce biogas (CH4 and CO2), syngas (H2,
CO2, and CO), liquid biofuels (ethanol and biodiesel), or pure hydrogen.
This chapter presents the results of critical analysis of published data on applications and the potentiality of the bioenergy production from biomass treatment
products (renewable sources). In particular, the chapter is divided into two parts


Bioenergy production from second- and third-generation feedstocks

561

focused on the production processes from different biomass feedstocks. In fact, nowadays, it is possible to consider the production processes of biofuels as a function of
the raw materials and the resulting experimental conditions of the processes.
These generations are divided into three parts for two specific reasons: (a) the
different type of substrate and (b) the biofuel product (as it is shown in
Table 17.1). In the first generation, the substrate consists mainly of seeds, grains,
or simple sugars, and biofuel (mainly bioethanol) (Dias et al., 2012) is produced
by fermentation of starch or sugars; in the second generation, the substrate is mainly
composed of lignocellulosic biomass, and biofuels produced are mainly bioethanol
and biobutanol (via enzymatic hydrolysis), methanol, and biodiesel (by thermochemical processes) (Biomass Research, 2009); in the third generation, the substrates are algae, and biofuels produced are mainly biodiesel, bioethanol, and
biohydrogen (from green and blue algae) (Leite et al., 2013).
The main criteria of feedstock choose is price, hydrocarbon content, and biodegradability. Simple sugars are preferred as substrate for bioenergy production because
they can be easily and quickly decomposed by microorganisms. However, from the
economical viewpoint, feedstocks containing pure sugars are comparably expensive,
for that reason: lignocellulosic biomass is the most profitable source for bioenergy
production. However, it should be noted that for more than half a century various
materials have been suggested as feedstock for the bioenergy production. As introduced previously, these materials can be divided on three generations.
The first generation of feedstock consists in simple sugars or more complex sugars
as corn or potato starch that undergo a treatment that makes them available for subsequent conversions as shown in Fig. 17.1.
Sustainable path from this foodstuff was to use acetone-butanol-ethanol (ABE)
fermentation.

The biosynthesis of the main products of this synthesis (acetone, ethanol, and butanol) shows the same metabolic pathway from glucose to acetyl coenzyme A (acetylCoA), but it triggers in several subsequent processes. There are three major classes of

Summarization of substrates and products in first-,
second-, and third-generation biofuels

Table 17.1

Biofuels
First generation

Second generation

Third generation

Substrate,
Seeds, grains, or sugars
Product,
Bioethanol

Substrate,
Lignocellulosic biomass
Product,
Bioethanol,
biobutanol,
biodiesel

Substrate,
Algae
Product,
Bioethanol,

biodiesel,
biohydrogen


562

Bioenergy Systems for the Future

CH2OH

CH2OH

O

CH2OH

O

OH

OH

OH

O

OH

O

OH

OH

O

OH

HO
HO

OH
300–600

O
OH
OH

OH

Fig. 17.1 Conversion of complex sugars to simple sugars.

products during fermentation process: (i) solvents (acetone, ethanol, and butanol),
(ii) organic acids (acetic, lactic, and butyric acids), and (iii) gases (CO2 and H2)
(Datta and Zeikus, 1985).
First generation of the starting materials for bioenergy production has the main pitfalls: The feed is foodstuff; therefore, alternative processes based on nonfood organic
substrates should be applied for noncompetitive way of these production processes.
This comes with the second and the third generations of starting materials that will
be discussed in this review chapter.

17.2

ABE process

As introduced above, the principal production process for the bioenergy production is
the ABE.
Nowadays, this process is adapted for the optimization of the two products that are
considered the fuels of the future, biobutanol and bioethanol. This process tends to
transform simple sugars in acetone, butanol, ethanol (in stoichiometric ratio of
6:3:1), H2, CO2, and a mixture of butyric, lactic, and acetic acid (as by-products)
(Liu et al., 2005) as shown in Fig. 17.2.
The alcohols produced in this way could be used as biofuel or reagents for subsequent chemical processes. In the field of biofuels, biobutanol, resulting from the ABE
process, is considered a valid substitute for bioethanol. The advantages related to the
use of biobutanol compared with bioethanol (the main product of the first-generation
fuels) are multiple both from the point of view of energy, compatibility with the
motors and with the distribution systems, and from the point of view of agroforestry
resources.
n-Butanol was produced starting from 1916 mainly as a solvent to feedstock. Compared with the methanol and ethanol, it is a more complex alcohol with significant
advantages: higher heat value (biobutanol has 110,000 BTU/gal, while bioethanol
CH2OH

CH2OH

O

CH2OH

O

OH

OH

O

OH
OH

O
OH

OH

O

OH

OH
300–600 OH

HO
HO

O
OH

Acetone + Butanol + Ethanol + H2 + CO2

OH

Fig. 17.2 From cellulose to acetone, butanol, and ethanol.


Bioenergy production from second- and third-generation feedstocks

563

only has 84,000 BTU/gal), low volatility, higher viscosity, and high concentrations in
mixing with other fuels from petroleum distillates, and it allows a reduction of NOx
emissions (Chen et al., 2009).
The limitation of the ABE process could be a self-inhibition caused by some
bacteria. The process could become more efficient through a genetic modification
of bacteria used or a specific search for other bacteria strain more tolerant to the
production cycle. The highest production capacity of n-butanol by bacterial fermentation is of 3.0 wt%.
The most common substrate for the ABE fermentation is lignocellulosic material
(mainly starch or simple sugars for the production of the first-generation biofuels),
which are converted to glucose following acid/enzyme hydrolysis. But at the same
time, by means of other pretreatments (as described below), it can still produce
glucose, the starting substrate of the ABE fermentation. Therefore, the ABE process
leads to the formation of n-butanol for both substrate composed of simple sugars (first
generation) and substrates composed of lignocellulosic biomass (second generation).
Despite the fact that simple sugars are more easily used to convert into biofuels, their
cost does not allow a large-scale use in industry. The use of biomass (second generation) has the dual advantage of being cost-effective than compared with the
first-generation feedstock and of being able to transform biomasses. Additionally,
lignocellulosic biomass can be supplied on a large-scale basis from different low-cost
raw materials such as municipal and industrial wastes and wood and agricultural
residues (Cardona and Sanchez, 2007).

17.2.1 From substrate to biofuel in ABE process
Lignocellulosic materials constitute a substantial renewable substrate for bioethanol

production in the ABE process. These cellulosic materials also contribute to environmental sustainability (Demirbas, 2003).
Lignocellulose is composed of three parts: cellulose (30%–50%), hemicellulose
(15%–35%), and lignin (10%–20%) (Petersen, 1984). Lignin and cellulose are very
difficult components to degrade, although both are rather heterogeneous polymers
and differ considerably depending on their origin. Lignin is an aromatic and rigid biopolymer with a molecular weight of 10,000 Da bonded via covalent bonds to xylans
(hemicellulose portion) conferring rigidity and high level of compactness to the plant
cell wall (Mielenz, 2001). Hemicellulose is an amorphous and variable structure
formed of heteropolymers including hexoses (D-glucose, D-galactose, and
D-mannose) and pentose (D-xylose and L-arabinose) and may contain sugar acids
(uronic acids), namely, D-glucuronic, D-galacturonic, and methylgalacturonic acids
(McMillan, 1994; Ranjan and Moholkar, 2013). Pentoses and hexoses are relatively
easy to hydrolyze, but in raw material, these molecules are protected from hydrolysis
by a complex linkage with lignin and cellulose. Glucose (simple sugars), hemicellulose, and cellulose are converted into pyruvate through three different pathways.
Glucose (6C) is, initially, phosphorylated to glucose-6-phosphate, which is subsequently converted to pyruvate (3C) via Embden-Meyerhof-Parnas (EMP) pathway
through some intermediates:


564

Bioenergy Systems for the Future

Glucose ! Glucose À 6 À phosphate ! Pyruvate

(17.1)

Other fermentation substrates contain hemicellulose or cellulose (e.g., fibrous biomass such as rice straw or wheat straw). Hemicellulose is converted in xylose, via
pentose phosphate pathway (PPP), and produces fructose-6-phosphate (intermediate
in the pathway that starts from glucose), and after, via EMP pathway, it can be
converted in pyruvate:
Hemicellulose ! Xylose ! PPP pathway ! Fructose À 6 À phosphate

! EMP pathway ! Pyruvate

(17.2)

When the substrates contain cellulose, instead, become glucose via cellulose hydrolysis that follow the metabolic pathway in the same manner as it earlier stated (Ranjan
and Moholkar, 2012),
Cellulose ! Glucose ! Pyruvate

(17.3)

In all the described cases, the final product of the first part of the reactions is the pyruvate that allows to produce ethanol, acetone, and n-butanol, as described in Fig. 17.3.

Pyruvate
Pyruvate Ferredoxin
Oxidoreductase
Aldehyde/alcohol
dehydrogenase
Acetate

Acetyl-CoA
Tiolase

Ethanol (1)
AcetoacetylCoA:acetate
/butyrate:CoA
transferase

Acetoacetyl-CoA

Acetone (6)

1. NADH+3-Hydroxybutyryl-CoA
Dehydrogenase;
2. Crotonase;
3. NADH + butyryl-CoA
dehydrogenase

Butyrate

Butyryl-CoA

Aldehyde/alcohol
dehydrogenase

Fig. 17.3 Schematic representation of the ABE process.

n-Butanol (3)


Bioenergy production from second- and third-generation feedstocks

565

At this point, the pyruvate-ferredoxin oxidoreductase (PFOR) enters into function
that cleaves pyruvate resulting from glycolysis, in the presence of coenzyme A, to produce carbon dioxide and acetyl-CoA, by converting the oxidized ferredoxin simultaneously in its reduced form (Menon and Ragsdale, 1997). In order to have a
quantitative view, the ferredoxin of clostridia produces 1 mol of each acetyl-CoA
and CO2 per mole of ferredoxin reduced. This process sees the consequent transfer
of two electrons (Uyeda and Rabinowitz, 1971).
Conversion of acetyl-CoA to acetate is permitted by the enzyme phosphate
acetyltransferase and acetate kinase, whereas conversion of butyryl-CoA to butyrate

is catalyzed by the enzyme phosphate butyltransferase and butyl kinase.
The next step of the process occurs in acid condition (solventogenic phase), and the
products of the preceding acidogenic phase are reassimilated and converted to acetone
and n-butanol.
The enzyme catalyzing this conversion is Co-A transferase, which converts CoA
from acetoacetyl-CoA either to acetate forming acetyl-CoA or to butyrate resulting in
butyryl-CoA.
Out of these, acetyl-CoA can be converted to acetone, butanol, and ethanol,
whereas butyryl-CoA can only be converted to butanol (Ranjan and Moholkar, 2012).

17.3

Second generation feedstocks

Despite the advantages of the conversion of simple sugars or starches (i.e., biodegradability), the first-generation biofuels have the highest carbon footprint compared with other generations of biofuel. The production technologies adopted for
the production of first-generation biofuel are inclusive of transesterification process
for biodiesel production and fermentation process for bioethanol production.
However, the physical characteristics of the raw biomass could greatly affect the
efficiency of the conversion processes. To overcome this problem, more complicated lignocellulosic biomass processing technologies, such as thermochemical
and biological conversion processes, are employed to produce second-generation
biofuel (Liew et al., 2014).
These biofuels, also called simply biofuels, are produced by processing biomass
and include bioethanol and biodiesel that can be used in vehicles and in industrial
process.
In fact (as shown in Fig. 17.4), despite the substrates are different, the conversion in
acetone, butanol, and ethanol is the same, once transformed into simple sugars. This
conversion is managed by ABE fermentation in both generation feedstocks.
Therefore, the conversion of lignocellulose to monomeric fermentable sugars in the
nature is a quite prolonged process. In order to receive enough amounts of fermentable
sugars, it is necessary to use pretreatment methods for the destruction of interconnections in the lignocellulosic biomass and cellulose and hemicellulose hydrolysis.

Owing to the structural complexity of the lignocellulosic matrix, biofuel production from biomasses requires at least three major unit operations including
pretreatment, hydrolysis, and fermentation.


566

Bioenergy Systems for the Future

1st Generation

Starch
(corn, potato)

2nd Generation

Sugars
(sugarcane)

Lignocellulosic
biomasses

Pretreatment

Simple sugars

ABE fermentation

Acetone

Butanol

Ethanol

Fig. 17.4 Conversion of different types of feedstocks into acetone, butanol, and ethanol.

17.3.1 Pretreatment of lignocellulosic biomasses
In general, there are four typical pretreatment processes: physical, biological, chemical, and combinatorial pretreatment (physiochemical and biochemical) conversion
(Agbor et al., 2011). The choice of the pretreatment method mainly depends on
physical-chemical properties of lignocellulosic biomass, and it is fundamental for
optimal successful hydrolysis and, consequently, to transform lignocellulosic polymer
units in the monomeric units of simple sugars. The overall efficiency of the pretreatment process is correlated to a good balance between low inhibitor formation
and high substrate digeribility. The goal of any pretreatment is characterized by
several criteria: reducing the degree of polymerization of the lignocellulosic chain,
preserving the pentose (hemicellulose) fractions, limiting the formation of degradation products that inhibit the growth of the fermentative microorganism, and minimizing energy demands and limiting cost (Council National Research, 2000).

17.3.1.1 Physical pretreatment
These methods can be of two types: mechanical comminution and pyrolysis. The
objective of the mechanical pretreatment is a reduction of particle size and crystallinity of lignocellulose in order to increase the specific surface the degree of polymerization. This can be produced by a combination of chipping, grinding, or milling


Bioenergy production from second- and third-generation feedstocks

567

depending on the final particle size of the material (10–30 mm after chipping and
0.2–2 mm after milling or grinding) (Alvira et al., 2010; Mosier et al., 2005). The
power requirement of mechanical comminution depends on the final particle size
and the waste biomass characteristics (Cadoche and Lopez, 1989). Instead, pyrolysis
treated biomasses at temperature greater than 300°C; cellulose rapidly decomposes to
produce gaseous products and residual char (Kilzer and Broido, 1965). The decomposition is much slower, and less volatile products are formed at lower temperatures.

Mild acid hydrolysis (H2SO4 1 N, 97°C, 2.5 h) of the residues from pyrolysis pretreatment has resulted in 80%–85% conversion of cellulose for reducing sugars with
more than 50% glucose (Fang et al., 1987; Balat, 2011).

17.3.1.2 Chemical pretreatment
Chemical pretreatment employs different chemicals such as acids, alkalies, and oxidizing agents. Among these methods, dilute acid pretreatment using H2SO4 is the most
widely used. Depending on the type of chemical used, pretreatment could have different
effects on lignocellulose structural components. Alkaline pretreatment, ozonolysis,
peroxide (both techniques that used oxidizing agents), and wet oxidation (WO) pretreatments are more effective in lignin removal, whereas dilute acid pretreatment is more
efficient in hemicellulose solubilization (Galbe and Zacchi, 2002).

17.3.1.3 Physical-chemical pretreatment
The solubilization of lignocellulose components depends on temperature, pH, and
moisture content. In lignocellulosic materials such as wheat straw, hemicelluloses
are the most thermal-chemically sensitive fraction. Hemicellulose compounds start
to solubilize into the water at temperature higher than 150°C, and among various components, xylan can be extracted the most easily (Sun and Cheng, 2002; Hendriks and
Zeeman, 2009). There are different types of solubilization of hemicelluloses by
physical-chemical production. Every type employs the characteristics of pressure
and temperature. The most useful method uses the explosion of CO2 to separate
the hemicellulose, that is, to reduce polymeric chains of glucosidic compounds to
most simple and fractionable sugar. Conventional mechanical methods require
70% more energy than physicochemical pretreatments to achieve the same amount
of sugar reduction. These methods are useful principally for agricultural residues,
but they are less effective for softwoods due to the low content of acetyl groups in
the hemicellulosic portion (Balat, 2011; Clark and Mackie, 1987).

17.3.1.4 Biological pretreatment
Biological pretreatment comprises using microorganisms, such as brown-, white-, and
soft-rot fungi, and seems to be, in our opinion, more effective than the other pretreatments. The abovementioned pretreatment methods are harsh and cost-energy intensive; on the contrary, biological pretreatment processes are mild and environmental
friendly.

568

Bioenergy Systems for the Future

Microbial pretreatment consists of a solid-state fermentation process in which
microorganisms grow on the lignocellulosic biomass selectively degrading lignin
(and in some cases hemicellulose), while cellulose is expected to remain intact.
For their heterotrophic character, these organisms are not able to produce the sugars
necessary for the production of biofuels in an autonomous way such as bacteria or
algae (third generation), but they are the perfect helpers in the degradation of lignocellulosic substrates and lipid accumulation.
The main fungi used as a pretreatment in the conversion of lignocellulosic biomass
into fermentable sugars are white-, brown-, and soft-rot fungi. Brown rots mainly
attack cellulose, whereas white- and soft-rot fungi are usually preferred for the high
selectivity in lignin degradation over cellulose loss (Wan and Li, 2012).
Lignin degradation by white-rot fungi, the most effective for biological pretreatment of lignocellulosic materials, occurs through the action of lignin-degrading
enzymes such as peroxidases and laccases (Kumar et al., 2009).
White-rot microbes typically secrete lignin peroxidases (as exposed below), along
with various types of glycosyl hydrolases that cleave the C-C lignin backbone in the
presence of hydrogen peroxide. Other enzymes involved in aerobically catalyzed
lignin degradation include Mn-dependent peroxides, laccases (monophenol oxidase),
and superoxide dismutase (Leonowicz et al., 1999).
In the oxidation part of lignin, the ligninolytic enzymes (LE) are laccase (LAC)
(EC 1.10.3.2), lignin peroxidase (LiP) (EC 1.11.14), and manganese peroxidase
(MnP) (EC 1.11.13) (Leonowicz et al., 1999; Novotny´ et al., 2004; Wan and Li, 2012).
Laccase is a copper binder enzyme, with four copper atoms in the active sites,
which utilizes molecular oxygen to carry out reactions of oxidation with phenolic
rings to produce phenoxy radicals; in particular, it catalyzes the removal of an electron
and a proton from phenolic hydroxyl and aromatic amino groups, to form free
phenoxy radicals and amino radicals (Hatakka, 1994; Leonowicz et al., 2001; Wan

and Li, 2012) as shown in Fig. 17.5.
LiP is a hemeprotein that needs hydrogen peroxide H2O2 from other enzymes to be
active. It catalyzes the oxidation of nonphenolic aromatic lignin moieties and similar
compounds, by one-electron oxidation of the aromatic ring (Leonowicz et al., 1999,
2001; Wan and Li, 2012; Wesenberg et al., 2003). The role of LiP in ligninolysis could
be the further transformation of lignin fragments that are initially released by MnP
(Wesenberg et al., 2003) (Fig. 17.6).
MnP is glycosylated glycoproteins with an iron protoporphyrin IX prosthetic group
that oxidizes different phenolic compounds, thanks to the oxidation of Mn2+ to Mn3+
(Leonowicz et al., 1999, 2001; Wan and Li, 2012; Wesenberg et al., 2003). The final
effect of these enzymes is to initiate wood decay and facilitate the penetration of
hydrolytic enzymes into cellulosic and hemicellulosic substrates. The enzymatic reaction should be described by the same mechanism of the LiP (Fig. 17.6).
Some of the best white-rot fungi in the degradation of lignin are Phanerochaete
chrysosporium, Ceriporiopsis subvermispora, and Daedalea flavida. (Maurya
et al., 2015; Wan and Li, 2012). Biological pretreatment by white-rot fungi has been
added together with organosolv pretreatment in an ethanol production process by
simultaneous saccharification and fermentation (SSF) from beech wood chips


Bioenergy production from second- and third-generation feedstocks

Reduced
laccase

Cu+

HO O

+
O Cu

H H

Cu2+O Cu2+
H
Cu+
H2O

Cu+

Cu+
HO O
Cu2+ Cu2+
O

Cu2+
H O
Cu2+ Cu2+
O
H
Cu2+

Peroxide-level
intermediate

H O
Cu2+ Cu2+
O

Cu2+O Cu2+

H
Cu2+

Native
intermediate

Resting
enzyme

Cu+

H2O

4 AH

4 A•

Cu2+
H O

Cu+

Cu2+O Cu2+
Resting
enzyme

Native
intermediate

Cu2+

Cu2+
H O

H
Cu2+

Peroxide-level
intermediate

Cu+

O2

Cu+

H
Cu+

569

H

H
Cu2+

+
O Cu
H
Reduced

Cu+ laccase

Fig. 17.5 Laccase utilization of molecular oxygen to carry out reactions of oxidation with
phenolic rings to produce phenoxy radicals.
Native
peroxidase
N

N

3+

Compound I

[R-OOH]
H2O2

O

H2O
N

Fe
N
N

N

4+

Fe
N

•+

N

O2– •
[R-OH]
H 2O
Product, radical
(R• + H+ or Mn3+)

Substrate
(RH or Mn2+)

H+
O
N

N

Fe
N

4+

O

Substrate

(RH or Mn2+)

N

N

Compound III

H2O

H2O2

N

4+

Fe
N

N

Product, radical
(R• + H+ or Mn3+)

Compound II

Fig. 17.6 LiP and MnP utilization of hydrogen peroxide H2O2 from other enzymes to be active.


570

Bioenergy Systems for the Future

(Itoh et al., 2003). Another approach is to use minimally treated mushroom spent straw
(MSS) as a feedstock for downstream thermochemical and biological processing.
The advantages of biological pretreatment include low-energy requirement and
mild environmental conditions. However, the rate of hydrolysis in most biological
pretreatment processes is very low.

17.3.2 Hydrolysis
Hydrolysis is performed to break down the complex structure of cellulose and hemicellulose into monomers (simple sugars). Glucose is obtained from cellulose, while
hemicellulose produces a mixture of pentoses and hexoses. Hydrolysis is carried
out using either mineral acids (acid hydrolysis) or enzymes (enzymatic hydrolysis).
In acid hydrolysis, lignocellulosic biomass (LCB) is treated with mineral acids
(e.g., sulfuric acid and hydrochloric acid) for a definite period of time at the specific
temperature to break cellulose and hemicellulose into monomer sugars as it follows:
ðC6 H10 O5 Þn + H2 O ! nC6 H12 O6

(17.4)

Hydrolysis without preceding pretreatment yields typically <20%, whereas yields
after pretreatment often exceed 90% (Hamelinck et al., 2005).
Further, acid hydrolysis is classified into two groups: concentrated acid hydrolysis
and dilute acid hydrolysis. On the other hand, enzymes (cellulase and hemicellulase)
are used as biocatalyst in enzymatic hydrolysis (Brethauer and Wyman, 2010;
Taherzadeh and Karimi, 2007).
Three main groups of cellulolytic enzymes were determined in white-rot fungi:
(i) Endo-1,4-β-glucanases (EDG; EC 3.2.1.4) are involved in the initial cellulose
breakdown, by an attack to the amorphous regions of cellulose, forming new free
chain ends, more accessible for cellobiohydrolases; (ii) cellobiohydrolases (CBH;

EC 3.3.1.91) are exocellulase enzymes responsible for cellobiose formation, hydrolyzing preferably β-1,4-glycosidic bonds from chain ends; and (iii) β-glucosidases
(BGL; EC 3.2.1.21) hydrolyze soluble cellobiose and cellodextrins to glucose
(Dashtban et al., 2009; Goodell et al., 2008; Sun and Cheng, 2002). In addition, there
are many other enzymes that attack hemicellulose, such as xylanase, glucuronidase,
acetylesterase, and β-xylosidase (Duff and Murray, 1996; Sun and Cheng, 2002).
In literature, strains of Trichoderma are considered among the best degrading fungi
in cellulase enzyme production, with other genera such as Chrysosporium, Penicillium,
and Acremonium (Gusakov, 2011; Zhang et al., 2014a,b).

17.3.3 Fermentation process
Wood cellulose ethanol production from biomasses refers to the use of special fermentation microorganisms to metabolize the abovementioned six (hexoses) and five (pentoses) carbon sugars.
In the fermentation process, the hydrolyzed sugars are mixed in a bath of water and
microorganisms. This microorganisms ferments sugars into bioethanol (as shown in


Bioenergy production from second- and third-generation feedstocks

571

the reaction) via EMP pathway (Lin and Tanaka, 2006) under anaerobic conditions
and controlled temperature as seen in the ABE process:
C6 H12 O6 ! 2C2 H5 OH + 2CO2

(17.5)

There are three types of microorganisms that can be used for the production of
bioenergy, yeasts, bacteria, and fungi. Table 17.2 shows some examples of microorganisms and their chemical-physical pretreatment conditions for an optimal fermentation. It also shows the carbon source of the microorganisms (the substrate where the
microorganisms grow).
In particular, the best known microorganisms for ethanol production are the yeast
Saccharomyces cerevisiae (i.e., the principal yeast used in the brewery and wine

industries and metabolizes glucose by EMP pathway) and the bacterium Zymomonas
mobilis (that metabolizes glucose through Entner-Doudoroff (ED) pathway)
(Claassen et al., 1999). They have, respectively, an ethanol yield of 130.12 and
99.78 g/L. The yeast S. Cerevisiae remains the major industrial ethanol producer
(Zaldivar et al., 2001), because it is generally recognized as safe (GRAS) microorganism that can be produced by fermentation up to the 20% (v/v) ethanol from carbon
(mainly C6 carbon sugars) (Cot et al., 2007). However, a major limitation, which
raises a serious industrial challenge, is the inhibition of the fermentation process
by accumulation of ethanol (Bayrock and Ingledew, 2001; Casey and Ingledew,
2008; Hahn-H€agerdal et al., 2007). Instead, the bacterium Z. mobilis cannot ferment
all forms of simple sugars but only glucose, fructose, and sucrose. For this reason, it is
not well suited for all the feedstocks in the substrate.
S. cerevisiae and the Z. mobilis offer high ethanol yields (90%–97% of the theoretical one) and high ethanol tolerance, up to ca. 10% (w/v), in fermentation medium
(Talebnia et al., 2010).
Another suitable microorganism that can be used in ethanol production is the bacterium Escherichia coli; the engineering form can give higher yields in bioethanol
production for the ability to ferment a wide spectrum of sugars.
Also the filamentous fungi, such as Neurospora crassa or Zygosaccharomyces
rouxii, can produce bioethanol but with a poor yield (9.9 g/L). The reason is not
yet entirely clear, but most probably, this low yield is due to the low resistance of these
fungi to higher concentration of ethyl alcohol formed in the batch.

17.3.4 SSF and SHF process
The SSF and SHF processes turn out to be important processes for the production of
ethanol from lignocellulosic substrates (the most common and popular are eucalyptus)
and herbaceous substrates (sorghum, bagasse, and wheat straw) (Badhan et al., 2007).
In fact, the cellulosic materials are natural complexes at higher carbon content in
the form of plant biomass. However, the production of ethanol from lignocellulosic
raw materials is more difficult than from sugar or starch, although precise studies have
shown that over 100 types of microorganisms can metabolize sugar with five carbon
atoms to produce ethanol, including bacteria, fungi, and yeast.

Yeasts

Bacteria

Fungi

572

Table 17.2

Different yields of ethanol in some of main yeasts, bacteria, and fungi
Ethanol
yield (g/L)

Reference

Glucose + sucrose
(0.28)

48–65

130.12

Ji et al. (2012)

55.5

Pure cellulose
(0.05)

25–50

15.20

Karimi et al.
(2006)

30

6.0

Soybean molasses
(0.2)

18

29.3

Letti et al.
(2012)

30

4.0

44–48

99.78

Ma et al. (2016)

30

6.3

Glucose
(0.2)
Glucose
(0.148)

46.0

First
aerobic
step
28
Second
anaerobic
step
37
35

5.0

ATCP
(0.02)

48

Lawford and
Rousseau
(1991)
Deshpande
et al. (1986)

5.0

Pure cellulose
(0.02)

168

/

Glucose
(0.2)

108

T (°C)

pH

Saccharomyces
cerevisiae mutant
3013
Saccharomyces
cerevisiae
Thermosacc®

Zymomonas
mobilis
NRRL 806
Zymomonas mobilis
ZMA7-2
Escherichia coli
ATCC 11303

30

5.5

38

Zygosaccharomyces
rouxii ATCC 12572

9.9

30

Groleau et al.
(1995)

Bioenergy Systems for the Future

Fermentation
time (h)

Microorganism

Neurospora crassa
NCIM 870

Carbon source and
concentration (g/
mL)


Bioenergy production from second- and third-generation feedstocks

573

Substrate

Pretreatment
SSF

Ethanol

Slurry
conditioning
SHF

Fig. 17.7 SHF and SSF processes for the production of ethanol.

The aim of the first pretreatment (i.e., using explosion stream with dilute sulfuric
acid or SO2) is to solubilize hemicellulosic sugars rendering the remaining cellulose
available for enzymatic hydrolysis. To convert the residual cellulose and hemicellulose into monomeric sugars (Parekh et al., 1988; Schell et al., 2003; Tucker et al.,
2003), pretreatment processes are implemented to improve the digestibility with partial solubilization or degradation of hemicellulose and lignin (Jan and Chen, 2003).

The next steps to obtain bioethanol are enzymatic hydrolysis and fermentation that
may occur separately (SHF) or simultaneously (SSF) (Fig. 17.7).
The advantages of SHF surely reside in the possibility of control and optimization of
the temperature of enzymatic hydrolysis and fermentation (Thomas-Pejo et al., 2008).
The enzymatic hydrolysis is performed by cellulase (endoglucanase and
exoglucanase normally) that break down cellulose into two glucose molecules by
means of the ß-glucosidase (Olofsson et al., 2008). However, the activity is inhibited
by endoglucanase cellobiose while the ß-glycosidase from glucose (Palmqvist and
Hahn-H€agerdal, 2000).
An optimal fermentative microorganism should be able to utilize both hexose and
pentose simultaneously with minimal toxic end-product formation. By means of the
SSF and of the SHF, the limits are exceeded preventing the inhibition by glucose information for the production of ethanol, optimizing the conversion of cellulose (Martin
et al, 2008).
The optimum temperature for saccharification is about 55°C while 30°C for the
fermentation.
Also the agitation requires an intermediate condition and is usually carried out at
150 rpm, and the microorganism used mainly in this process is the S. cerevisiae (Adsul
et al., 2004).
Surely, these processes and in particular the SSF are consolidated techniques and
without any problem from the point of view of the yields and of the reactions, but in
terms of economy, yet, many constraints have to be solved. Then, to actually reach the
fiber capable of producing ethanol, the industry has a long way to go.


574

17.4

Bioenergy Systems for the Future

Third generation feedstocks

The third-generation biofuels (also called advanced biofuels) are sourced from nonfood crops (mainly algae), but the resulting fuel is indistinguishable from its petroleum counterparts (Fenton and Ohuallachain, 2012).
On the basis of current technology projections, third generation is considered to be
a viable alternative energy resource devoid of the major drawbacks (i.e., food-fuel
competition) associated with first- and second-generation biofuels (Singh et al., 2011).
Algae utilize enormous amounts of CO2 for their growth and remove CO2 from
power plant emissions, convert biomass via photosynthesis, and liberate more oxygen
to the atmosphere. The algal biomass can be transformed into different types of
biofuels according to three types of production processes (as shown in Fig. 17.8), thermochemical processes, biological processes, and chemical reaction.

3.1
Algal biomass

3.3
Biological
conversion

Processes

Pyrolysis

Gasification

Liquefaction

ABE fermentation

Anaerobic digestion

Photo biological

Transesterification

Bio-oil charcoal

Fuel gas

Bio-oil

Ethanol, acetone,
butanol

CH4, H2

H2

Biodiesel

3.4
Chemical
reaction

Products

3.2
Thermochemical
conversion

Fig. 17.8 Schematic representation of main processes and products from algal biomass.

Bioenergy production from second- and third-generation feedstocks

575

17.4.1 The feedstock in the third generation: the algae
Algae can be divided into macroalgae and microalgae. All these organisms are characterized by rapid growth on saline water, municipal wastewater, coastal seawater,
and land unsuitable for farming (Chen et al., 2011; Pittman et al., 2011).
The total annual world production of algae biomass is about 12 Mt dry basis (around
16 Mt wet basis) for macroalgae and about 9200 t dry basis for microalgae, which were
harvested from wild habitats and aquaculture farms (Chen et al., 2015; Jung et al., 2013;
Vassilev and Vassileva, 2016). The amount of the mass-cultivated macroalgae has continuously increased over the last 10 years at an average of 10% (Vassilev and Vassileva,
2016; Jung et al., 2013). About 98% of commercial algae biomass production is currently with open ponds because this cultivation seems to be the most economical
and preferable way (Vassilev and Vassileva, 2016; Chen et al., 2015).

17.4.1.1 Macroalgae
Macroalgae (also known as seaweed) are multicellular aquatic organisms characterized by having low levels of cellulose and lipid and no lignin content in their structure
but high levels of structural polysaccharides (Allen et al., 2015; Ghadiryanfar et al.,
2016; Goh and Lee, 2010). Macroalgae are comparatively large and photoauxotrophic
organisms that are able to grow up to 60 m in length (Raheem et al., 2015).
Macroalgae are classified mainly into three major groups according to the thallus
color derived from photosynthetic pigmentation variations, namely, green
(Chlorophyta), red (Rhodophyta), and brown (Phaeophyta) (Chen et al., 2015;
Demirbas, 2010; Vassilev and Vassileva, 2016).

17.4.1.2 Microalgae
Microalgae are unicellular photosynthetic microorganisms that are highly productive
and are able to produce large amounts of biomass more efficiently than current cultivation practices for terrestrial crops. They have sized of <400 and of 1–30 μm in
diameter (Vassilev and Vassileva, 2016). Microalgae have the possibility to convert

algal biomass, water, and CO2 by sunlight energy (as it can be seen in Section 17.4.3)
into various forms of bioenergy products.
The photosynthetic efficiency of microalgae in engineered systems can reach
4%–5% of the solar energy compared with 1%–2% for terrestrial plants (Shilton
and Guieyesse, 2010).
These conversions are much more efficient than those of crop plants. On the one
hand, there are many advantages for microalgae compared with lignocellulosic feedstocks: (1) fast growing, (2) the need of lower amounts of water per kilogram of
biomass produced, (3) grown in salt water, (4) the ability to sequester CO2 from flue
gas of industrial installations, and (5) the yield of a large amount of lipids and starch
suitable for the production of biodiesel, bioethanol, and others. Biologists have categorized microalgae in a variety of classes, mainly distinguished by their pigmentation,
life cycle, and basic cellular structure. The most important classes or categories of
microalgae in terms of their abundance are (1) diatoms (Bacillariophyceae), (2) green


576

Bioenergy Systems for the Future

(Chlorophyceae), (3) blue and blue-green cyanobacteria (Cyanophyceae), (4) golden
(Chrysophyceae), and (5) red (Rhodophyceae) algae (Demirbas, 2010; Vassilev and
Vassileva, 2016; Ziolkowska and Simon, 2014).
It is estimated that 50000 species of microalgae exist; however, only few were
practically used (Vassilev and Vassileva, 2016). The most important microalgae
for each class are reported in Table 17.3.
The differences reported in table between the various types of microalgae are in
term of total lipid percent, the range in term of percentage from C16:0 to C18:3
(long-chain fatty acids). This parameter is the lipid produced from microalgal species
that usually contains C16 and C18 fatty acids, which is similar to that of vegetable oils
and suitable for biodiesel production (Harrington, 1986; Ho et al., 2010; Miao and Wu,
2006). C16 and C18 are fatty acid groups that occupied up to 86% of total fatty acids

when cultured in the nutrient-deficient medium. This C16/C18 content is markedly
higher than that obtained from cultivation under the nutrient-rich (67%) and
nitrogen-deficient (78%) conditions (Ho et al., 2010). In addition, microalgae production has the potential to utilize CO2 emissions and offers potential for a carbon neutral
biofuel.

17.4.2 Thermochemical processes
In thermochemical processes, the biomass is gasified, liquefied, or heated (according
to the production process) to obtain a wide range of products: H2, CO, CO2, CH4, light
hydrocarbons biochar, and biotar (that are biohydrocarbons) (Kapdan and Kargı,
2006) as described in the following reaction:
Biomass ! H2 + CO + CH4 + other products

(17.6)

Thermochemical conversion technique can further be divided into gasification and
pyrolysis (as described in Fig. 17.9).

17.4.2.1 Pyrolysis
Pyrolysis is a thermochemical process in which biomass is converted into biochar,
bio-oil, and syngas at high temperatures in the absence of oxygen. The pyrolysis processes occur in the range of 400–1200°C. Although the product yield depends
upon various operating parameters, generally, low temperature and high residence
time favor the char production (Tripathi et al., 2016). Depending on the values of
these two conditions (temperature and residence time, whose experimental values
are reported in Table 17.4), the pyrolysis can be further classified into slow pyrolysis,
fast pyrolysis, and flash pyrolysis.
Slow pyrolysis is principally used for the production of char, but liquid and gaseous
products are also formed in a small quantity ( Jahirul et al., 2012).
Instead, the fast pyrolysis is a procedure where the biomass is heated up rapidly to a
temperature of 850–1250°C. A typical fast pyrolysis produces 60%–75% of liquid
product (tar and oils that remain in liquid form at room temperature like acetone

Methane yield and biomass composition of some microalgae

Species

Protein (%)

Lipid (%)

Carbohydrate (%)

Methane yield
(mL g21)

Chlorella vulgaris
Chlamydomonas
reinhardtii
Dunaliella tertiolecta

41.51
45.70

15.67
22.40

20.99
—

403

587

61.32

2.87

21.69

24

39.00

20.00

17.00

204

30.38

4.66

13.41

287

Nannochloropsis
oculata
Scenedesmus obliquus

References
Wang et al., 2013; L€
u et al., 2013
Kebelmann et al., 2013; Mussgnug
et al., 2010
Shuping et al., 2010; Lakaniemi et al.,
2011
Liu et al., 2012; Buxy et al., 2013

Bioenergy production from second- and third-generation feedstocks

Table 17.3

Chen et al., 2014; Mussgnug et al.,
2010

577


578

Bioenergy Systems for the Future

Biomass
ΔT
In presence of O2

ΔT

Pirolysis

Gassification

Biooil

Biochar

CO2 + H2

Fig. 17.9 Products obtained from pyrolysis and gasification in thermochemical processes.

Table 17.4

Pyrolysis classes

Temperature (°C)
Residence time (s)
Pressure (MPa)
References

Slow pyrolysis

Fast pyrolysis

Flash pyrolysis

550–950
300–550
0.1
Tripathi et al.,

2016

850–1200
0.5–10
0.1
Bridgwater et al.,
1999;
Tripathi et al., 2016

900–1200
<1
0.1
Li et al., 2013;
Tripathi et al.,
2016

and acetic acid (Ni et al., 2006)), 15%–25% of biochar (and almost pure carbon plus
other inert materials), and 10%–20% of noncondensable gaseous products (H2, CH4,
CO, CO2, and other gases depending on the organic nature of biomass and pyrolysis
(Bridgwater, 2006)), while the flash pyrolysis can be considered as an improved form
of fast pyrolysis: in this procedure, the temperature required for the degradation of the
components of biomass is archived by heating it with a very high rate of the order of
1000°C/s.

17.4.2.2 Gasification
Gasification is a process in which carbonaceous content of the biomass is converted
into the gaseous fuel in the presence of gaseous medium like oxygen, air, carbon dioxide, steam, or some mixture of these gases at elevated temperature (between 700°C
and 900°C) (Tripathi et al., 2016).
As in the pyrolysis procedure, also the gasification process uses high temperatures,
but in gasification process, the combustion takes place in the presence of O2.

Bioenergy production from second- and third-generation feedstocks

579

This process has challenges toward large-scale development, that is, low hydrogen
production and high tar content in the syngas (Chen et al., 2016; Ni et al., 2007; Shen
and Yoshikawa, 2013).
The unwanted tar may cause the formation of tar aerosols and polymerization to a
more complex structure, which are not favorable for hydrogen production through
steam reforming. Several research groups (Aznar et al., 1996; Ekstrom et al., 1996;
Orıo et al., 1996) have focused their studies to maximize the hydrogen quantity produced and to minimize that of tar.
Under typical gasification conditions, oxygen levels are restricted to less than 30%
of that required for complete combustion, and CO and H2 are the major products
(Ni et al., 2006).
In the literature, there are several works that indicate the temperatures necessary to
reduce underivatized mono-, di-, and poly-nuclear aromatics (which constitute the tar)
to light gases. It has reported (Corella et al., 1999; Milne et al., 1998) that tar could be
thermally cracked at temperature above 1000°C. Such authors carried their research
on biomass gasification under similar conditions but varying the gasifying agent. In
fact, the use of some additives (such as dolomite) inside the gasifier helps tar reduction
(Corella et al., 1999). It is a cheap disposable catalyst that can significantly reduce the
tar content of the product gas from a gasifier. Dolomite generally contains 30 wt%
CaO, 21 wt% MgO, and 45 wt% CO2; it also contains minerals such as SiO2,
Fe2O3, and Al2O3. The surface areas of the various types also differ as do the pore
sizes and distributions.

17.4.3 Biological processes
The biological process of energy conversion of algal biomass into other fuels includes

alcoholic fermentation (such as the ABE process), anaerobic digestion, and photobiological hydrogen production. All these biological processes are essentially dependent
on the presence of enzymes suitable to convert the lignocellulosic biomass in so-called
molecules of C1 block (such as CH4, CO, and CO2), N2, and hydrogen. In this paragraph, exclusively, the photobiological conversion for the production of hydrogen will
be discussed in detail (see Fig.17.10).
3.3.1 Direct photolysis

3.3.2 Indirect photolysis
3.3
Biological
conversion

Photo biological
production

3.3.3 Photo-fermentation

H2

3.3.4 Dark fermentation

3.3.5 Integrated processes

Fig. 17.10 Schematic representation of different types of photobiological production involved
in hydrogen biosynthesis.


580

Bioenergy Systems for the Future

This process has the great advantage that the reaction can run at low temperature
and, above all, does not produce a range of harmful by-products to the environment. In
particular, with the presence of certain enzymes, it is possible to sectorialized the
transformation of biomass in the specific production of hydrogen. The enzymes that
allow this transformation are mainly of nitrogenase and hydrogenase ([FeFe]hydrogenase and [NiFe]-hydrogenase) (Hallenbeck, 2009).
Nitrogenase enzymes are formed in two subunits: (i) reductase subunit (an Fe-S
protein) and (ii) dinitrogenase complex (Mo-Fe-S protein). This enzyme is responsible
for the transfer of electrons from the external donor to the dinitrogenase complex of
the enzyme (Eroglu and Melis, 2011a,b).
The nitrogenase enzyme is responsible for the reduction of protons into molecular
hydrogen, which occurs simultaneously with the reduction of dinitrogen reaction
(catalyzed by the Mo-Fe-S protein) that leads to the formation of two molecules of
ammonia (Allakhverdiev et al., 2010); moreover, the stoichiometric factors that affect
the reaction of ammonia and hydrogen development are mainly governed by the metal
factor (Mo, Fe, and V) linked to the nitrogenase enzyme site as described by the
following reactions:
Mo À nitrogenase : N2 + 8H + + 8eÀ ! 2NH3 + H2

(17.7)

Fe À nitrogenase : N2 + 8H + + 8eÀ ! 2NH3 + H2

(17.8)

V À nitrogenase : N2 + 8H + + 8eÀ ! 2NH3 + H2

(17.9)

However, in the absence of dinitrogen substrate, nitrogenase enzymes exclusively
catalyze hydrogen productions from protons and high potential-energy electrons with

an expenditure of ATP energy.
In fact, nitrogenase enzymes use MgATP (2 ATP/e-), and low potential electrons
are derived from reduced ferredoxin (Fd) to reduce a variety of substrates.
Reduced Fd (a small Fe-S protein that can accept or discharge electrons) acts as an
electron donor to an enzyme, which reversibly catalyzes the reduction of protons (H+)
to molecular hydrogen according to the reaction (Basak and Das, 2007; Das and
Veziroglu, 2008):
2H + + 2FdÀ $ H2 + 2Fd

(17.10)

In the absence of other substrates, nitrogenase continues to turn over, reducing protons
to hydrogen (Hallenbeck and Benemann, 2002) according to the reaction
2eÀ + 2H + + 4 ATP ! H2 + 4 ADP + 4 Pi

(17.11)

where ADP and Pi refer to adenosine diphosphate and inorganic phosphate, respectively (Ni et al., 2006).
Nitrogenase is a slow enzyme (about 5 sÀ1) with a turn over time per electron. Each
electron-transfer step between Fe protein and MoFe protein involves an obligatory


Bioenergy production from second- and third-generation feedstocks

581

cycle of association and dissociation of the protein complex (Howard and Rees, 1996).
Furthermore the turn over necessitate the biosynthesis of enormous quantities of the
Fe protein and of the MoFe protein. Thus, considering the low turnover number, the
considerable energy inputs necessary for biosynthesis and the requirement for ATP for

catalysis, nitrogenase is not a very metabolically effective way to produce H2.
Instead, the hydrogenase enzymes are divided in two parts: [NiFe]-hydrogenases
(that catalyze the oxidation of hydrogen) and [FeFe]-hydrogenases (that catalyze the
proton reduction) (Capon et al., 2009) according to the following reaction:
2eÀ + 2H + $ H2

(17.12)

Both enzymatic transformations for the production of hydrogen may be dependent or
independent from the light (Das, 2009), depending on if they derive energy either
directly from light energy or indirectly by consuming photosynthetically derived
carbon compounds, respectively.
Independent processes from light are anaerobic processes that are called dark
fermentation processes, while the light-dependent processes can be or photolysis or
rather photolytic hydrogen production from water by algae or cyanobacteria (also
known as direct photolysis) or photofermentation. Under anaerobic conditions, hydrogen is produced as a by-product during the conversion of organic compounds (algae)
into organic acids, which are then used for methane generation (Kapdan and
Kargı, 2006).
Instead, it has been proved that integrated process of dark and photofermentation
has maximum efficiency for the production of biohydrogen.

17.4.3.1 Direct photolysis
Photosynthesis involves the absorption of light by two distinct photosynthetic systems
operating in series: a water-splitting and O2-evolving system (photosystem I or PSI)
and a second photosystem (PSII) (Das and Veziroglu, 2001). Absorption of light in the
form of photons by PSI (700 nm) and/or PSII (680 nm) generates a strong oxidant that
can oxide water into protons, electrons, and O2 (Oh et al., 2011). The electrons reduce
protons to form H2 according to the following reaction:
hν

2H2 O ! 2H2 + O2

(17.13)

The generated hydrogen ions are converted into hydrogen gas in the medium with
electrons (donated by reduced Fd) by hydrogenase enzyme (blue-green algae such
as Cyanobacteria) present in the cells. Light energy absorbed by PSII generates electrons that are transferred to Fd using light energy absorbed by PSI (Das and Veziroglu,
2001, 2008), as shown in Fig. 17.11.
Since hydrogenase is sensitive to oxygen, it is necessary to maintain the oxygen
content at a low level under 0.1% so that hydrogen production can be sustained
(Zhang et al., 2014a,b).


582

Bioenergy Systems for the Future

H2
hv

2e–
Blue-green algae
(Hydrogenase)

PSI / PSII
Fd

2H2O

2H+

O2

Fig. 17.11 Schematic representation of the direct photolysis.

The advantage of this technology is that the primary feed is water, which is inexpensive and available almost everywhere (Holladay et al., 2009), but the O2 produced
from microalgae inhibits [Fe]-hydrogenase and, consequently, the H2 production.
Thus, the O2 sensitiveness of the hydrogenase enzyme reaction remains the key problem of this biohydrogen production system (Hallenbeck and Benemann, 2002).

17.4.3.2 Indirect photolysis
In the indirect photolysis, H2 is produced from electrons that are derived from the carbohydrate catabolism (Prince and Kheshgi, 2005) using mainly green algae and
Cyanobacteria (that maintain low concentration of O2) (McKinlay and Harwood,
2010). This process is designed to limit the action of O2 on the [Fe]-hydrogenase.
As shown in Fig. 17.12, the H2 production is spatially separated from photosynthesis
through sulfur depletion/repletion (Kruse and Hankamer, 2010).
Stage 1 (+O2)

Stage 2 (−O2)
H2

hv
2e−
PSI / PSII
Fd

2H2O

O2

Cell material

(glucose)

2e−
Fermentation
Fd

CO2

Cell material

Fig. 17.12 Schematic representation of the indirect photolysis.

Blue-green algae
(hydrogenase)

2H+


Bioenergy production from second- and third-generation feedstocks

583

In stage 1, the CO2 is fixed by oxygenic phototrophs for biosynthesis (sugars)
(McKinlay and Harwood, 2010) according to the reaction
hν

12H2 O + 6 CO2 ! C6 H12 O6 + 6 O2

(17.14)

In stage 2, sugars can be fermented to H2 and CO2 as shown in the following reaction:
H2 ase

C6 H12 O6 + 6 H2 O ƒƒ! 12 H2 + 6 O2

(17.15)

It has also been demonstrated that the depletion of photosynthetic O2 improved the
anaerobic H2 production because photosynthesis and carbohydrate metabolism were
both active (Marquez-Reyes et al., 2015).

17.4.3.3 Photo-fermentation
Photofermentation process is an extremely hopeful method due to the gentle reaction
conditions, high hydrogen production, use of solar energy, and conversion of organic
waste to hydrogen. Photosynthetic bacteria play a key role in the photofermentation
process, and their performance determines the utilization range and conversion
efficiency of substrate to hydrogen. Thus, the screening of bacteria with a remarkable
capacity for hydrogen production is crucial to the photofermentation hydrogen
production process (Liu et al., 2015). This production process converts volatile fatty
acids (VFA) (such as acetic acid) in H2 and CO2 using photosynthetic nonsulfur (PNS)
bacteria under anaerobic conditions (Argun and Kargi, 2011; Levin et al., 2004) as
described in Eq. (17.16):
PNS

CH3 COOH + 2H2 O ƒ! 4H2 + 2 CO2

(17.16)

The PNS bacteria (such as Rhodobacter sphaeroides O.U001 and Rhodobacter
capsulatus) (Argun and Kargi, 2011; Das and Veziroglu, 2001; Kapdan and Kargı,

2006) are mainly made of hydrogenase enzymes and nitrogenase (Das and
Veziroglu, 2001); however, nitrogenase is the major enzyme responsible for the
molecular H2 production under anaerobic conditions (Dasgupta et al., 2010).
This reaction is not spontaneous requiring external energy input in the form of
light; in fact, in this anaerobic photosynthesis, solar energy (in the range between
400 and 1000 nm) (Argun and Kargi, 2011) is captured to produce ATP and highenergy electrons that reduce ferredoxin. ATP and reduced ferredoxin drive proton
reduction to hydrogen by PNS as described in the schematic representation in
Fig. 17.13.
The advantages of this technology are the excellent conversion of organic acid
wastes to H2 and CO2 (expressed by the yield coefficient that is the ratio of the amount
of produced H2 to the consumed carbon source) and the potential waste treatment
credits. In fact, according to previous research data, the yield coefficient for the formation of H2 is up to 80% of the theoretical yield (as shown by reaction (17.15) in