Posada Duke-Manizales PDF

Design and Analysis of
Technological Schemes for Glycerol

Conversion to Added Value Products
John Alexander Posada Duque
Universidad Nacional de Colombia

Facultad de Ingeniera y Arquitectura, Departamento de Ingeniera Elctrica, Electrnica y
Computacin
Manizales, Colombia
2011
Diseo y Evaluacin de los

Esquemas Tecnolgicas para la
Conversin de Glicerol en Produtos
de Valor Agregado
Universidad Nacional de Colombia Sede Manizales

Computacin
Manizales, Colombia
Design and Analysis of

Technological Schemes for Glycerol
Conversion to Added Value Products
Thesis submitted in partial fulfillment of the requirements for the degree of:
Doctor of Philosophy in Engineering
Advisor:
Ph.D., M.Sc, Chemical Engineer Carlos Ariel Cardona Alzate
Research line:
Chemical and Biotechnological Process Engineering
Research group:
Chemical, Catalytic and Biotechnological Process
Universidad Nacional de Colombia

Computacin
Manizales, Colombia
2011
______________________
A mi madre y mi hermana por su apoyo,
a Patricia por su valiosa presencia.
Acknowledgment
I would like to express thanks to God and to my mother for the most essential reason, the
life. I also would like to express sincere thanks to my advisor, Dr. Carlos Ariel Cardona
Alzate, who has been for more than six years the most important influence not only in my
career but also in my life. Kind thanks to Dr. Ramon Gonzalez for receiving me at his
laboratory during the internship. Special thanks to my research fellows, Luis Rincon,
Julian Quintero, and Javier Naranjo, for their friendship and unconditional help. And
thanks to Patricia Arevalo and to my friends.
Thanks to the National University of Colombia for the financial support, to the Research
Office of National University of Colombia branch Manizales for the financial support in the
internship in United States, to the Research and Extension Projects Office of National
University of Colombia branch Manizales for the financial support in air tickets to United
States, to the Department of Electricity, Electronic and Computational Engineering of
National University of Colombia branch Manizales for the financial support for attending to
congresses.
Resumen y Abstract
Resumen
Uno de los principales problemas relacionados con la creciente industria del biodiesel es
la sobreproduccin de glicerol, que se obtiene en una relacin en peso de 1/10 (glicerol/
biodiesel). Lo que ha llevado a que su precio de venta caiga en un orden de magnitud.
As, la gran cantidad de glicerol co-producido puede ser usada como una materia prima
renovable y de bajo costo para producir compuestos qumicos y combustibles. Aqu se
analiza la conversin qumica y bioqumica de glicerol hacia productos de valor agregado
basado en criterios tecno-econmicos. Entonces se consideraron nueve productos
finales (va qumica: gas de sntesis, acrolena y 1,2-propanodiol; va bioqumica: etanol,
1,3-propanodiol, cido D-lctico, cido succnico, cido propinico y poly-3-hidroxibutirato). Adems, un total de 27 esquemas tecnolgicos fueron diseados, simulados y
evaluados econmicamente utilizando Aspen Plus y Aspen Icarus Process Evaluator.
Como una conclusin, una plataforma de biorefinerias basada en glicerol fue obtenida
para la produccin rentable de combustibles fsiles y bioclsticos.
Palabras clave: Conversin de glicerol, diseo de procesos, simulacin de procesos,

evaluacin de procesos, biorefinerias basadas en glicerol.
VI
Glycerol Conversion to Added Value Products
Abstract
An important concern related to the growing biodiesel industry is the over-production of
raw glycerol as by-product, which is obtained in a weight ratio of 1/10 (glycerol/biodiesel).
This fact had led to a 10-field drop of its sale price. Thus, the large amount of byproduced glycerol can be used as low-cost and renewable feedstock in order to produce
chemicals and fuels. Here, the chemical and bio-chemical conversion of glycerol to
added-value products was analyzed based on techno-economic criteria. In this way, nine
final products (for chemical conversion: syn-gas, acrolein, and 1,2-propanediol; while for
fermentative conversion: ethanol, 1,3-propanediol, D-lactic acid, succinic acid, propionic
acid, and poly-3-hydroxybutyrate) were considered. And a total 27 technological schemes
were designed, simulated, and economically assessed, using Aspen Plus and Aspen
Icarus Process Evaluator. As a conclusion a glycerol-based platform for biorefineries was
obtained for the profitable production of fuels, chemicals, and bio-plastics.
Keywords:
Glycerol
conversion;
process
assessment; glycerol-based biorefineries.
design;
process
simulation;
process
Contents
VII
Table of Contents
ACKNOWLEDGEMENTS
RESEMEN
ABSTRACT
TABLE OF CONTENTS
LIST OF FIGURES
LIST OF TABLES
Pg.
IV
V
VI
VII
X
XIII
1. Introduction
1.1. Application field and Motivation
1.2. Thesis objectives
1.3.
Thesis structure
References
1
2
5
5
7
2. Chapter 2: The glycerols world

2.1. Overview
2.2. Biodiesel industry
2.3. Glycerol market and its oversupply problem
2.4. Glycerol as raw material
References
9
9
10
12
13
14
3. Chapter 3: Methodology for processes design and analysis

3.1. Processes design
3.2. Processes simulation
3.3. Processes assessment
References
17
17
20
22
24
4. Chapter 4: Separation and purification of glycerol

4.1. Commercial qualities of glycerol
4.2. Effect of the feedstock for biodiesel production on glycerol composition
4.3. Conventional purification process
4.4. Alternative purification process
25
25
27
28
29
VIII

4.5. Simulation of the glycerol purification process
4.6. Economical assessment for glycerol purification processes
4.7. Conclusions
References
31
33
34
35
5. Chapter 5: Chemical conversion of glycerol

5.1. Oxidation
5.2. Reduction
5.3. Etherification
5.4. Pirolysis and gasification
References
36
36
39
41
41
43
6. Chapter 6: Biochemical conversion of glycerol

6.1. 1,3-Propanediol
6.2. Ethanol
6.3. Poly-3-hydroxybutirate
6.4. D-Lactic acid
6.5. Succinic acid
6.6. Propionic acid
References
47
47
48
51
53
54
58
59
7. Chapter 7: Study cases for chemical conversion of glycerol

7.1. Generalities
7.2. Acrolein production
7.3. Hydrogen production
7.4. 1,2-propanediol production
7.5. Economic assessment
7.6. Conclusions
References
69
69
70
73
76
78
81
81
8. Chapter8: Study cases for biochemical conversion of glycerol

8.1. 1,3-Propanediol production
8.2. Ethanol production
8.3. PHB production
8.4. D-Lactic acid production
8.5. Succinic acid production
8.6. Propionic acid production
8.7. Economic assessment
8.8. Conclusions
References
85
85
104
115
124
138
148
153
167
169
Contents
IX
9. Chapter 9: Experimental setup for glycerol fermentation to PHB

9.1. Generalities
9.2. Materials and methods
9.3. Analytical Methods
9.4. Results and discussion
References
183
183
186
187
189
192
10. Chapter 10: Conclusions
194
11. Chapter 11: List of Publications
199
Figures
List of Figures
Page
Figure 1.1. World biodiesel production and capacity
Figure 1.2. Global biodiesel production by feedstock
Figure 3.1. Hierarchical decomposition according to the "onion diagram"
19
Figure 3.2. The process design method based on the so called breadth-first
20
Figure 4.1. Flowsheet of conventional schemes for glycerol purification
29
Figure 4.2. Flowsheet of the Ambersep BD50 process
30
Figure 4.3. Simplified flowsheet for raw glycerol purification
31
Figure 5.1. Possible products for glycerol oxidation
38
Figure 6.1. Schematic representation of glycerol degradation process on the part

of Escherichia coli, on non fermentative process.
49
Figure 6.2. Main metabolic pathways for fermentative degradation of glycerol

by Escherichia coli.
50
Figure 6.3. Pathways involved in the microaerobic utilization of glycerol in E. coli
54
Figure 6.4. Products that can be synthesized from succinic acid
55
Figure 6.5. Pathways involved in the micro aerobic utilization of glycerol
57
Figure 7.1. Simplified flowsheet for acrolein production by glycerol dehydration
71
Figure 7.2. Simplified flowsheet for hydrogen production by gasification
74
Figure 7.3. Simplified flowsheet for 1,2-propanediol production by hydrogenolysis
77
Figure 8.1. Hysteresis loops and multiple steady states
89
Figure 8.2.a) 1,3-propanediol volumetric productivity, (the column in the right side
gives the scale). b) Region of multiplicity of steady states, optimal productivity
for each dilution rate, global optimal productivity, and wash-out line.
90
Figures
XI
Figure 8.3. 1,3-Propanediol productivity and concentration in the second

fermentation stage
91
Figure 8.4. Volumetric productivity in the second fermentation stage using the
optimal dilution rate obtained by the model 1 for the first fermentation stage,
(the column in the right side gives the scale).
92
Figure 8.5. Product of productivities of both fermentation stages using the optimal
dilution rate obtained by the model 1 for the first fermentation stage, (the column in
the right side gives the scale).
93
Figure 8.6. Acetylation reaction of 1,3-propanediol with iso-butyl aldehyde to

2-iso-propyl-1,3-dioxane
95
Figure 8.7. Simplified flowsheet for 1,3-propanediol production from raw glycerol
98
Figure 8.8. Residue map curves for the reactive system
99
Figure 8.9. Direct separation with fed 0.377645/0.6223552iP13DO/Water
100
Figure 8.10. P/W ratio, Direct Separation (XF: 0.377645/0.622355-2iP13DO/water)
101
Figure 8.11. iso-Volatility curve (Wateriso-Butyraldehyde2-iso-Propil-1,3-Dioxane) 101

Figure 8.12. Simplified flowsheet of fuel ethanol production from glycerol at
88 wt % and 98 wt %.
104
Figure 8.13. Stages for ethanol production from sugar cane, corn, and crude
glycerol
107
Figure 8.14. Simplified flowsheet for ethanol production from: (A) sugar Cane
and (B) Corn
108
Figure 8.15. Flowsheet for the integrated process of combined biodiesel and
bioethanol production
112
Figure 8.16. Flowsheets for PHB production from glycerol (88 or 98 wt %)
120
Figure 8.17. Scheme for the simulation procedure to synthesize PHB from crude
glycerol
124
Figure 8.18. Complex formed during the reactive extraction process of

D-lactic acid
133
Figure 8.19. Simplified flowsheet for D-lactic acid production from raw glycerol
134
Figure 8.20. Reaction complexes of succinic acid, formic acid and acetic acid
with TOA
144
Figure 8.21. Simplified flowsheet for succinic acid production from raw glycerol
144
XII
Figure 8.22. Simplified flowsheet for propionic acid production from raw glycerol
150
Figure 9.1. Accumulation profile I of Bacillus megaterium
189
Figure 9.2. Accumulation profile II of Bacillus megaterium
190
Figure 9.3. Accumulation profile III of Bacillus megaterium
190
Figure 9.4. Accumulation profile IV of Bacillus megaterium
191
Tables
XIII
List of Tables
Page
Table 3.1. Used costs and prices for the economic assessment
23
Table 4.1. Quality specifications for the main qualities of glycerol
25
Table 4.2. Fatty acid profile of vegetable and used oils
27
Table 4.3. Composition of the glycerol layer obtained by decantation during

the biodiesel production from different feedstocks
28
Table 4.4. Simulation results for raw glycerol purification process
32
Table 4.5. Purification costs (PC) of raw glycerol (US$/L)
33
Table 7.1. Simulation results for dehydration process from glycerol
72
Table 7.2. Simulation results for gasification process from glycerol
74
Table 7.3. Simulation results for hydrogenolysis process from glycerol
78
Table 7.4. Production costs for glycerol conversion to added-value
79
Table 7.5. Percentage of Production costs for glycerol conversion to added-value
79
Table 8.1. Results summary for each optimization model
94
Table 8.2. Fermentation results for the three considered scenarios
96
Table 8.3. stoichiometric reactions for each scenario and each fermentation stage
97
Table 8.4. Singular Points ** - Acetilation System of 1,3-PD* with 2iP13DO*
99
Table 8.5. Summary of the main simulation results for 1,3-propanediol production
from glycerol
102
Table 8.6. Data representing the behavior of the downstream process
104
Table 8.7. Simulation results for fuel ethanol production from glycerol
106
Table 8.8. Main input data and operation conditions used in the simulation process
113
XIV
Table 8.9. Main process streams for ethanol production from lignocellulosic biomass 114
Table 8.10. Process conditions for glycerol fermentation
116
Table 8.11. PHB Extraction Methods
118
Table 8.12. Process conditions for PHB recovery: Downstream Process I
121
Table 8.13. Process conditions for PHB recovery: Downstream Process II
121
Table 8.14. Process conditions for PHB recovery: Process III
122
Table 8.15. Downstream processes for lactic acid recovery from a fermentation broth 131
Table 8.16. Base information for the glycerol fermentation to D-lactic acid
132
Table 8.17. Stoichiometry for glycerol fermentation to D-Lactic Acid by Engineered

E. coli
134
Table 8.18. Summary of the main simulation results for D-lactic acid production
process
135
Table 8.19. Data representing the behavior of the downstream process for D-lactic
acid production
Table 8.20. Base information for the glycerol fermentation to succinic acid
137
Table 8.21. Stoichiometry for glycerol fermentation to succinic acid by Engineered

E. coli
141
141
Table 8.22. Removal efficiency (%) of the carboxylic acids from the fermentation 142
broth.
Table 8.23. Summary of the main simulation results for succinic acid production
process
145
Table 8.24. Data representing the behavior of the downstream process for succinic
acid production
147
Table 8.25. Stoichiometry of the fermentation process for each scenario
149
Table 8.26. Summary of the main simulation results for prpionic acid production
process.
151
Table 8.27. Data representing the behavior of the downstream process for propionic 153
acid production
Table 8.28. Economic results for raw glycerol conversion to 1,3-propanediol: Cost
(USD$/kg) and Share (%)
154
Table 8.29. Bioconversion costs (BCCs) for fuel ethanol production form raw glycerol 166
Tables
XV
Table 8.30. Global production costs (GPCs) for fuel ethanol production from
raw glycerol
157
Table 8.31. Discriminated costs for integrated biodiesel and raw-ethanol production
from oil palm
158
Table 8.32. Total PHB production costs from crude glycerol through raw glycerol
(88 wt %) and pure glycerol (98 wt %).
159
Table 8.33. Main producers of PHA in the world
160
Table 8.34. Economic results for raw glycerol conversion to D-lactic acid: Cost
162
Table 8.35. Economic results for raw glycerol conversion to succinic acid: Cost
164
Table 8.36. Economic results for raw glycerol conversion to propionic acid: Cost
(USD$/kg) and Share (%).
166
Table 9.1. Some microorganisms PHB producer from different agroindustrial wastes 185
Table 9.2. Comparison between experimental results for glucose and glycerol
fermentation to PHB
191
1. Introduction
Fossil sources have diminished significantly because they have been used as the main
raw material for the current economy and life style. For instance, large scale products
such as transportation fuels and daily use components are obtained from petrochemical
industry. Furthermore, the increasing demand of fossil sources from developing
economies (like China and India) and speculations about oil reserves availability have
caused high crude oil prices. In fact, experts predict the end of cheap oil in 2040 at the
latest [1], which are currently and again above USD$ 100 per barrel. This economic issue
added to the environmental conscience, which is focus on the problems derived from
pollution and accumulation of greenhouse gases, have taken to develop alternative
technologies in order to produce sustainable fuels and chemicals using renewable
resources. In this way, biodiesel and bioethanol are the most important technological
platforms for liquid fuels production.
Although biofuels such as biodiesel and bioethanol represent a renewable, convenient,

and environmental friendly alternative for fossil fuels substitution, they also cause
concerns in relation to their economic viability. Implementation of biorefineries as an
additional process to the biofuels production is an interesting alternative to both overcome
the limited profitability of these technologies and use the generated sub-products.
Therefore, the concept of biorefinery could be especially advantageous if the conversion
of by-products or wastes to added-value products is considered [2].
Glycerol as the main by-product on biodiesel production is obtained at high concentration

in a weight ratio of 1/10 (glycerol/biodiesel). Moreover, the growing market of biodiesel
has generated a glycerol oversupply, where its production increased 400% in a two years
period and consequently the glycerol commercial price fell down near to 10 fold during the
same period of time [3]. As a result of the low prices of glycerol, traditional producers such
as Dow Chemical, and Procter and Gamble Chemicals, stopped its production [4].
Since glycerol sales have represented an important profitability for biodiesel industry, it is
reasonable that low prices of glycerol could impact the economy of biodiesel producers
negatively. For that reason, the correct exploitation of glycerol as raw material should be
focused on its transformation to added-value products. Thus, the use of glycerol is a highpriority topic for managers and researchers related to biofuels production. In this sense,
the establishment of glycerols biorefineries able to co-generate added-value products is
an excellent opportunity not only to raise the profitability but also to produce other
chemicals from a biobased raw material.
In order to analyze the glycerol conversion possibilities, this highly functional molecule
has been identified as a potential raw material for organic synthesis of many
intermediates and chemical products. Chemically glycerol can be transformed by many
ways such as oxidation, hydrogenolysis, etherification, pyrolysis, and gasification. Thus,
different kinds of products such as acrolein, 1,2-propanediol, polyglycerols, syn gas,
among many others compounds can be chemically obtained. On the other hand, because
of glycerol is a structural component of many lipids, it can also be biochemically
transformed to added value compounds. Some products of glycerol fermentation are: 1,3propanediol, ethanol, propionic acid, citric acid, lactic acid, poly-3-hydroxybutirate, and
biosurfactants. Then, due to the wide variety of potential products from glycerol, its
biorefineries are an excellent commercial opportunity. In this way, it is necessary to
determine the most appropriate alternative for glycerol transformation. In this study, the
process design and the assessment of different technological schemes for glycerol
transformation to added-value components is systematically performed considering
technologic and economic indicators.
1.1 Application field and motivation

Biodiesel production sector is a dynamic industry with a rapid global market growth. For
instance, over the past decade the biodiesel production was governmentally driven aiming
to the development of large scale industries. Thus, Europe took the lead with more than
1.6 mill Tons of biodiesel produced in 2002 (at capacities of approx. 2.1 mill Tons), while
in the USA approx. 40.000 T were produced [5]. Furthermore, in 2008 the global biodiesel
production reached more than 11.1 mill Tons (see Figure 1.1), representing around 1 % of
all diesel consumption of the USA and between 2-3 % of the total transportation
1. Introduction
consumption in Europe [6]. Even though, Europe represents 80% of global biodiesel
production and consumption, the U.S. is increasing its production at a faster rate than
Europe, while Brazil is expected to surpass the U.S. and European biodiesel production
by the year 2015 [6].
35
30
25
20
Production
Capacity
15
10
5
0
2001
2002
2003
2004
2005
2006
2007
2008
2009
Figure 1.1. World biodiesel production and capacity.
Currently, new economic and environmental concerns are leading to create governmental
incentives targeting to a combination of: reduction of petroleum imports and increase of
production and consumption of renewable fuels. In this way, Europe, Brazil, China, and
India each have targets to replace 5% to 20% of total diesel with biodiesel [6]. In addition,
if governments promote the development of second generation biofuels (and their
production using alternative and non-food feedstocks) throughout investment and politics,
the prospects for biodiesel market will be early reached. Figure 1.2 shows the expected
biodiesel production from different feedstocks where the share in total biodiesel
production from edible vegetable oil could decrease from almost 90% to about 75% by
2019. This expected change is due to the development of biodiesel production from
jatropha mainly in India and to the increasing use of animal fats to produce biodiesel in
the USA. Also, biomass based biodiesel could represent almost 6.5% of total biodiesel
production by 2019.
Figure 1.2. Global biodiesel production by feedstock
Under the above described situation, high quantities of raw glycerol are continuously
produced since it is obtained in ratio of 9 wt % respect to the produced biodiesel. Then, in
order to avoid both economic and environmental drawbacks related to the use and
disposal of glycerol, new applications for glycerol must be proposed. Even though, the
most traditional applications of glycerol have been related to its use as additive in: food,
tobacco, pharmaceuticals and medicine, and for the synthesis of trinitroglycerine, alkidic
resins, and polyurethanes, one of the most attractive alternatives for glycerol utilization is
as feedstock for producing added-value compounds such as: bioplastics, platform
chemicals, and fuels. Thus, because of both the low prices and high availability of
glycerol, this compound could be a great opportunity to make money through biorefineries
built adjacent to the biodiesel production plant.
From a chemical view of point, glycerol is a highly versatile molecule with two primary
hydroxyl groups and a secondary hydroxyl group which offers different reaction
possibilities. Meanwhile from a biochemical view of point, the glycerol molecule is
abundant in nature in the form of triglycerides (a chemical combination of glycerol and
fatty acids) which are the major constituents of nearly all vegetable oils and animal fats.
Thus, the high functionality and occurrence in nature of glycerol allow it to be transformed
by a chemical route or a fermentative way, as it was above indicated.
1. Introduction
In this way, the most important possibilities for glycerol transformation to added-value
compounds are here reviewed and methodologically assessed by mean of processes
engineering tools such as: process design, process simulation, and economic evaluation.
And finally all the analyzed possibilities are systematically compared.
1.2 Thesis objectives

This thesis aims to design and assess technological schemes for the conversion of raw
glycerol obtained during the biodiesel production to added-value products, in order to
identify the best alternatives from a technical and economic view of point. Thus, this
research required: (i) to identify and select the most promissory possibilities for glycerol
transformation, (ii) to simulate and assess the chosen technological schemes and
scenarios for the several identified potential products from glycerol, and (iii) to compare
these technological schemes based on economic criteria.
1.3 Thesis structure

This thesis presents the results of different studies that have been already published or
are under review for their publication. The thesis is accordingly divided into the following
chapters:
Chapter 2: The glycerols world
This chapter introduces to the reader with the current status of glycerol as the by-product
on biodiesel production and discusses the glycerol problem related to its oversupply.
Additionally, the main uses of glycerol as additive and its market are also presented.
Finally, the glycerol conversion possibilities are described.
Chapter 3: Methodology to design and analyze processes based on simulation tools.
This chapter details the used methodology for the process design, processes simulation,
and process assessment in both cases, i.e., chemical and fermentative conversion of
glycerol.
Chapter 4: Separation and purification of glycerol
This chapter presents the requirements for the most important commercial qualities of
glycerol, as well as the influence of the feedstock used for biodiesel production on the
glycerol layer. Additionally, conventional and non-conventional processes for raw glycerol
purification are discussed. Finally, the purification costs of raw glycerol up to different
commercial qualities are obtained based on simulation and economic assessment tools.
Chapter 5: Chemical conversion of glycerol
This chapter reviews the alternatives for chemical conversion of glycerol by different
reaction ways such as: oxidation, reduction (hydrogenolysis), etherification, pirolysis, and
gasification. Conversion levels, yields, selectivities, and productivities are also presented.
Chapter 6: Biochemical conversion of glycerol
This chapter reviews the alternatives for fermentative conversion of glycerol by different
strains. Fermentation products such as: 1,3-propanediol, ethanol, lactic acid, succinic
acid,
propionic
acid,
poly-3-hydroxybutyrate,
and
biosurfactants
are
discussed.
Additionally, conversion levels, yields, selectivities, and productivities are presented.
Chapter 7: Cases of study for chemical conversion of glycerol
This chapter presents the flowsheets, simulation results, and economic assessments for
the chemical conversion of glycerol to: acrolein, 1,2-propanediol, and hydrogen.
Chapter 8: Cases of study for biochemical conversion of glycerol
This chapter presents the flowsheets, simulation results, and economic assessments for
the fermentative conversion of glycerol to: 1,3-propanediol, ethanol, D-lactic acid, succinic
acid, propionic acid, and poly-3-hydroxybutyrate.
Chapter 9: Experimental setup
This chapter shows the experimental setup performed for poly-3-hydroxybutyrate

production from glycerol using two strains: cupriavidus necator and bacillus megaterium.
1. Introduction
Chapter 10: Conclusions
This chapter contains the general conclusions of the thesis and also presents the
contributions made during this work. Finally, some recommendations for future works are
given.
Chapter 11: List of publications and submitted papers
This chapter shows the published results throughout scientific meeting, papers, book
chapters, invited book chapters, and books. Also, a list containing the submitted papers
was included.
References
[1] Posada J.A., Orrego C.E., Cardona C.A. 2009. Biodiesel production: Biotechnological
approach. International Review of Chemical Engineering (I.Re.Che.), 1(6):571-580.
[2] Yazdani S.S. and Gonzalez R., 2007. Anaerobic fermentation of glycerol: A path to
economic viability for the biofuels industry. Current Opinion in Biotechnology, 18:213219
[3] Posada J.A., Cardona C.A., 2010. Anlisis de la refinacin de glicerina obtenida como
co-producto en la produccin de biodiesel (Validation of glycerin refining obtained as a
by-product of biodiesel production). Ingeniera y Universidad 14:2-27.
[4] Posada J.A., Cardona C.A., Cetina D.M., Orrego C.E., 2009. Bioglicerol como materia
prima para la obtencin de productos de valor agregado (Bioglycerol as raw material to
obtain added value products). En: Cardona C.A. (ed). Avances investigativos en la
produccin de Biocombustibles (Reasearching advances for biofuels production).
Manizales: Ed. Universidad Nacional de Colombia sede Manizales. p. 103-127. ISBN:
978-958-44-5261-0.
[5]
Worldwide
review
on
biodiesel
production.
Austrian
Biofuels
Institute,
www.biodiesel.at, 2003.
[6] Biodiesel 2020: Global Market Survey, Feedstock Trends and Market Forecasts.
2. The Glycerols World

This chapter describes the relationship between the market of both glycerol and biodiesel,
and it also discusses the influence of the growing biodiesel production on the commercial
prices of glycerol. Additionally, the potential of raw glycerol for biorefineries developing
using it as a main feedstock is presented. Finally, an overview on the possibilities of
glycerol transformation by chemical and biochemical routes is given.
2.1 Overview
The glycerol molecule (1,2,3-propanetriol) is a highly reactive tri-alcohol which has two
primary and a secondary hydroxyl groups. Some physical characteristics are: water
soluble, colorless, odorless, viscous, and hygroscopic; with a specific gravity of 1.261 g
mL-1, melting temperature of 18.2 C, and a boiling temperature of 290 C (accompanied
by decomposition). Chemically, glycerol is able for reacting with a stable alcohol under
most operational conditions, and it is basically non-toxic to human health and
environment. The key of its usefulness is the particular combination among its
physicochemical properties, compatibility with other substances, and easy handling. Due
to these particular properties set, glycerol has found more than 1500 end-uses or large
volume applications.
Glycerol is a commodity chemical obtained mainly as by-product in the oleochemical and

biodiesel industry; meanwhile glycerin is the commercial name for mixtures containing
high quantities of glycerol. This molecule is one of the most versatile substances known
due to its unique combination of physical and chemical properties, which allows it to be
used in multitude of products and additionally it is often used as: humectant, plasticizer,
emollient, thickener, solvent, dispersing medium, lubricant, sweetener, and antifreeze.
Glycerol is naturally combined with triglycerides in all animal fats and vegetable oils,
representing about 10% of these materials. This component is derived from fats and oils
10
during fatty acids and soap production, or by the transesterification process with alcohols
for biodiesel synthesis. Although glycerol can also be produced synthetically by
petrochemical processes from epichlorohydrin and using propylene as raw material, such
processes are no longer conducted at the industrial level [1].
The glycerols world has a complex behavior since it is by-produced with biodiesel, and in
addition its price is related to the no-predictable network of both its supply and demand.
This is a typical behavior for a commodity used as additive in many applications and now
being used as raw material for the production of platform chemicals, bioplastics, and
biofuels. Here the most important topics related to the glycerol industry are elucidated.
2.2 Biodiesel industry

Biodiesel is defined as a clean burning fuel used for diesel engines, manufactured from
renewable sources (vegetable oils, animal fats, or used cooking oils) and short chain
alcohols (methanol, ethanol, or butanol), to produce a methyl, ethyl or butyl esters fatty
acids mixture. A vegetable oil usually contains up to 14 different kinds of fatty acids [2].
Most of biodiesel production processes were developed in the early 40s, during World
War II by explosives manufacturers searching for a simpler way to obtain glycerol. Now,
biodiesel is commercially produced from agricultural products such as rapeseed, soy
bean, and palm oils. Also, other high fatty acid feedstocks such as: used frying oil, grease
trap waste oil, and waste tallow or lard, have been used. Several variables as local
availability, cost, government support, and fuel performance must be analyzed in order to
choose the best feedstock, since biodiesel production costs are highly dependent upon
the feedstocks price.
Biodiesel is a fuel with low viscosity and pour point, non-toxic, and biodegradable, which
is also cleaner than diesel. Biodiesel is mainly composed by a mixture of fatty acid alkyl
esters (FAAE), which can be produced from vegetable oils, wasted cooking oils, and
animal fats. Thus it is considered as renewable fuel source. Recently biodiesel has been
promoted as a way for enhancing energy independence, promoting rural development,
and reducing green house gas emissions.
11
Biodiesel can be produced through the reaction between feedstock oil with either
methanol or ethanol. Oils solubility in methanol is lesser than in ethanol, and rate reaction
is mass transfer limited and methanol makes higher equilibrium conversion due to higher
reactive intermediate methoxide. Most of the biodiesel is produced currently using
methanol, which is petrochemically obtained. This dependence on methanol could be
considered as non renewable basis. On this way, different efforts to produce biodiesel
from ethanol are carried out to generate a renewable process [3-5]. Also ethanol could be
a renewable alternative to produce biodiesel because it can be obtained from glycerol
which could be also obtained during the same biodiesel process [3-6].
Transesterification process can be carried out by two ways, chemically or biocatalytically

catalyzed. Chemical catalysis has other two alternatives, alkali- and acid- catalysis.
Industrial production of fuel biodiesel is performed by methanolysis using alkaline
catalysts, and high conversion levels in short reaction times are reached. However this
way has several drawbacks: free fatty acids (FFAs) and water interfere with the reaction
generating fatty acid alkaline salts (soaps). Soaps should be removed by washing water,
which also removes glycerol, methanol (MeOH), and catalyst. Also alkaline catalyst has to
be removed from the product. Raw glycerol as by-product should be treated as a waste
material making the glycerol recovery difficult, and the alkaline wastewater requires
treatment. It is also an energetically intensive process [7-8]. On the other hand, in acid
catalysis process, sulfuric and sulfonic acid are preferred because these carry out high
alkyl esters yields. But elevated reaction temperatures (>100C) and reaction times (ca 50
h) to complete conversion are required.
Commonly, a catalyst is used to improve reaction rate and yield, and an alcohol excess is
utilized to shift the equilibrium towards the products side. Among the used catalysts are
alkalis (NaOH, KOH, sodium and potassium alcoxides, carbonates, etc), acids (sulfonic
acids, HCl, H3PO4, H2SO4, zeolites), enzymes (lypases), and whole cells.
Acid catalysis produce high alkyl esters yield. High reaction temperatures and reaction
times to obtain complete conversion are required. Basic catalysis is a quick reaction, with
high yields, which take place under moderate conditions in comparison with acid
catalysts, but chemical transesterification using an alkali-catalysis has several drawbacks
like soap formation by saponification, and difficult recovery of glycerin by emulsion
12
development. In contrast, biocatalysts allow synthesis of specific alkyl esters, easy

recovery of glycerol, and transesterification of glycerides with high free fatty acid content
[9], and its main disadvantages are the biocatalyst cost and lower reaction rates [10-12].
Lipase enzymes have been used in biodiesel production in free form or immobilized on
some different materials such as ceramics, kaolinites, silica, etc. [13]. In general
immobilization enhances the stability of lipase due to the ability of the support material to
retain just the right quantity of water for the enzyme to remain active. Different reactive
mixtures containing water have been analyzed. For example, immobilized Rhizopus
delemar and Rhizomucor miehei lipases efficiently catalyze alcoholysis with long-chain
fatty alcohols even in the presence of 20% water [14]. However enzymatic methods have
not been industrialized because the enzymes have high price and instability [7, 11-12].
Biodiesel production could be fully sustainable if ethanol is produced from glycerol, which
is the by-product in biodiesel production. Also, enzymatic transesterification can be
carried out using ethanol with low water content or azeotropic ethanol, without affecting
considerably the biodiesel production. Genetically modified E. coli [6] and E. aerogenes
[4-5] have been reported to ferment crude glycerol or pure glycerol to ethanol. In order to
close the renewable biodiesel production in an integrated biotechnological system the
follow structure is analyzed in the next section: aqueous-ethanol as raw material,
biocatalysts use, and biological transformation of glycerol to ethanol.
2.3 Glycerol market and its oversupply problem

Until 2003 supply of raw glycerol in the market remained relatively stable, when the
production of biodiesel started increasing in the USA [15]. Since then, the availability of
crude glycerol has been almost doubled, while its demand has remained almost
unchanged. Thus, combined effect of supply excess and limited demand of raw glycerol
led to low sale prices. Although pure glycerol is an important feedstock in many industrial
sectors, raw glycerol must be refined by large scale biodiesel producers using traditional
separation processes to remove impurities such as fatty acids, alcohol, and catalyst.
Some of these processes are filtration, chemical additions, and fractional vacuum
distillation. Generally processes are expensive and economically unfeasible for small and
medium scale plants [16].
13
Traditional commercial applications of glycerol are related to its use as an additive or raw
material. The industrial sectors who consume glycerol are: pharmaceutical (18%),
personal care (toothpaste and cosmetics 16%), polyether/polyols manufacture (14%),
food (11%), triacetin (10%), alkyd (8%), snuff (6%), detergents (2%), cellophane (2%),
and explosives (2%). The remaining share (11%) is used in the manufacture of lacquers,
varnishes, inks, adhesives, plastic synthetics, regenerated cellulose, and other industrial
uses [17].
Annually nearly 160000 tons of glycerol is used for technical applications and it is
expected an annual growth rate of 2.8%. Raw glycerin supply in the market remained
relatively stable until 2003, when biodiesel production started to increase in the U.S. and
the E.U. [15]. Then, availability of raw glycerin has almost doubled, and its demand has
remained largely unchanged. This excess supply and limited demand has taken to low
glycerol prices, but although refined glycerin prices have decreased in the last years; the
strongest impact has been suffered by raw glycerin, and thus its sale prices plummet
quickly [18].
Since 2006, the glycerol oversupply forced to biodiesel producers to receive sales prices
of 2 cents per pound or even lower prices for the raw product. But at mid-2007, reached
prices were between 6 and 10 cents per pound [19]. On the other hand, refined glycerin
prices have had a similar behavior, with prices as low as 20-30 cents per pound,
depending on the quality and purity [18-19]. In this sense the raw glycerin market will
remain weak while large amounts of this raw component being available. Therefore
glycerol is nowadays a key problem in biodiesel production, and its low sale price could
convert this by-product in a residue, then the biodiesel producers must be found
alternative uses to avoid the continue falling on the glycerol price.
2.4 Glycerol as raw material

Development of biorefineries based on raw glycerol to produce high-value compounds is
necessary in the biodiesel industry to overcome the economic glycerol drawback. The
simplest alternative to increase the value of raw glycerol is refining it to technical glycerin,
food or pharmaceutical grade, although to synthesize value-added components by
chemical o fermentative via are alternatives with higher potential. Chemically glycerol can
14
be transformed to: oxidation products on metallic catalysts as Pt, Pd, Au, using promoters
as Bi and Pb; glycols by hydrogenolysis on Ru, Cu and Pt catalysts; polyglycerols by
etherification on zeolites and mesoporous materials; and syngas by pyrolysis and
gasification.
Also, due to glycerol is abundant in nature and produced by yeasts during osmoregulation
to decrease extracellular water activity [20], its wide occurrence allows to different kinds of
microorganisms metabolizing glycerol as a sole carbon and energy source, and then this
may substitute traditional carbohydrates, such as sucrose, glucose and starch, in some
industrial fermentation processes [21-23]. Glycerol can be transform by fermentative via
to 1,3-propanediol, dihydroxyacetone, succinic acid, propionic acid, ethanol, citric acid,
pigments, polyhydroxyalcanoate, and biosurfactants. [24]. The following sections review
the main technological topics related to glycerol transformation by chemical and
biochemical via.
References
[1] McCoy M. Glycerine Surplus. Chem. Eng. News. 2006, 84(6):7-8
[2] Tyson, S.K., Biodiesel Handling and Use Guidelines. 2001, U.S. Department of
Energy. NREL/TP-580-30004: CO. USA. p. 17.
[3] da Silva, G.P., Mack, M., Contiero, J., Glycerol: A promising and abundant carbon
source for industrial microbiology, Biotechnol Adv 27(1) (2009), 30-39.
[4] Yazdani, S.S., Gonzalez, R., Engineering Escherichia coli for the efficient conversion
of glycerol to ethanol and co-products, Metab Eng 10(6) (2008) 340-351.
[5] Ito, T., Nakashimada, Y., Senba, K., Matsui, T., Nishio, N., Hydrogen and ethanol
production from glycerol-containing wastes discharged after biodiesel manufacturing
process, J Biosci Bioeng 100 (2005) 260265.
[6] Dharmadi, Y., Murarka, A., Gonzalez, R., Anaerobic fermentation of glycerol by
Escherichia coli: a new platform for metabolic engineering, Biotechnol Bioeng 94 (2006)
821829.
15
[7] Fukuda, H., Kondo, A., Noda, H., Biodiesel fuel production by transesterification of oils,
J Biosci Bioeng 92(5) (2001) 405-416.
[8] Shimada, Y., Watanabe, Y., Sugihara, A., Tominaga, Y., Enzymatic alcoholysis for
biodiesel fuel production and application of the reaction to oil processing, J. Mol. Catal. B:
Enzym. 17(3-5) (2002) 133-142.
[9] Shah, S., Sharma, S., Gupta, M.N., Biodiesel preparation by lipase-catalyzed
transesterification of jatropha oil, Energ Fuel 18 (2004) 154-159.
[10] Fukuda, H., Kondo, A., Noda, H., Biodiesel fuel production by transesterification of
oils, J Biosci Bioeng 92(5) (2001) 405-416.
[11] Xu, Y., Du, W., Liu, D., Zeng, J., A novel enzymatic route for biodiesel production
from renewable oils in a solvent-free medium, Biotechnol Lett25 (2003) 1239-1241.
[12] Shimada, Y., Watanabe, Y., Samukawa, T., Sugihara, A., Noda, H., Fukuda, H.,
Tominaga, Y., Conversion of vegetable oil to biodiesel using immobilized Candida
antarctica lipase, J. Am. Oil Chem. Soc. 76 (1999) 789-793.
[13] Yagiz, F., Kazan, D., Akin, N., Biodiesel production from waste oils by using lipase
immobilized on hydrotalcite and zeolites, Chem. Eng. J. 134 (2007) 262-267.
[14] Shimada, Y., Watanabe, Y., Sugihara, A., Tominaga, Y., Enzymatic alcoholysis for
biodiesel fuel production and application of the reaction to oil processing, J. Mol. Catal. B:
Enzym. 17(3-5) (2002) 133-142.
[15] Ott, L., Bicker, M., Vogel, H., 2006. Catalytic dehydration of glycerol in sub- and
supercritical water: a new chemical process for acrolein production. Green Chem. 8, 214220.
[16] Posada, J.A. and C.A. Cardona, Anlisis de la refinacin de glicerina obtenida como
co-producto en la produccin de biodiesel. Ingeniera y Universidad, 2010. 14(1)
[17] Pagliario, M. and M. Rossi., The future of Glycerol: New usages for a versatile raw
Material. 2008, Cambridge: RSC Publishing.
16
[18] Rahmat, N., A.Z. Abdullah, and A.R. Mohamed, Recent progress on innovative and
potential technologies for glycerol transformation into fuel additives: A critical review.
Renewable and Sustainable Energy Reviews. 14(3): p. 987-1000.
[19] Ito, T., et al., Hydrogen and Ethanol Production from Glycerol-Containing Wastes
Discharged after Biodiesel Manufacturing Process. J. Biosci. Bioeng., 2005. 100(3): p.
260-265
[20] Wang, Z., et al., Glycerol production by microbial fermentation: A review.
Biotechnology Advances, 2001. 19(3): p. 201-223.
[21] Solomon, B.O., et al., Comparison of the energetic efficiencies of hydrogen and
oxychemicals formation in Klebsiella pneumoniae and Clostridium butyricum during
anaerobic growth on glycerol. Journal of Biotechnology, 1995. 39(2): p. 107-117.
[22] Barbirato, F. and A. Bories, Relationship between the physiology of Enterobacter
agglomerans CNCM 1210 grown anaerobically on glycerol and the culture conditions.
Research in Microbiology. 148(6): p. 475-484.
[23] Menzel, K., A.P. Zeng, and W.D. Deckwer, High concentration and productivity of
1,3-propanediol from continuous fermentation of glycerol by Klebsiella pneumoniae.
Enzyme and Microbial Technology, 1997. 20(2): p. 82-86.
[24] da Silva, G.P., M. Mack, and J. Contiero, Glycerol: A promising and abundant carbon
source for industrial microbiology. Biotechnology Advances. 27(1): p. 30-39.
3. Methodology for Processes Design and Analysis

This chapter describes a methodological procedure in order to design and assess
technological schemes for the conversion of raw glycerol to added-value products. This
methodology uses a strategy based on knowledge which employs both heuristic rules and
researchers experience. Also it is equally applied to chemical or biochemical processes,
as well as conventional technologies or integrated process. Also, directions are given to
perform both steps: the process simulation and the process assessment.
3.1 Processes design

This thesis aims to design and assess technological schemes for the conversion of raw
glycerol obtained during the biodiesel production to added-value products. Thus, different
possibilities of glycerol conversion to added-value products should be first indentified
based on the reported literature. In this way, two main routes for glycerol transformation
are available, chemical conversion and fermentative transformation. Glycerol can be
chemically transformed by many ways such as: oxidation, hydrogenolysis, etherification,
pyrolysis, and gasification. In this sense, many catalysts such as: Pt, Pd, Au, Ru, Cu, Pt,
zeolites, and mesoporouses materials, have been widely reported for glycerol conversion.
Otherwise, many wild and metabolically engineered strains have been analyzed for the
glycerol uptake as substrate in order to produce a wide spectrum of metabolites such us:
1,3-propanediol, ethanol, poly-3-hydroxibitirate, lactic acid, propionic acid, succinic acid,
and rhamnolipids.
In order to achieve this objective it was required to: (i) classify all the information available
on glycerol transformation by chemical or fermentative routes; (ii) organize these
information based on the specific used way for either route, chemical (e.g., oxidation,
hydrogenolysis, etherification, pyrolysis, and gasification) or fermentative (e.g., production
of: 1,3-propanediol, ethanol, poly-3-hydroxibitirate, lactic acid, propionic acid, succinic
18
acid, and rhamnolipids); (iii) compare and analyze each transformation possibility based
on operational criteria such as: conversion, yield, and productivity; and (iv) choose the
conversion possibilities with the higher potential to be commercialized. This first stage
corresponds to both the literature review and the choosing of the most attractive
possibilities for glycerol conversion to added-value components. Moreover, it was found
that not only pure glycerol has been used as feedstock to its transformation but also crude
glycerol has been widely analyzed. Thus, in order to homogenize the feedstock used for
this study, raw glycerol obtained from the biodiesel production process was considered as
the unique feedstock. In this way, the influences of several feedstocks used for biodiesel
production were analyzed on the composition of the glycerol layer and an average
composition for the raw glycerol stream was chose. Due to this raw glycerol stream
contains low quantity of glycerol, a purification process was analyzed in order to obtain
the three most important qualities of commercially available glycerol. Under this view of
point, only one feedstock (i.e., raw glycerol) is always considered and different qualities of
glycerol (crude glycerol, technical glycerol and USP glycerol) can be used for its
transformation. Additionally, the fact of work with raw glycerol as the unique feedstock,
allows considering any designed process as an adjacent biorefinery to the biodiesel
production process.
Because of many final products and transformation routes are considered though out this
study, each alternative requires both a specific process analysis and a process design
using a strategy based on knowledge.
The synthesis of technological schemes by mean of a strategy based on knowledge

allows generating systematically alternatives which consider the specific characteristics of
each process. Thus, it is possible to design technological configurations of high
performance
considering
mainly
techno-economic
criteria.
Here,
the
traditional
hierarchical decomposition methodology based on the onion diagram (see Figure 3.1.) for
process design is applied [1]. This sequential procedure allows designing and comparing
different alternatives for the same objective.
The process design starts analyzing the reaction step which is the fundamental stage in
this study, and then the analysis continues to the external layer of the onion diagram
adding stages such us: the separation and recycle system according to the Figure 3.1.
3. Methodology
19
This heuristic and hierarchical methodology emphasizes in both the decomposition and
analysis of different process alternatives, allowing a quick selection of technological
configurations that are often close to the best solution. Furthermore, the nature of this
approach, allows discarding many configurations easily which in general do not lead to
"good" designs. In addition, tiered design allows the use of process simulators and thus
the process diagram can be completed in an evolutionary manner. This methodology has
been applied primarily to processes of chemical or petrochemical industry.
Figure 3.1. Hierarchical decomposition according to the "onion diagram"
On the other hand, for the downstream process design the method so called breadth-first
was applied in order to analyze different alternatives for the products recovery. This
method allows both screening the best alternative for a specific purpose of the
downstream process, and evaluating of process alternatives at the next level of
hierarchical decomposition (see Figure 3.2).
Most of the alternatives for glycerol conversion to added-value compounds are analyzed
during first hierarchical decomposition levels (1 and 2) by mean of the economic potential
criteria which also involves operational variables such as conversion and yield. Thus, the
alternatives with the highest economic potential are selected to continue the synthesis of
technological schemes, while the alternatives showing unfavorable economic potential are
20
discarded. Thus, base structures are obtained for the chosen conversion possibilities
which are later complemented through detailed process information for the main stages of
processing.
Figure 3.2. The process design method based on the so called breadth-first
The last hierarchical levels of analysis (3-6) require more detailed information for the main
process variables which are performed by mean of sensitivity analysis and subsequent
rigorous economic evaluation. Details of the processes simulation and economic
assessment are given below.
3.2 Processes simulation

Aspen Plus (Aspen Technology, Inc., USA) is the main used tool for defining, structuring,
specifying, and simulating the technological schemes for either chemical or biochemical
conversion of glycerol to added-value components.
Information required for simulating the most basic technological schemes such as:
physical and chemical properties, parameters of design, and operation of processing
units, are mainly obtained from secondary sources (e.g., articles, technical reports,
databases, patents, among others). Then, the most complex and detailed technological
schemes are obtained by mean of rigorous simulations which involve sensitivity analysis
and search of optimal operation conditions.
Because of both the petrochemical character of Aspen Plus and its modular-sequential
approach, there are not available kinetic models describing the biotechnological
processes such as fermentations or enzymatic reactions. Therefore, it is required to work
with the available interface between Aspen Plus and Excel. Additionally, the study of
3. Methodology
21
complex fermentation kinetics describing inhibition phenomena, in some cases requires

generating more complex calculation routines using other kind of softwares (e.g.,
MatLab).
Although several possibilities for glycerol conversion to added-value products have been
reported, a few publications describe kinetic models fitted good enough. Therefore, the
stoichiometric approach is here considered as a completely valid and relevant approach
for analyzing the reaction stage of different technological schemes.
On the other hand, specific compounds involved in the different processes of raw glycerol
conversion to added-value products such as: free fatty acids, alkyl esters, proteins, salts,
cell mass strains, enzymes, and other complex molecules produced by reactive-extractive
process are not available on the Aspen Plus Database. Thus, these compounds should
be created for each simulation as follows: conventional (by mean of group contribution
methods), solids (e.g., biomass), or non-conventional (e.g., enzymes).
All processes are designed and analyzed using the same calculation base which is 1000
Kg/h of raw glycerol always fed to the glycerol purification process. As the simulation
results, mass and energy balances are obtained for the technological schemes. Thus, it is
possible to obtain requirements of additional raw material, solvents, utility fluids, and
energy.
The analysis of conventional separation methods in the distillation process was carried
out with the help of the corresponding modules of the process simulators. For this, both
short-cut methods and rigorous models available in the simulation package were
employed. For simulation of the different technologies involving the operation of
distillation, the short-cut method DSTWU incorporated in the package Aspen Plus was
applied. This method uses the equations and correlations of Winn-Underwood-Gilliland in
order to provide an initial estimation of the minimum number of theoretic stages, minimum
reflux ratio, location of the feed stage, and components distribution. The rigorous
calculation of the operating conditions in the distillation columns was performed using the
module RadFrac based on the equilibrium method that employs the MESH equations
(Mass balance equations, phase Equilibrium equations, Summation of the compositions,
and Heat balance equations) using the inside-out algorithm. Residue curve maps were
22
used for the conceptual design of the distillation schemes applying the principles of
topological thermodynamics (analysis of the statics) [2]. Sensitivity analyses were
performed in order to study the effect of the main operating variables (reflux ratio,
temperature of the feed stream, ratio between the distillate and the feed, etc.) on the
biodiesel purity and the energy consumption of this operation. The final result is the
determination of operating conditions that allow developing energetically efficient
processes. The objective of this procedure was to generate the mass and energy
balances from which the requirements for raw materials, consumables, service fluids and
energy needs are calculated.
On the other hand, because of the significant differences involved in the reaction of
glycerol to different products, the reaction conditions are specific for each technological
scheme. Besides, the downstream process is designed based on the products distribution
obtained after the reactive stage. Thus, detailed information about reaction stage and
downstream process is given according to each case of study.
Estimation of the energy consumption is performed based on the results of the mass and
energy balances generated during the simulation process. Thus, the thermal energy
required in the heat exchangers and re-boilers was taken into account, as well as the
electric energy needed by pumps, compressors, mills, and other equipments. The energy
demand was calculated from the mass and energy balances generated by the simulator.
The balances included the energy consumption of reboilers and condensers used in
distillation columns, and the energy consumption of the reactors.
3.3 Processes assessment

The capital and operating costs were calculated using the software Aspen Icarus Process
Evaluator (Aspen Technologies, Inc., USA). This software estimates the capital costs of
process units as well as the operating costs, among other valuable data, utilizing the
design information provided by Aspen Plus and the data introduced by the user for
specific conditions such as project location among others. Also, analyses are based on
the strategy designed by Cardona et al [3-7] for process assessment.
3. Methodology
23
This analysis was estimated in US dollars for a 10-year period at an annual interest rate
of 16 %, considering the straight line depreciation method and a 33% income tax [8]. The
cost for raw glycerol, crude glycerol, and refined glycerol as well as the labor cost for
operatives and supervisors, and the prices for electricity, water and low pressure vapor
are showed in the Table 3.1. Additionally, the commercial price for other required
compounds such us raw materials and solvents are listed in the Table 3.1.
Table 3.1. Used costs and prices for the economic assessment
Costs
Value
Units
Operatives
2.14
UDS$/h
Supervisors
4.29
UDS$/h
0.03044
UDS$/kwh
Water
1.252
UDS$/m3
Low pressure vapor
8.18
UDS$/Ton
Raw glycerol
132.45
UDS$/Ton
Crude glycerol (85 wt %)
540.84
UDS$/Ton
Refined glycerol (98 wt %)
706.41
UDS$/Ton
Succinic acid
2492.2
UDS$/Ton
Lactic acid
1552.2
UDS$/Ton
Acetic acid
591.8
UDS$/Ton
Dichloromethane
850
UDS$/Ton
Trioctylamine
2550
UDS$/Ton
Methanol
290
UDS$/Ton
1-Octanol
1835
UDS$/Ton
XX
UDS$/Ton
1,3-Propanediol
1766
UDS$/Ton
Propionic acid
1220
UDS$/Ton
DES
3050
UDS$/Ton
Glucose
480
UDS$/Ton
PHB
3500
UDS$/Ton
Propionic acid
1800
UDS$/Ton
Electricity
Iso-butylaldehyde
24
References
[1] Smith R.M. The nature of chemical process design and integration. Chemical Process:
Design and Integration. John Wiley & Sons: Hoboken, NJ, USA, (2005), pp 1-15.
[2] Pisarenko Y.A., Serafimov L.A., Cardona C.A., Efremov D.L., Shuwalov A.S., Reactive
distillation design: analysis of the process statics. Reviews in Chemical Engineering 17
(4), 2001.
[3] Cardona, C.A., Snchez, O. J., Energy consumption analysis of integrated flowsheets
for production of fuel ethanol from lignocellulosic biomass. Energy. 2006. 31:2447-2459.
[4] Cardona, C.A., Snchez, .J., Fuel ethanol production: Process design trends and
integration opportunities. Bioresource Technology. 2007. 98 2415-2457.
[5] Quintero, J.A., Montoya, M.I., Snchez, O.J., Giraldo, O.H., Cardona, C.A., Fuel
ethanol production from sugarcane and corn: Comparative analysis for a Colombian case.
Energy. 2008. 33:385-399.
[6] Cardona, C.A., Gutirrez, L.F., Snchez, O. J., In Energy Efficiency Research
Advances (D. M. Bergmann, ed.). 2008. Pp:173-212. Nova Science Publishers,
Hauppauge, NY, USA.
[7] Gutirrez, L.F., Snchez, .J., Cardona, C.A., Process integration possibilities for
biodiesel production from palm oil using ethanol obtained from lignocellulosic residues of
oil palm industry. Bioresource Technology. 2009. 100:1227-1237.
[8] Posada J.A., Cardona C.A., Rincn L.E., Sustainable biodiesel production from palm
using in situ produced glycerol and biomass for raw bioethanol. In 32nd symposium on
biotechnology for fuels and chemicals. Clearwater Beach, Florida. April 19-22. 2010.
4. Separation and Purification of Glycerol

This chapter presents commercial, technical, and technological aspects related to the
glycerol purification process such as the most important commercially qualities of glycerol,
the influence of the feedstock used for biodiesel production on the glycerol layer, and the
conventional and non-conventional purification processes. The most important qualities of
commercial glycerol are: crude glycerol (80-88 wt %), technical glycerol (98 wt %), and
refined glycerol (USP or FCC grades, 99.7 wt %). Thus, a flowsheet able to purify raw
glycerol up to these three qualities was designed, simulated, and economic assessed.
Simulation results showed that is possible to reach the quality requirements while
economic results showed that is a profitable process. Also, recovering of anhydrous
methanol at 99 wt % could represent an additional incoming for the purification process
which could reduce the purification costs among 19 to 26 %. Simulation process is carried
out using Aspen Plus software, while the economic evaluation is performed by Aspen
Icarus Process Evaluator package.
4.1 Commercial qualities of glycerol

Most of the marketed glycerol is manufactured to satisfy the strict requirements of the
United States Pharmacopeia (USP) and the Food Chemicals Codex (FCC) (The Soap
and Detergent Association). However, technical grades of glycerol which have not been
certified as USP or FCC are also available in the market. The three main qualities of
glycerol commercially available depend on their purity, these are: raw glycerol, technical
glycerol, and refined glycerol (USP or FCC grade). Raw glycerol usually has between 40
and 88 wt % of glycerol, and contains high amount of methanol, soaps, and salts. This
glycerol is commonly obtained as by-product on biodiesel production. Technical glycerol
is a high purity product where most of its pollutants have been totally removed. This
glycerol is free of methanol, soaps, salts, and other components. Refined glycerol is a
pharmaceutical quality product which can be used in foods, personal care, cosmetics,
26
pharmaceutical products, and other special applications. Also, these products must
complete the specifications of Pharmacopeia of the USA (USP 30) and the Food and
Drug Administration (FDA) of the USA. Table 4.1 shows the main quality specifications
and the thresholds for the pollutants present in this glycerol [1].
Table 4.1. Quality specifications for the main qualities of glycerol

Properties
Raw
Glycerol
Technical
Glycerol
Refined Glycerol
(USP)
Glycerol Content
40-88%
98.0% Min
99.70%
2.0% Max
N/A
N/A
Moisture
N/A
2.0% Max
0.3% Mx.
Chlorides
N/A
10 ppm Max
10 ppm Mx.
Color
N/A
40 Max (Pt - Co)
10 Max. (APHA)
Specific Gravity
N/A
1.262 (@25C)
1.2612 Min
Sulfate
N/A
N/A
20 ppm Mx
Analysis
N/A
N/A
99.0 - 101.0%
(dry base)
Heavy Metals
N/A
5 ppm Mx.
5 ppm Mx.
Chlorates
Components
N/A
30 ppm Mx.
30 ppm Mx.
Ignition Residues
N/A
N/A
100 ppm Mx.
Fatty acids and

Esters
N/A
1.00 Mx
1000 Mx
12.0% Max
5.0% Mx
0.5% Mx
pH (solution 10%)
4.0 - 9.0
4.0 - 9.1
N/A
Organic Residues
2.0% Mx
2.0% Mx
N/A
Ash
Water
4. Glycerol Purification
27
4.2 Effect of the feedstock for biodiesel production on

glycerol composition
Raw glycerol has a very low value in the market because of its impurities. Also,
composition of glycerol highly depends on both the family of used raw material and the
process conditions for biodiesel production. This fact occurs because the chemical
compositions of the feedstocks used for biodiesel production could change significantly.
Fats and oils usually contain more than ten types of fatty acids, which have between 12
and 22 carbons. But, often the higher proportion of fatty acids has between 16 and 18
carbons. Although these fatty acids are saturated, monounsaturated or polyunsaturated
[2], different degrees of saturation affect the properties of the biodiesel fuel. Thus, a
"perfect" biodiesel should be only obtained from monounsaturated fatty acids.
The composition profile of fatty acids was presented by He and Thompson [3] for six
vegetable oils (i.e., IdaGold mustard, PacGold mustard, rapeseed, canola, crambe, and
soybean) and for waste vegetable oil (WVO) used as feedstocks on biodiesel production
as shown in Table 4.2. Additionally, based on the reported information by He and
Thompson [3], the composition of glycerol layer was calculated for each used feedstock
for biodiesel production. The results are shown in Table 4.3.
Table 4.2. Fatty acid profile of vegetable and used oils [3]
Composition (wt %)
Fatty acids
IdaGold PacGold Rape Canola Soybean Crambe WVO
Palmitic (16:0)
2,8
3,1
2,9
4,5
10,7
18,7
Estearic (18:0)
1,6
1,8
4,3
0,9
6,3
Oleic (18:1)
24,8
23,9
13,7
60,7
24,9
17,9
40,5
Linoleic (18:2)
10,3
21,6
11,8
19,1
51,6
8,1
28
Linolenic (18:3)
9,4
9,9
7,5
9,5
7,3
4,5
1,5
Eicosic (20:1)
10,7
12,1
8,7
1,8
0,2
3,7
---
Erucic (22:1)
34,7
22,1
48,5
0,9
---
54,1
---
MW Average
(Kg/Kmol)
946,3
924,6
968,5 882,1
872,8
978,5
867,2
28
Table 4.3. Composition of the glycerol layer obtained by decantation during the biodiesel
production from different feedstocks
Oil
Component
IdaGold PacGold Colza Canola Soja
Methanol (wt %)
32,59
32,68
28,20 25,07
26,06 23,17
11,72
Glycerol (wt %)
60,05
61,39
59,94 60,38
61,67 65,01
46,41
NaOCH 3 (wt %)
2,62
2,82
2,27
2,24
2,56
2,69
1,99
Proteins (wt %)
0,13
0,18
0,06
0,05
0,05
0,46
0,14
Fats (wt %):
1,94
1,08
8,88
11,68
7,17
8,42
36,41
Palmitic (16:0)
0,054
0,030
0,249 0,327
0,201 0,236
1,020
Estearic (18:0)
0,019
0,011
0,089 0,117
0,072 0,084
0,364
Oleic (18:1)
0,480
0,269
2,203 2,896
1,779 2,087
9,030
Linoleic (18:2)
0,200
0,112
0,915 1,203
0,739 0,867
3,750
Linolenic (18:3)
0,182
0,102
0,835 1,098
0,674 0,791
3,423
Eicosic (20:1)
0,207
0,116
0,951 1,250
0,767 0,901
3,896
Erucic (22:1)
0,672
0,376
3,082 4,052
2,489 2,921
12,635
2,67
1,85
0,64
2,48
3,33
Ash (wt %)
0,58
Crambe WVO
0,26
4.3 Conventional purification process

At laboratory scale the purification of the system containing biodiesel, glycerol, soaps, and
salts (mainly sodium methoxide, NaOCH3), is preformed using separation funnels, which
allow to soaps being remained in the crude glycerol layer. Layer containing esters must
be heated up to 85 C in order to recover the unreacted methanol. While industrially raw
glycerol is refined through a filtration process, followed by mixing with chemical additives
which allow the precipitation of salts and finally different qualities of commercial glycerol
are obtained by a vacuum fractional distillation process.
29
Distillation is the most commonly used method for glycerol purification. This technology
produces high purity glycerol at high yields. However, the glycerol distillation is an energy
intensive process because of its high heat capacity, requiring a high supply of energy for
vaporization [4]. Ion exchange has also been used to purify raw glycerol [5], but this
technique is not economically viable from an industrial view of point due to the high
content of salts. Also, when contents of sales are above 5 wt % which is tipically found in
the glycerol stream obtained from the biodiesel industry, the chemical regeneration cost of
these resins becomes very high. Figure 4.1 shows the flow diagram for the two above
described conventional techniques for glycerol purification.
Purification by
vaccum distillation
P-7
Concentration
by evaporation
Filtration
Purification by
ionic exchange
Evaportation and refining

of crude glycerol
Figure 4.1. Flowsheet of conventional schemes for glycerol purification.
4.4 Alternative purification processes

A commercially available technology for raw glycerol purification obtained during the
biodiesel production was jointly developed by Rohm and Haas, a provider of functional
polymers by ion exchange technologies and catalysts, and by Novasep Process, a
supplier of purification solutions which includes chromatography, ion exchange,
membranes, crystallization, and evaporation. The process is the so called Ambersep
BD50 [6]. In principle, this process uses a chromatographic separator in order to remove
large amount of salts and free fatty acids. Refined stream is then processed in an
evaporator / crystallizer unit, which removes the salts in a crystalline form. This fact
30
avoids the effluents production in the glycerol purification plant. Thus, a glycerol stream at
a purity of 99.5 wt % is obtained. But if a high quality glycerol is required, (e.g., 5 to 10
parts per million of salt content) it is possible to use a ion exchange demineralization unit.
This process has lower energy requirements compared to the traditional distillation
process. The block diagram for the Ambersep BD50 process is shown in Figure 4.2 which
illustrates the different steps for the raw glycerol purification process.
Crude
Pre-Heating
Heating up to 90C
Filtration
Heating up to 90C
Degasification
Filtration
Refined
Degasification
Crystallization NaCl
CHR
Effluent
Light Water
Secondary
Glycerol
NaCl
Cooling at 40C
(Optional)
IEX (Optional)
Concentration
Refined
Glycerol
Figure 4.2. Flowsheet of the Ambersep BD50 process
31
4.5 Simulation of the glycerol purification processes

Based on the calculated compositions for the glycerol layer obtained from different
feedstocks (see Table 4.3.), the profile compositions obtained from IdaGold mustard
represents the average values among the first use oils analyzed. Thus, this stream was
chosen to design the purification process of the raw glycerol.
Figure 4.3.a. shows the simplified flowsheeet for raw glycerol purification to 88 wt %
(crude glycerol) and to 98 wt % (technique glycerol). In order to obtain glycerol at 99.7 wt
% (glycerol USP grade), it is required a further refining process throughout a ion
exchange resin which removes the triglycerides still contained in the mixture, as shown in
Figure 4.3.b.
Water
waste 1
Methanol
Water
RII-1
Water
waste 2
Glicerina
USP
Raw
Glycerol
Solids
Organic
Phase
Aqueous
Glycerol
Glycerol
Adsorbato
Figure 4.3. Simplified flowsheet for raw glycerol purification. a) purification at 88 and 98
wt %. b) Purification at 99.7 wt %. 1. First evaporation column, 2. Neutralization tank, 3.
Centrifuge, 4. Decantation tank, 5. Second evaporation column, 6. Distillation column. 7:
Ionic exchange resine.
The raw glycerol stream is initially evaporated, where 90 % of methanol at 99 wt % is

recovered. Also, since glycerol is the unique impurity present in the recovered stream, this
steam of anhydrous methanol is appropriate to be reused in the transesterification
process. Bottom stream obtained from Evaporator I is neutralized using an acid solution.
Then both salts produced during the neutralization process and remaining ashes and
proteins are retired by centrifugation. The clarified product obtained from the centrifuge is
washed with water using a weight ratio of 2.4 (water/glycerol stream). Thus, 50% of the
triglycerides remaining in the mixture are withdrawn with a glycerol lost of 1.8 %. The
resulting aqueous glycerol stream and free of salts, solids, and protein but with a low
content of both methanol and triglycerides, is subjected again to an evaporation process
which removes more than 90% of water and most of the remaining methanol, with a
32
glycerol lost of 0.2%.Thus, the glycerol purity reached is 80 wt %. Then, the glycerol
stream is purified through a distillation column to reach the required purity, either 88 wt %
or 98 wt %. Although in all cases the used flowsheet is the same, the operational
conditions change depending on the required purity.
In order to obtain glycerol at USP grade, the process conditions adjusted for glycerol at 98
wt % are in general preserved, but both the reflux ratio and the ratio of distillate/feed are
increased for the distillation tower. Also, a final refinement stage through an ion exchange
resin is required to remove 95 % of the triglycerides contained still in the mixture. Table
4.4 summarizes the simulation results obtained for the purification processes of raw
glycerol. Also, it can be observed that the obtained products meet the quality
requirements shown in Table 4.1.
Table 4.4. Simulation results for raw glycerol purification process.

Streams
Variable
Temperature (C)
Raw
Glycerol
Methanol
Glycerol
at 88%
Glycerol
at 98%
Glycerol
at 99,7%
25
144,2
104,7
189,2
204
973,3
301,981
665,25
596,595
586,179
Mass flow free of ash (kg/hr)

0,014
Mass fraction:
Triglycerides
0,02
0,015
0,016
0,001
Methanol
0,335
0,99
Water
0,105
0,004
0,002
Glycerol
0,617
0,01
0,88
0,98
0,997
NaOCH 3
0,027
4,3 ppm
4,3 ppm
1,62 ppm
Protein
0,001
0,2 ppm
0,2 ppm
75 ppb
27,6
0,003
0,003
0,00015
Mass flow of ash (kg/hr)
33
4.6 Economical assessment for glycerol purification

processes
Because of the fed glycerol stream contains 32.6 wt % of methanol it is required to
consider two different scenarios. The first one considers that the withdrawn methanol is
not recovered while the second scenario considers that the withdrawn methanol from the
raw glycerol stream is recycled and reused as feedstock during the transesterification
process since this stream is composed of 99 wt % methanol and 1 % glycerol. Thus,
under the light of the second scenario, methanol is considered as a by-product stream
which has an economical value. Economic assessment results for raw glycerol purification
to 88, 98, and 99.7 wt % are shown in Table 4.5. The purification costs (PC) for each
purification process are in the first column discriminated by raw materials, utilities,
operating labor, maintenance and operating charges, plant overhead, general and
administrative costs, capital depreciation, and co-products credit. The second column
contains the share of each item by each purification process.
Table 4.5. Purification costs (PC) of raw glycerol (US$/L)

Item
% CP
(US$/L)
CP Glycerol % CP CP Glycerol % CP CP Glycerol

(88%)
88 wt %
98 wt %
(98%) 99.7 wt %
Raw materials
0.05539
24.78
0.05539
23.55
0.05539
23.11
Utilities
0.03741
16.73
0.07290
31.00
0.13544
56.51
Operating labor
0.01889
8.45
0.01889
8.03
0.02173
9.07
Maintenance
0.00721
3.22
0.00793
3.37
0.00979
4.08
Operating charges
0.00472
2.11
0.00520
2.21
0.00543
2.27
General costs
0.01305
5.84
0.01436
6.11
0.01576
6.57
Administrative costs
0.01093
4.89
0.01179
5.01
0.01257
5.24
Capital depreciation
0.07595
33.97
0.07595
32.30
0.08983
37.48
Co-products credit
0.05900
-26.39 0.06574
-25.05 0.06691
CT without sale of metanol
0.22356
0.26241
0.34593
CT with sale of metanol
0.16456
0.19666
0.27902
(99.7%)
-19.34
34
Due to the low commercial prices of raw glycerol obtained from the biodiesel production,
raw materials represent less than 25% of the total purification cost. The capital cost
accounts the most share of the purification cost, being it about 35%. Moreover, an
increase in the glycerol purity represents an increase in the utilities cost, reaching up to
56.5% of total purification cost when glycerol at 99.7% is obtained. On the other hand, the
recovery of anhydrous methanol at 99 wt % represents a significant reduction in the total
purification cost, with a decreasing between 19 26 % of the total costs.
The purification cost of raw glycerol obtained from biodiesel production was reported by
Johnson and Taconi [7] as 0.15 USD$/lb or 0.26 USD$/L. This value is close to the total
purification cost obtained for glycerol at 98% at the scenario that no considers the sale of
anhydrous methanol as co-product. This scenario is the most standard analyzed since it
is a technical quality of glycerol with no by-products production.
Commercial sale prices for different qualities of glycerol are as follows: 0.28 USD$/L for
glycerol at 88 wt %, 1.39 USD$/L for vegetable glycerol at 98 wt %, 1.11 USD$/L for
tallow glycerol at 98 wt % and 3.48 USD$/L for glycerol USP grade or at 99.7 wt %. For
the assessed production scale, the purification and refining of raw glycerol is profitable
since the purification costs are lower than their selling prices.
4.7 Conclusions
Commercially three qualities of glycerol were identified as the most important ones. Crude
glycerol with a purity ranging from 80-88 wt %, technical glycerol mainly found at 97 wt %,
and refined glycerol (USP or FCC grades) at 99.7 wt %. These three types of glycerol
differ significantly in the content of water, fatty acid residues, esters, and other organic
wastes. Also, some differences were found for the use of diverse feedstocks for biodiesel
production on the composition of the glycerol layer. Although, most of the first use oils
lead to not big differences in the glycerol layer, a completely different behavior was
observed for the glycerol obtained from WVO represented by low concentration of
glycerol and methanol with a high content of fats. On the other hand, based on the
traditional purification of glycerol, a flowsheet able to purify raw glycerol up to the three
commercial qualities above described was designed, simulated and economically
assessed. Results showed that not only quality requirements were successfully obtained
35
but also for the analyzed purification scale all the processes were profitable. Thus, a
homogenized raw material and purification process was obtained in order to continue the
analysis of different possibilities of glycerol transformation to added-value products.
References
[1] Posada, J.A. and C.A. Cardona, Anlisis de la refinacin de glicerina obtenida como
co-producto en la produccin de biodiesel. Ingeniera y Universidad, 2010. 14(1)
[2] Beln-Camacho, D.R, Snchez, E.D., Garca, D., Moreno-lvarez, M.J., Linares O.
Caractersticas fisicoqumicas y composicin en cidos grasos del aceite extrado de
semillas de tomate de rbol (Cyphomandra betacea Sendt) variedades roja y amarilla.
Grasas y Aceites. 2004, 55(4):428-433
[3] Thompson, J.C., He, B.B., Characterization of crude glycerol from biodiesel production
for multiple feedstocks. Appl. Eng. Agric. 2006, 22(2):261-265.
[4] Posada, J.A., Cardona, C.A., Rincn L.E., Sustainable biodiesel production from palm
using in situ produced glycerol and biomass for raw bioethanol. En: Society for Industrial
Microbiology. 32nd symposium on biotechnology for fuels and chemicals. Clearwater
Beach, Florida. April 19-22. 2010.
[5] Berriosa, M., Skelton, R.L., Comparison of purification methods for biodiesel. Chem.
Eng. J. 2008, 144:459-465.
[6] AMBERSEP BD50 Technology. <www.amberlyst.com/glycerol.htm> [Consulted: 2909-2009].
[7] Johnson, D.T., Taconi, K.A., The Glycerin Glut: Options for the Value-Added
Conversion of Crude Glycerol Resulting from Biodiesel Production. Environ. Prog. 2007,
26(4):338-348.
5. Chemical Conversion of Glycerol

This chapter presents different ways of glycerol transformation to added-value products.
Different reactions are described, such as: (i) oxidation on metallic catalysts like Pt, Pd,
Au, and on promoters as Bi and Pb; (ii) hydrogenolysis to glycols on Ru, Cu and Pt
catalysts; (iii) etherification to polyglycerols on zeolites and mesoporous materials; (iv)
pyrolysis and gasification, where the objective is to produce syn-gas.
Glycerol is a potentially important feedstock for biorefineries, available as a byproduct in

the biodiesel production by transesterification of vegetable oils or animal fats. Also, due to
its high functionality, there are many transformation ways to produce added-value
compounds using glycerol as sole feedstock for its conversion. On the other hand, new
uses for glycerol need to be found since the biodiesel production cost vary inversely with
the glycerol cost.
The high differences between the price of raw glycerol and refined glycerol, added to its
chemical versatility have carried out an intense research for developing alternative uses
and practical technologies to utilize the raw glycerol. In this sense chemical possibilities of
glycerol transformation are reviewed as follows. Thus, several transformation possibilities
to added value products have been found by chemical or biochemical ways.
5.1 Oxidation
Glycerol oxidation on metallic catalysts is carried out by mean of oxidative
dehydrogenation mechanism on the metal surface [1]. First step is the alcohol
dehydrogenation, followed by the oxidation of intermediate formed [2]. Due to the
potential complexity of products distribution (Figure 5.1) the selectivity control on the
process oxidation is key [3].
38
The main derived oxygenated products from glycerol (GLY), are: glyceric acid (GLYAC),
dihydroxyacetone (DHA), hydroxypyruvic acid (HYPAC), tartaric acid (TARAC), mesoxalic
acid (MESOXAC), oxalic acid (OXALAC), besides some intermediates as glyceraldehyde
(GLYAL), glycolic acid (GLYCAC), and glyoxylic acid (GLYOXAC) as is shown in the
Figure. 5.1.
Figure 5.1. Possible products for glycerol oxidation
The most studied metallic catalysts are palladium (Pd), platinum (Pt), and gold (Au),
although the main disadvantage of Pd and Pt are their deactivation with the reaction time
increment [2]. To improve the activity, selectivity, and stability of the reactive system,
promoters are used on Pt and Au for redox reactions; there are particularly heavy metals
from groups IV (lead, Pb) and V (bismuth, Bi) [4].This fact allows preventing the products
over-oxidation on the metal surface, avoiding the products degradation until total oxidation
to carbon dioxide, also promoters favors the secondary alcohols oxidation. Primary
alcohols are oxidized to carboxylic acids (GLYAC, TARAC and HYPAC via DHA) in
absence of promoters or under basic pH, and secondary alcohols are selectively oxidized
on Pt-Bi metallic catalyst at acid pH (DHA, HYPAC via GLYAC and MESAC) [1-2].
Gallezot et al. [1, 5-8], Hutchings et al [9-11], Prati et al [12-18], Claus et al [2, 19] and
Davis [20-22] have studied the selective glycerol oxidation on mono - or bi - metallic
catalysts of Pd, Pt, and Au, using oxygen as oxidizer agent. Gallezot et al showed that
5. Chemical Conversion
39
GLYAC and TARAC are obtained under basic pH, while HYPAC is obtained under not
very acid pH via DHA and, DHA and HYPAC are obtained under acid pH via GLYAC and
MESAC [4, 6, 8, 23]. Total glycerol conversion is achieved for Pd and Pt catalysts with
selectivities of 70% and 35% to GLYAC and HYPAC respectively. Also, for Pt-Bi catalyst
selectivities of 83%, 74%, 37%, and 39% to TARAC, HYPAC, DHA, and MESOXAC are
obtained respectively, with conversions upper to 75%, except for MESOXAC which was
53%. On the other hand, proofs carried out with activated coal (AC) as support showed
that 5% Pd/CA catalyst has higher potential redox than 5% Pt/CA [4], and for Au catalyst
was found that activity and selectivity increase when the particle diameter diminishes.
Hutchings et al [3, 9-11] and Prati et al [12-18, 24] studied the glycerol reaction on Au
catalysts. Au supported on carbon (Au/C) is extremely selective to GLYAC (>82%), with
conversion higher to 60% [3]. Also, in systems at basic pH the selectivity to GLYAC is
increases with both, pH and oxygen pressure. Bi metallic catalysts of Pd-Au take to total
GLY conversion with high GLYAC selectivity (>45%), which was increased when bimetallic catalysts were immobilized on graphite.
GLY oxidation by Au catalysts supported on graphite, activated coal, and carbon

nanoparticles was studied by Claus et al [2, 19] , who found that the last one support is
the most chemically active, also confirmed the dependence among selectivity to GLYAC
and particle size. Other mono- and bi-metallic nanoparticles of Au-Pd were evaluated by
Davis et al [20-22] for GLY oxidation in liquid phase. The highest turnover frequency
(TOF) was exhibits for the Au mono-metallic catalyst and the highest selectivity to GLYAC
was reached by Pd. Also, activity and selectivity for bimetallic catalyst Au-Pd was
dependent of the Au quantity.
5.2 Reduction
Glycerol reduction produces mainly 1,2- propileneglycol (12-PG), 1,3-propileneglycol (13PG), ethyleneglycol (ETGLY), and other by-products such as lactic acid (LACAC), acetol
(ACET), acroleine (ACRO), besides degradation products such as propanol (PROPOH) ,
methanol (METOH), methane (MET), and carbon dioxide (CO2) [25]. Among glycerol
reduction products the most important is propyleneglycol because of its high functionality
which can be used in unsaturated polyester resins, functional fluids (antifreezes and heat
40
transfer), pharmaceuticals, foods, cosmetics, liquid detergents, tobacco humectants,

flavors and fragrances, personal care, paints, and animal feed.
Several technological schemes for propyleneglycol production from glycerol have been
patented [26-29] in which are used different catalysts such as copper, zinc, ruthenium,
cobalt, magnesium, molybdenum, nickel, palladium and platinum; under a widely
operation conditions for pressure (2000 5000 psi) and temperature (200 350 C). On
the other hand Shanks and Lahr [30, 31] studied the interactions among reactants and
catalyst for dehydrogenation/hydrogenation process with Ru supported on activated
carbon (5% wt Ru/CA), thus pH effects, competitive adsorption, and products degradation
(ethylene glycol and propylene glycol) were analyzed under high pressure, meddle
temperature, and high glycerol concentration (1450 psi, 205 C and 10 wt % of glycerol).
Due to the high catalytic activity of Ru was found that ethyleneglycol and
propyleneglycerol degradation rate is independent of the initial glycol concentration,
although propylene glycol is less competitive than ethylene glycol to active sites. Also,
while selectivity to propyleneglycol is independent of pH, selectivity to ethyleneglycol
increases at low basic conditions.
Lahr and Shanks [30] purposed a model for glycerol reduction, in which glycerol is
adsorbed and dehydrogenated reversibly on the metallic catalyst where glyceraldehyde is
formed, which is then desorbed and could react through four different way in a basic
media: (i) retro-aldol mechanism to produce glycol aldehyde as precursor of ethylene
glycol; (ii) oxidation and subsequent descarboxylation to produce also glycol aldehyde;
(iii) dehydration to 2- hydroxypropionaldehyde as precursor of propylene glycol; or (iv)
degradation to unwanted side products which is also a possible way to produce the
glycols precursors. Finally, the respective precursors are hydrogenated to glycols. A new
mechanism for glycerol reduction under moderate operation conditions was proposed by
Dasari et al [25] where hydroxyacetone is formed by dehydrogenation of glycerol, which
after react with hydrogen to produce propylene glycol and water.
The highest selectivities to propylene glycol have been reported for Cu-based catalysts
which exhibit low selectivities to ethylene glycol and other degradation by-products [25].
While Ru- and Pd- based catalysts have low selectivities to propylene glycol (< 50%
generally) because of competitive hydrogenolysis where C-C and C-O bonds are taken to
41
an excessive degradation to produce lower alcohols and gases [25, 32]. In general terms
glycerol conversion is significantly increased by temperature, while yield has a maximum
near to 200C due to the degradation products which occurs at high temperature. Also,
propyleneglycol selectivity can be improved increasing the water contend in the glycerol
mixture which reduce the glycerol conversion, but however the net yield increase.
5.3 Etherification
Glycerol etherification takes to polyglycerols which are oxygenated compounds used as
surfactants, lubricants, cosmetics, and food preservatives. Polyglycerols have low
polymerization level, and these can be obtained in lineal, cyclic, or branched chains, but
researching effort s are focused on selective production of di- and/or tri-glycerols.
Selectivity of glycerol etherification is similar to pseudo-polymerization where generally a
mixture of lineal and cyclic polyglycerols is obtained, especially in presence of
homogeneous catalysts such as sodium, potassium or carbonate hydroxide [32-34].
Etherification selectivity in the first reaction step on acid catalysts is not really controlled
and a mixture of di- to hexa- glycerols (lineal or cyclic), esters of polyglycerol, and
acroleine as by-products is obtained. Although selectivity in the first step could be slightly
improved modifying the pseudo-pore size in the mesoporous materials [35]. On the other
hand, glycerol conversion was improved by Na2CO3, although low selectivities to di- and
tri-glycerols were as obtained. Then alkaline exchange zeolites were studied and
selectivity was increased [32]. Incorporation on mesoporous catalytic structure of
elements such as Al, Mg, and La, modifying only the activity, and selectivity is hold almost
constant. Clacens et al [35] found that impregnation method takes to materials most
stable and selective than incorporation method. Among the impregnated materials La is
the most active but its selectivity was the worst; a positive behavior was found to Mg
which is highly selective
5.4 Pyrolysis and gasification

Pyrolysis process produces liquid fuels at temperatures between 400 600 C, and gas
products at temperatures upper to 750 C. Although gasification process is similar to
42
pyrolysis the main difference is that gasification is carried out in presence of oxygen like:
air, or pure oxygen, or vapor.
Reactions catalyzed by protons or hydroxyl ions can be performed under almost- or

super-critical water conditions (P> 22.1 MPa and T> 647 K) because of water is not only a
solvent it is also a catalyst due to the self-dissociation which takes to formation of
hydroxyl ions and protons. Under these conditions two competitive ways have been
identified. The first one consists in a series of ionic reactions which occur at high pressure
and/or low temperature. The second is a degradation reaction of free radicals, which
occurs at low pressure and/or high temperature. On the other hand, temperature
increases the reaction rate until critical temperature is reached, then reaction rate
decreases drastically related to subcritical conditions.
The main products of glycerol degradation are: methanol, acetaldehyde, propionaldehyde,

acroleine, allylic alcohol, ethanol, formaldehyde, carbon monoxide, carbon dioxide, and
hydrogen. Acetaldehyde and formaldehyde formation increase with the pressure which
indicates that these compounds are mainly formed by the ionic reaction, while methanol
and allylic alcohol formation decrease with the pressure which indicates that these
compounds are formed by the free radicals way [36]. Formation of gasses products
happens to high temperature; also gases formation decrease with the pressure, this
indicates a production by a reaction mechanism of free radicals. Gases formation occurs
at high temperature; also gases formation decrease with the pressure which indicates that
these are produced by a free radicals mechanism.
Syngas is the main product of pyrolysis and gasification processes. Syngas is a mixture of
hydrogen (H2) and carbon monoxide (CO). A wide range of conversions and selectivities
have been reported depending on the operational conditions such as temperature,
pressure, and glycerol concentration [36-41], also the pollutants presence for raw
glycerol, such as methanol and KOH [41]. Low glycerol concentration and high
temperature takes to high CO2 concentrations in the gas product and the most products
remain in the liquid phase [42]. On the other hand when temperature is increased the H2
and CO2 production are improved, which takes to a low CO concentration in the gas
product.
43
Nitrogen (N2) is used like carrier gas for glycerol pyrolysis. High amounts of N2 takes to
high liquid phase yield and gas production diminished. This process has a yield of 93% to
syngas (H2 + CO) at 800 C like showed Valliyappan [41]. On the other hand gasification
is carried out with vapor without any carried. Total glycerol conversion was reported for a
initial mixture of 50 wt % of glycerol by Valliyappan from pure and raw glycerol [41].
References
[1] Gallezot, P., Selective oxidation with air on metal catalysts. Catalysis Today, 1997.
37(4): p. 405-418.
[2] Demirel-Glen, S., M. Lucas, and P. Claus, Liquid phase oxidation of glycerol over
carbon supported gold catalysts. Catalysis Today, 2005. 102-103: p. 166-172.
[3] Hutchings, G.J., Catalysis by gold. Catalysis Today, 2005. 100(1-2): p. 55-61.
[4] Garcia, R., M. Besson, and P. Gallezot, Chemoselective catalytic oxidation of glycerol
with air on platinum metals. Applied Catalysis A: General, 1995. 127(1-2): p. 165-176.
[5] Fordham, P., et al., Selective catalytic oxidation with air of glycerol and oxygenated
derivatives on platinum metals, in Studies in Surface Science and Catalysis. 1996,
Elsevier. p. 161-170.
[6] Fordham, P., et al., Selective oxidation with air of glyceric to hydroxypyruvic acid and
tartronic to mesoxalic acid on PtBi/C catalysts, in Studies in Surface Science and
Catalysis. 1997, Elsevier. p. 429-436.
[7] Fordham, P., M. Besson, and P. Gallezot, Selective oxidation with air of glyceric to
hydroxypyruvic acid and tartronic to mesoxalic acid on PtBi/C catalysts. Stud. Surf. Sci.
Catal., 1997. 108: p. 429-436.
[8] Fordham, P., M. Besson, and P. Gallezot, Selective Catalytic Oxidation of Glyceric
Acid to Tartronic and Hydroxypyruvic Acids. Applied Catalysis A: General, 1995. 133: p.
L179-L184.
[9] Carrettin, S., et al., Selective oxidation of glycerol to glyceric acid using a gold catalyst
in aqueous sodium hydroxide. Chem. Commun., 2002. 7: p. 696-697.
44
[10] Carrettin, S., et al., Oxidation of glycerol using supported gold catalysts. Top. Catal.,
2004. 27: p. 131-136.
[11] Carrettin, S., et al., Oxidation of glycerol using supported Pt, Pd and Au catalysts.
Phys. Chem. Chem. Phys., 2003. 5: p. 1329-1336.
[12] Bianchi, C.L., et al., Selective oxidation of glycerol with oxygen using mono and
bimetallic catalysts based on Au, Pd and Pt metals Catalysis Today, 2005. 102-103: p.
203-212.
[13] Dimitratos, N., et al., Effect of particle size on monometallic and bimetallic (Au, Pd)/C
on the liquid phase oxidation of glicerol. Catal. Lett., 2006. 108: p. 147.
[14] Dimitratos, N., F. Porta, and L. Prati, Au, Pd (Mono and Bimetallic) Catalysts
Supported on Graphite Using the Immobilization Method Synthesis and Catalytic Testing
for Liquid Phase Oxidation of Glycerol. Applied Catalysis A: General, 2005. 291: p. 210214.
[15] N. Dimitratos, A.V., D. Wang, F. Porta, D. Su, L. Prati,, Pd and Pt catalysts modified
by alloying with Au in the selective oxidation of alcohols. Journal of Catalysis, 2005.
244(1): p. 113-121.
[16] Prati, L. and F. Porta, Oxidation of alcohols and sugars using Au/C catalysts - Part 1.
Alcohols. Appl. Catal., A., 2005. 291(199-203).
[17] Prati, L. and M. Rossi, Chemoselective catalytic oxidation of polyols with dioxygen on
gold supported catalysts. Stud. Surf. Sci. Catal., 1997. 110: p. 509-516.
[18] Wang, D., et al., Single-phase bimetallic system for the selective oxidation of glycerol
to glycerate. Chem. Commun., 2006. 18: p. 1956-1958.
[19] Demirel, S., et al., Use of Renewables for the Production of Chemicals: Glycerol
Oxidation over Carbon Supported Gold Catalysts. Appl. Catal. B: Environmental, 2007.
70: p. 637-643.
[20] Ketchie, W.C., et al., Influence of gold particle size on the aqueous-phase oxidation
of carbon monoxide and glycerol. Journal of Catalysis, 2007. 250: p. 94-101.
45
[21] Ketchie, W.C., M. Murayama, and R.J. Davis, Selective oxidation of glycerol over
carbon-supported AuPd catalysts. Journal of Catalysis. , 2007. 250: p. 264-273.
[22] Ketchie, W.C., M. Murayama, and R.J. Davis, Promotional Effect of Hydroxyl on the
Aqueous Phase Oxidation of Carbon Monoxide and Glycerol over Supported Au
Catalysts. Topics in Catalysis, 2007. 44: p. 307-317.
[23] Fordham, P., M. Besson, and P. Gallezot, Selective oxidation with air of glyceric to
hydroxypyruvic acid and tartronic to mesoxalic acid on PtBi/C catalysts. Stud. Surf. Sci.
Catal. , 1997. 108: p. 429-436.
[24] Porta, F. and L. Prati, Selective oxidation of glycerol to sodium glycerate with goldon-carbon catalyst: an insight into reaction selectivity. Journal of Catalysis, 2004. 224(2):
p. 397-403.
[25] Dasari, M.A., et al., Low-Pressure Hydrogenolysis of Glycerol to Propylene Glycol.
Applied Catalysis A: General, 2005. 281: p. 225-231.
[26] Casale, B. and A.M. Gomez, Method of hydrogenating glycerol. 1993: U.S. Patent N.
5,214,219.
[27] Casale, B. and A.M. Gomez, Catalytic method of hydrogenating glycerol. 1994: U.S.
Patent N. 5,276,181.
[28] S. Ludwig, E.M., Preparation of 1,2-propaned. 1997: U.S. Patent N. 5,616,817.
[29] Tessie, C., Production of propanediols. 1987: U.S. Patent N. 4,642,394.
[30] D. G. Lahr, B.H.S., Kinetic Analysis of the Hydrogenolysis of Lower Polyhydric
Alcohols: Glycerol to Glycols. Ind. Eng. Chem. Res. , 2003. 42: p. 5467-5472.
[31] Lahr, D.G. and B.H. Shanks, Effect of Sulfur and Temperature on RutheniumCatalyzed Glycerol Hydrogenolysis to Glycols. Journal of Catalysis, 2005. 232: p. 386394.
[32] Barrault, J., et al., Catalysis and Fine Chemistry. Catalysis Today, 2002. 75: p. 177181.
46
[33] Gerald, J., S. Werner, and D. Helmut, Process for the Preparation of Diglycerol
and/or Polyglycerol, S.W. GMBH, Editor. 1993: U.S. Patent N. 5 243 086.
[34] Lutz, J., et al., Process for the production of diglycerol, H. KGAA, Editor. 1998: U.S.
Patent N. 5 710 350.
[35] Clacens, J.M., Y. Pouilloux, and J. Barraultn, Selective Etherification of Glycerol to
Polyglycerols over Impregnated Basic MCM-41 type Mesoporous Catalysts. Applied
Catalysis A: General, 2002. 227: p. 181-190.
[36] W. Buhler, E.D., H.J. Ederer, A. Kruse, C. Mas, , Ionic Reactions and Pyrolysis of
Glycerol as Competing Reaction Pathways In Near- and Supercritical Water. Journal of
Supercritical Fluids, 2002. 22: p. 37-53.
[37] Matsumura, Y., et al., Biomass Gasification in Near- and Super-Critical Water: Status
and Prospects (Review). Biomass and Bioenergy, 2005. 29: p. 269-292.
[38] Mozaffarian, M., E.P. Deurwaarder, and S.R.A. Kersten, ECN-C--04-081 Green Gas
(SNG) Production by Supercritical Gasification of Biomass. November 2004.
[39] Antal, M.J., et al., Biomass Gasification in Supercritical Water. Ind. Eng. Chem. Res.,
2000. 39: p. 4040-4053.
[40] Xu, X., et al., Carbon-Catalysed Gasification
of
Organic
Feedstocks
in
Supercritical Water. Ind. Eng. Chem. Res., 1996. 35: p. 2522-2530.

[41] Valliyappan, T., Hydrogen or Syn Gas Production from Glycerol Using Pyrolysis and
Steam Gasification Processes, in Department of Chemical Engineering. 2004, University
of Saskatchewan Saskatoon, Saskatchewan
[42] Van Swaaij, W., Technical Feasibility of Biomass Gasification in Fluidized Bed with
Supercritical Water. 2003.
6. Biochemical conversion of glycerol

This chapter studies different possibilities for glycerol bioconversion to added value
products: 1,3-propanediol, ethanol, poly-3-hydroxybutirate, lactic acid, succinic acid,
propionic acid, and rhamnolipids. Also, the influence of the main process variables on the
fermentation behavior (conversion, selectivity, and products distribution) is discussed.
6.1 1,3-propanediol
In the early of 90s a biotechnological route which uses glycerol to produce 1,3propanediol by mean a fermentation process was developed [1]. 1,3-propanediol is a
commercially important compound because it can be used as adhesive, antifreeze,
cosmetics moisturizing, stabilizing detergents, and as additive for painting, printing inks,
and high pressure lubricants. Also it can be used as monomer for polyesters synthesis
such as polytrimethylene terephthalate (PTT) and polyethylene terephthalate (PET) which
can improve the chemical and mechanical properties in comparison with other similar
monomers.
Fermentative production of 1,3-propanediol (PD) under anaerobiosis takes place in two

parallel ways. In the first one, a fraction of glycerol is oxidezed by glycerol-dehydrogenase
(Glyc-DH) to dihydroxy-acetone (DHA), and then phosphorrylated by DHA kinase to enter
glycol-lysis. The remaining glycerol is then dehydrated to 3-hydroxypropionaldehyde
(3HPA)
by
glyceroldehydratase,
where
reduction
con-tinues
by
propanedioldehydrogenase (PPD-DH) and by a dependent NAD oxidorreduc-tase to 1,3propanediol [2-3]. 1,3-propanediol production can be performed biologically by several
bacterial strains such as Klebsiella pneumoniae, Citrobacter freundii, Enterobacter
agglomerans,
Clostridium
butyricum,
and
Clostridium
acetobutylicum
[4-5].
K.
pneumoniae and C. butyricum are commercially the most promising bacterial strains
because of their high yield, productivity, and resistance to both substrate and product
48
inhibition. Among these two bacteria, K. pneumoniae DSM-2026 has been presented as
one of the most appropriate bacterial strain for glycerol fermentation to 1,3-propanediol [6]
and it was selected as the main process microorganism in this article. The purpose of this
article is to analyze the glycerol fermentation to 1,3-propanediol by K. pneumoniae in one
and two continuous fermentation stages.
Studies performed under batch and fed-batch cultures have showed low productivities of
1,3-propanediol, about 2-3 g L-1h-1 with a maximum 1,3-propanediol concentration of 5060 g L-1. In continuous cultures the productivity can be increased, but the maximum
concentration reached is the half (about 30 g L-1) of the obtained under fed-batch or batch
culture conditions. Due to glycerol bioconversion to 1,3-propanediol is a complex
biological mechanism which is subject to inhibitions by substrate and products [7],
process analysis become an important tool to develop efficient configuration process that
allows obtaining the metabolite at high yield, concentration, and productivity. In this sense
Posada et al [8], studied four culture configurations (batch, fed batch, continuous, and two
continuous stages) for 1,3- propanediol production from glycerol, and each configuration
process was optimized.
6.2 Ethanol
E. coli has showed the ability for metabolizing glycerol in presence of an external electron
acceptor. Glycerol degradation process begins with the GlpF incorporation in the
cytoplasm. Later phosphorylation process is carried out, which is catalyzed by GlpK
kinasa. This phosphorylated carbohydrate (glycerol 3-phosphate) starts an oxidereduction process which is accelerated by different enzymes. The anaerobic process is
catalyzed by the dehydrogenases GlpC, GlpB, and GlpA, while the aerobic process is
catalyzed by GlpD. This dehydrogenation process produces dihydroxyacetone 3phosphate and finally glycolysis process takes place to obtain pyruvate (see Figure 6.1.).
Also microorganisms such as Klebsiella, Citrobacter, Enterobacter, Clostridium,
Lactobacillus, Bacillus, Propionibacterium, and Anaerobiospirillum have been reported for
glycerol degrading in fermentative way. Degradation process of these microorganisms is
strongly linked to 1,3-propanediol synthesis with Citrobacter freundii and Klebsiella
pneumonia. However these microorganisms present diverse problems for their industrial
use such as pathogenicity level, requirements of strict anaerobic conditions, and complex
6. Biochemical Conversion
49
cultivation media. In this way, it is necessary to search microorganisms able to metabolize

glycerol without pathogenic effects as occurs with E. coli. Also, E. coli can use glycerol as
carbon source without any external electron receiver. This process is regulated by GldA
dehydrogenase and DHAK dihydroxyacetone kinase for obtaining ethanol, succinate,
acetate, and formate (see Figure 6.2.) [9].
Figure 6.1. Schematic representation of glycerol degradation process on the part of

Escherichia coli, on non fermentative process.
Deletions in E. coli have been carried out to increase formiate and ethanol yields from
glycerol at a concentration of 10 g/L [10]. Thus, from glycerol dehydrogenase (gldA) and
dihydroxyacetone kinase (dhaKLM) over expression a yield of 95% to ethanol from
50
glycerol was achieved. Also, a genomic analysis was carried out for determining the
genes effect on the change from aerobic to anaerobic conditions in E. coli. A metabolic
characterization to evaluate succinate, acetate, formiate, lactate, and ethanol yields was
carried out [11].
Figure 6.2. Main metabolic pathways for fermentative degradation of glycerol by

Escherichia coli.
51
A mixture of ethanol and formiate can be produced by glycerol fermentation using

Klebsiella planticola isolated from the rumen [12]. Dharmadi et al [13], reported the
glycerol fermentation by E. coli, the authors have evaluated the pH-dependence and CO2
availability. Ito et al [14], showed that glycerol at 10 g/L was almost completely consumed
within 84 h; the main products were ethanol and succinic acid with molar yields of 86%
and 7%, respectively. According to the authors, E. coli is already a good biocatalyst for
glycerol conversion into ethanol and hydrogen.
E. aerogenes can be used for ethanol production at high yield from biodiesel wastes
containing glycerol. In this way, a synthetic medium containing biodiesel wastes of
glycerol at 80 mM was analyzed and glycerol was consumed in 24 h, producing 0.89 mol
of H 2 and 1.0 mol of ethanol per mol of glycerol [14].
6.3 poly-3-hydroxybutirate
Polyhydorxyalcanoates
are
attractive
substitute
biopolymers
for
conventional
petrochemical plastics which have similar physical properties to thermoplastics and

elastomers. PHAs are homo or heteropolyesters synthesized and stored intracellularly by
several Gram Negative bacteria [15]. PHAs can be produced from renewable resources
through a fermentation process under restricted growth conditions for nitrogen,
phosphorus, sulfurs and/or oxygen in the presence of an excess carbon source, and they
can also be completely biodegraded by many microorganisms [16]. PHAs are stored in
form of granules by bacteria and can account for up to 80% of the total bacterial dry
weight [17]. On the other hand, polyhydoroxybutyrates (PHBs) were the first type of PHAs
discovered and the most widely studied. PHB has similar properties to conventional
plastics like polypropylene or polyethylene, and it can be extruded, molded, spun into
fibers, made into films, and used to make heteropolymers with other synthetic polymers
[18-19].
Wild strains such as Cupriavidus necator [20], Methylobacterium rhodesianum or

recombinant microorganism such as E. coli recombinant [21, 22] can produce PHB using
glycerol as a carbon and energy source. Bacteria used for PHAs production can be
divided into two groups based on culture conditions. The first group requires limitation of
an essential nutrient such as N, P Mg, K, O or S, and excess of a carbon source; some
52
examples are B. megaterium, C. necator, A. eutrophus, P. extorquens, and Ps.

oleovorans. In the second group, nutrient limitation is not required and the polymer can be
accumulated during the growth phase [23]; some examples are E. coli recombinant, Az.
vinelandii recombinant, and A. latus. PHB producer strains which use glycerol as the
carbon source are in the first group of bacteria. Polyhydorxyalcanoates are attractive
substitute biopolymers for conventional petrochemical plastics which have similar physical
properties to thermoplastics and elastomers.
PHAs are stored in the form of granules by bacteria and can account for up to 80% of the
total bacterial dry weight [24]. On the other hand, polyhydorxybutyrates (PHBs) were the
first type of PHAs discovered and the most widely studied. PHB has similar mechanical
properties to conventional plastics like polypropylene or polyethylene, and it can be
extruded, molded, spun into fibers, made into films, and used to make heteropolymers
with other synthetic polymers [25].
The fermentation stage can be performed in different operational modes. Batch PHB
production is normally induced by co-culturing the cells [26] or by limiting them with
nitrogen availability using an excess of carbon source in the stationary phase [27]. To
induce the desired nutrient limitation and to achieve a high cell density, a fed-batch
process is the most commonly used method [28-29]. Thus, cell growth is maintained
without nutrient limitation until a desired concentration is achieved. Then, an essential
nutrient is limited to allow an efficient PHB synthesis. During this nutrient limitation stage
the residual cell concentration (i.e., the difference between cell concentration and polymer
concentration) remains almost constant and cell concentration increases only by
polymeric intracellular accumulation [30]. For bacteria requiring an essential nutrient
limitation a two-stage chemostat should be employed thus resulting in a 1.7 fold higher
productivity compared to the one-stage chemostat [30]. Culture performance is affected
by several variables including temperature, pH, fed carbon-to-nitrogen ratio, concentration
of substrates and trace elements, ionic strength, agitation intensity, and dissolved oxygen.
To substantially enhance the yield and productivity of many bioprocesses, optimization
[31-32] and control [33] of the fermentation conditions has been used.
53
6.4 D-Lactic acid

The exhibited heterofermentative behavior of glycerol metabolism under anaerobic and
microaerobic conditions by wild-type E. coli was recently reported [34-35]. And it was
found that significant amounts of ethanol, acetic acid, succinic acid, and formic acid were
produced, while a negligible amount of D-lactic acid was obtained. Besides the ability of
E. coli to metabolize glycerol under anaerobic and microaerobic conditions, the
corresponding pathways involved in the glycerol utilization were recently elucidated [36]
and under these conditions, ethanol was identified as the primary fermentation product.
Later, based on metabolic engineering strategies, an engineered E. coli for the efficient
conversion of glycerol to D-lactic acid in a minimal medium was reported [37]. Thus, the
homofermentative route to produce D-lactic acid was engineered by overexpressing the
pathways involved in the glycerol conversion to D-lactic acid and blocking the pathways
leading to the synthesis of by-products. In general terms, the enzymes involved in the
pathways for glycerol conversion to glycolytic intermediates (i.e., GlpK-GlpD and GldADHAK) and the enzyme involved in the pathway for D-lactic acid synthesis from pyruvic
acid were overexpressed (i.e., D-lactate dehydrogenase). Meanwhile, the by-products
formation was minimized by inactivation of enzymes such as: pyruvate-formate lyase
(pflB),
fumarate
reductase
(frdA),
phosphate
acetyltransferase
(pta),
and
alcohol/acetaldehyde dehydrogenase (adhE). Also, a mutation which blocks the aerobic

D-lactate dehydrogenase (dld) was introduced in order to prevent the utilization of Dlactic acid. The Figure 6.3 shows both the pathways involved in the microaerobic
utilization of glycerol in E. coli and the genetic modifications performed by metabolic
engineering strategies for gene overexpressions or disruptions.
Although lactic acid bacteria have been used for D-lactic acid production from
carbohydrate rich feedstocks, it has also been reported the use of alternative biocatalysts
which are mainly engineered Escherichia coli strains able to produce D- or L-lactic acid
[38-42]. But only a few papers have been published on the use of glycerol as carbon
source for lactic acid production [37, 43]. For instance, Hong et al. [43] compared eight
bacterial strains for lactic acid production from glycerol. Thus, the strain named AC-521
and a member of E. coli, showed the best performance for a fed-batch fermentation
process. On the other hand, Mazumdar et al. [37] engineered several E. coli strains by
overexpressing pathways involved in the conversion of glycerol to lactic acid and blocking
54
those leading to the synthesis of by-products as it was above described. In all cases they
used a minimal medium supplemented with sodium selenite, Na2HPO4, (NH4)2SO4,
NH4Cl, and 20 (or 40 or 60) g/l of pure (or crude) glycerol.
Figure 6.3. Pathways involved in the microaerobic utilization of glycerol in E. coli and
Genetic modifications supporting the metabolic engineering strategies employed by
Mazumdar et al [37]. Thicker lines (overexpression of gldA-dhaKLM, glpK-glpD, and ldhA)
or cross bars (disruption of pflB, pta, adhE, frdA, and dld). Broken lines illustrate multiple
steps. Relevant reactions are represented by the names of the gene(s) coding for the
enzymes.
6.5 Succinic acid

Succinic acid is a C4 dicarboxylic acid produced as both intermediate of the tricarboxylic
acid cycle (TCA) and one of the fermentation products of energy metabolism [44]. This
metabolite can be used for the manufacture of industrially important chemicals including
adipic acid, 1,4-butanediol, tetrahydrofuran, N-methyl pyrrolidinone, 2-pyrrolidinone,
succinate salts and gamma-butyrolactone (see Figure 6.4); and for the synthesis of
55
biodegradable polymers such as polybutyrate succinate (PBS) and polyamides

(Nylonx,4) and green solvents [45].
Figure 6.4. Products that can be synthesized from succinic acid.
Succinic acid is currently produced from crude oil by either catalytic hydrogenation of
maleic anhydride to succinic anhydride and subsequent hydration, or direct catalytic
hydrogenation of maleic acid [46]. The commercial price of petrochemically produced
succinic acid is about 5.98.8 USD$/kg depending on its purity. Also, for its production
from maleic anhydride, the raw material costs are about 1 USD$/kg of succinic acid [45].
Even though the production of chemicals based on succinic acid accounts to about
16.000 Ton/year [47], the market potential was estimated to be about 270,000 Ton/year if
succinic acid replaced maleic anhydride for all uses [48-49]. Thus, because of these
predictions, the dramatic raising in petroleum price, and the increasing environmental
concerns, the fermentative production of succinic acid from renewable resources has
recently received much attention. In this way several microorganisms including
56
Actinobacillus succinogenes [50-51], Anaerobiospirillum succiniciproducens [52-53], and

Mannheimia succiniciproducens [54], recombinant Escherichia coli strains [55-56] and
Corynebacterium glutamicum [57-58] have been found to produce succinic. Also, during
fermentative production of succinic acid some by-products such as acetic acid, formic
acid, lactic acid, and ethanol are also obtained. By products formation limits the possibility
of its fermentative production in industrial scale, since the succinic acid yield is reduced
and a more complex and costly downstream process is required [45, 59-60].
The biological production of succinate from glycerol occurs through a redox-balanced

pathway in the presence of excess carbon dioxide. Unlike glycerol, succinate production
from glucose is not redox balanced and can provide a maximum theoretical molar yield of
1.71 (carbon yield of 1.14) without external reducing power. Although ethanol and
succinate are the only two products resulting from redox-balanced pathways of glycerol
fermentation in E. coli [61-62], succinic acid is minor product [61]. In order to improve the
succinate production, Blankschien et al. [63] engineered an E. coli strain by blocking
pathways to competing metabolic products and thus leaving only the succinate pathway
achieving redox balance during glycerol utilization (see Figure 6.5.)
Glycerol dissimilation in E. coli to dihydroxyacetonephosphate (DHAP) can proceed

through two respiratory routes: the aerobic GlpKGlpD and the anaerobic GlpKGlpABC,
or through the fermentative route GldADhaKLM (see Figure 6.5.). The last one has been
reported to use glycerol efficiently under both anaerobic and microaerobic conditions [61,
64].
Because net ATP is typically not generated by substrate-level phosphorylation when

succinate is produced from glycerol in wild-type E. coli (i.e., through ppc, see below and
Fig. 6.5), use of the fermentative GldADhaKLM route is preferred because higher energy
NADH is generated in glycerol dissimilation through GldA as opposed to a reduced flavin
through GlpD or GlpABC [65-66]. However, DhaKLM uses phosphoenolpyruvate (PEP)
as a cofactor, impacting the metabolic nodes available for glycerol fermentation to
succinate (See Figure 6.5).
Production of succinate from glycerol involves fixing CO2 onto a 3-carbon intermediate,
which is stepwise converted to succinate by the reductive branch of the TCA cycle [67]
57
(See Figure 6.5). E. coli uses PEP carboxylase (ppc) as its main carboxylation enzyme for
succinate generation; however, this is not ideal as PEP levels will be decreased when the
fermentative route of glycerol dissimilation (GldADhaKLM) is used (See Figure 6.5). An
analogous argument can be made for the use of the primarily gluconeogenic PEP
carboxykinases (from E. coli or natural succinate producers) [67]. Succinate synthesis
from pyruvate, which is readily available, is limited because E. coli lacks a native
pyruvatecarboxylase (pyc) and the conversion of pyruvate to malate by the gluconeogenic
malic enzymes is not kinetically favored [67]. An effective way to retain the GldADhaKLM
route and generate succinate is to introduce a pyruvate carboxylase (pyc) into E. coli,
creating an efficient node for the step wise conversion of pyruvate to succinate.
Figure 6.5. Pathways involved in the micro aerobic utilization of glycerol and the
generation of phosphoenol pyruvate and pyruvate, which can be carboxylated to
intermediates leading to succinate [63].
58
Use of a heterologous pyruvate carboxylase (pyc) in E. coli to drive succinate production

from glycerol leaves one remaining obstacle, the lack of net ATP production by substratelevel phosphorylation. Such a complication can be effectively overcome by the use of
microaerobic conditions. ATP will be gained through oxidative phosphorylation resulting
from the reducing equivalents generated during the utilization of glycerol, including those
generated by the incorporation of glycerol into cell mass (i.e. cell mass is less reduced on
average than glycerol) [61] (See Figure 6.5)
6.6 Propionic acid

Propionic acid and its calcium, sodium, and potassium salts are widely used as
preservatives in animal feed and human foods, and propionic acid is also an important
chemical intermediate in the synthesis of cellulose fibers, herbicides, perfumes and
pharmaceuticals [68-69]. Currently, almost all propionic acid is produced by chemical
synthesis
from
petroleum
feedstocks.
The
acid
also
could
be
produced
by
propionibacteria via the dicarboxylic acid pathway with acetic acid and succinic acid as
byproducts [70-74], but low yield and productivity due to the inhibition of propionic acid on
cell growth and propionic acid synthesis [72, 75] is a problem. To alleviate the inhibition of
propionic acid on microbial growth and propionic acid synthesis, two approaches,
extractive propionic acid fermentation [76-78] and propionic acid production with propionic
acid-tolerant bacteria obtained via adaptive evolution [72, 79-80] have been developed.
Despite such advancements, current microbial propionic acid production cannot
economically compete petrochemical routes. Producing propionic acid from agricultural
and industrial wastes may make microbial propionic acid production economically
competitive. Glycerol is a main by-product of the biodiesel industry [81] and could thus be
a low-cost feedstock to produce propionic acid. While most studies on propionic acid
production by Propionibacterium acidipropionici have focused on glucose and whey
lactose [77, 82-85], some studied have explored glycerol as the carbon source [86-87],
and it was observed that glycerol might be advantageous since less acetic acid was
produced during the consumption of glycerol [70, 86].
Since optimal conditions for the use of glycerol in propionic acid production have not yet
been established, we optimized propionic acid production by propionic acid-tolerant P.
acidipropionici CGMCC 1.2230 with glycerol as the carbon source in batch cultures and
59
then scaled-up production in a 10 m3 fermentor using the optimized conditions. The

results obtained here may be helpful for industrial production of propionic acid.
References
[1] Ya-Nan, Z., C. Guo, and Y. Shan-Jing, Microbial production of 1,3-propanediol from
glycerol by encapsulated Klebsiella pneumoniae. Biochemical Engineering Journal, 2006.
32: p. 93-99.
[2] Barbirato, F., Soucaille P., and Bories. A.: Physiologic mechanisms involved in
accumulation
of
3-hydroxypropionaldehyde
during
fermentation
of
glycerol
by
Enterobacter agglomerans. Appl. Environ. Microbiol. 62, 4405-4409 (1996).

[3] Papanikolaou, S., Ruiz-Sanchez, P., Pariset, B., Blanchard, F., and Fick. M.: High
production of 1,3-Propanediol from industrial glycerol by a newly isolated Clostridium
butyricum strain. J. Biotechnol. 77, 191208 (2000).
[4] Gonzalez-Pajuelo, M., Meynial-Salles, I., Mendes, F., Andrade, J.C., Vasconcelos, I.,
and Soucaille, P.: Metabolic engineering of Clostridium acetobutylicum for the industrial
production of 1,3-Propanediol from glycerol. Metab. Eng. 7, 329-336 (2005).
[5] Ito, T., Nakashimada, Y., Senba, K., Matsui, T., and Nishio N.: Hydrogen and ethanol
process. J. Biosci. Bioeng.. 100, 260265 (2005).
[6] Zhao, Y.N., Chen, G., and Yao, S.J.: Microbial production of 1,3-propanediol from
glycerol by encapsulated Klebsiella pneumoniae. Biochem. Eng. J. 32, 9399 (2006).
[7] Ya-Qin, S., et al., Mathematical modeling of glycerol fermentation by Klebsiella
pneumoniae: Concerning enzyme-catalytic reductive pathway and transport of glycerol
and 1,3-propanediol across cell membrane. . Biochemical Engineering Journal, 2008. 38:
p. 22-32.
[8] Posada, J.A., J.J. Jaramillo, and C.A. Cardona, Glycerol Fermentation to 1,3Propanediol: Modeling, Simulation, and Optimization. . 1st International Conference on
Modelling and Simulations (ICOMOS 2010 - VF) VIRTUAL FORUM, July 12th - 26th,
2010.
60
[9] Murarka, A., et al., Fermentative utilization of Glycerol by Escherichia coli and its
implications for the production of fuels and chemicals. Appl. Environ. Microbiol., 2008. 74:
p. 1124-1135.
[10] Yazdani, S.S. and R. Gonzalez, Anaerobic fermentation of glycerol: a path to
economic viability for the biofuels industry. . Curr. Opin. Biotechnol., 2007. 18: p. 213-219.
[11] Nikel, P.L., et al., Poly(3-hydroxybutyrate) synthesis from glycerol by a recombinant
Escherichia coli arcA mutant in fed-batch microaerobic cultures. Appl. Microbiol.
Biotechnol., 2008. 77: p. 1337-1343.
[12] G.N.Jarvis, E.R.B. Moore, and J.H. Thiele, Formate and ethanol are the major
products of glycerol fermentation produced by a Klebsiella planticola strain isolated from
red deer. . J. Appl. Microbiol., 1997. 83: p. 166-174.
[13] Dharmadi, Y., A. Murarka, and R. Gonzalez, Anaerobic Fermentation of Glycerol by
Escherichia coli: A New Platform for Metabolic Engineering. Biotechnology &
Bioengineering, 2006. 94(5): p. 821-829.
[14] Ito, T., et al., Hydrogen and Ethanol Production from Glycerol-Containing Wastes
Discharged after Biodiesel Manufacturing Process. J. Biosci. Bioeng., 2005. 100(3): p.
260-265
[15] Mahishi LH, Tripathi G, Rawal SK. Poly(3-hydroxybutyrate) (PHB) synthesis by
recombinant Escherichia coli harbouring Streptomyces aureofaciens PHB biosynthesis
genes: Effect of various carbon and nitrogen sources. Microbiol Res 2003;158;1927.
[16] Steinbchel A. Perspectives for biotechnological production and utilization of
biopolymers: metabolic engineering of polyhydroxyalkanoate biosynthesis pathways as a
successful example. Macromol Biosci 2001;1;1-24.
[17] Khanna S, Srivastava AK. Recent advances in microbial polyhydroxyalkanoates.
Process Biochem 2005;40;607619.
[18] Savenkova L, Gercberga Z, Muter O, Nikolaeva V, Dzene A, Tupureina V. PHBbased films as matrices for pesticides. Process Biochem 2002:37;719-722.
[19] Ali AQ, Kannan TP, Ahmad A, Samsudin AR. In vitro genotoxicity tests for
polyhydroxybutyrate A synthetic biomaterial, Toxicol in Vitro 2008;22(1);57-67.
61
[20] Cavalheiro, J.M.B.T., et al., Poly(3-hydroxybutyrate) production by Cupriavidus

necator using waste glycerol. Process Biochemistry, 2009. 44(5): p. 509-515.
[21] Pablo, I.N., et al., Poly(3-hydroxybutyrate) synthesis from glycerol by a recombinant
Escherichia coli arcA mutant in fed-batch microaerobic cultures. . Appl. Microbiol.
Biotechnol., 2008. 77: p. 1337-1343.
[22] Mahishi, L.H., G. Tripathi, and S.K. Rawal, Poly(3-hydroxybutyrate) (PHB) synthesis
by recombinant Escherichia coli harbouring Streptomyces aureofaciens PHB biosynthesis
genes: Effect of various carbon and nitrogen sources. Microbiol. Res., 2003. 158: p. 1927.
[23] Lee, S.Y., Plastic bacteria? Progress and prospects for polyhydroxyalkanoate
production in bacteria. Tibtech, 1996. 14: p. 431-438.
[24]
Khanna,
S.
and
A.K.
Srivastava,
Recent
advances
in
microbial
polyhydroxyalkanoates. Process Biochem., 2005. 40: p. 607-619.

[25] Ali, A.Q., et al., In vitro genotoxicity tests for polyhydroxybutyrate - A synthetic
biomaterial. Toxicol in Vitro 2008. 22(1): p. 57-67.
[26] Ganduri VSRK, Ghosh S, Patnaik PR. Mixing control as a device to increase PHB
production in batch fermentations with co-cultures of Lactobacillus delbrueckii and
Ralstonia eutropha. Process Biochem 2005:40; 257-264.
[27] Khanna S, Srivastava AK. Computer simulated fed-batch cultivation for over
production of PHB: A comparison of simultaneous and alternate feeding of carbon and
nitrogen. Biochem Eng J 2006;27;197203.
[28] Shahhosseini S. Simulation and optimisation of PHB production in fed-batch culture
of Ralstonia eutropha. Process Biochem 2004:39;963-969.
[29] Khanna S, Srivastava AK. Optimization of nutrient feed concentration and addition
time for production of poly(-hydroxybutyrate). Enzyme Microb Technol 2006;39;1145
1151.
[30] Lee SY. Plastic bacteria? Progress and prospects for polyhydroxyalkanoate
production in bacteria. Tibtech 1996;14;431-438.
62
[31] Grothe E, Moo-Young M, Chisti Y. Fermentation optimization for the production of

poly(-hydroxybutyric acid) microbial thermoplastic. Enzyme Microb Technol 1999;
25:132141.
[32] Khanna S. Srivastava AK. Statistical media optimization studies for growth and PHB
production by Ralstonia eutropha. Process Biochem 2005:40;2173-2182.
[33] Dias JML,, Pardelha F, Eusbio M, Reis MAM, Oliveira R. On-line monitoring of PHB
production by mixed microbial cultures using respirometry, titrimetry and chemometric
modeling. Process Biochem 2009:44;419-427.
[34] Gonzalez, R., A. Murarka, Y. Dharmadi, and S. S. Yazdani. 2008. A new model for
the anaerobic fermentation of glycerol in enteric bacteria: trunk and auxiliary pathways in
Escherichia coli. Metab. Eng. 10:234245.
[35] Murarka, A., Y. Dharmadi, S. S. Yazdani, and R. Gonzalez. 2008. Fermentative
utilization of glycerol in Escherichia coli and its implications for the production of fuels and
chemicals. Appl. Environ. Microbiol. 74:11241135.
[36] Durnin, G., J. Clomburg, Z. Yeates, P. J. J. Alvarez, K. Zygourakis, P. Campbell, and
R. Gonzalez. 2009. Understanding and harnessing the microaerobic metabolism of
glycerol in Escherichia coli. Biotechnol. Bioeng. 103:148161.
[37] Mazumdar, S., Clomburg, J.M., and Gonzalez, R. 2010. Escherichia coli Strains
Engineered for Homofermentative Production of D-Lactic Acid from Glycerol. Applied and
Environmental Microbiology. 76: 43274336.
[38] Dien, B. S., N. N. Nichols, and R. J. Bothast. 2001. Recombinant Escherichia coli
engineered for production of L-lactic acid from hexose and pentose sugars. J. Ind.
Microbiol. Biotechnol. 27:259264.
[39] Okano, K., T. Tanaka, C. Ogino, H. Fukuda, and A. Kondo. 2010. Biotechnological
production of enantiomeric pure lactic acid from renewable resources: recent
achievements, perspectives, and limits. Appl. Microbiol. Biotechnol. 85:413423.
[40] Zhou, S., K. T. Shanmugam, and L. O. Ingram. 2003. Functional replacement of the
Escherichia
coli
D()-lactate
dehydrogenase
gene
(ldhA)
with
the
L(+)-lactate
63
dehydrogenase gene (ldhL) from Pediococcus acidilactici. Appl. Environ. Microbiol.

69:22372244.
[41] Zhou, S., T. B. Causey, A. Hasona, K. T. Shanmugam, and L. O. Ingram. 2003.
Production of optically pure D-lactic acid in mineral salts medium by metabolically
engineered Escherichia coli W3110. Appl. Environ. Microbiol. 69:399407.
[42] Zhu, Y., M. A. Eiteman, K. DeWitt, and E. Altman. 2007. Homolactate fermentation by
metabolically engineered Escherichia coli strains. Appl. Environ. Microbiol. 73:456464.
[43] Hong, A.A.; Cheng, K.K.; Peng, F.; Zhou, S.; Sun, Y.; Liu, C.M.; Liu, D.H. Strain
isolation and optimization of precess parameters for bioconversion of glycerol to lactic
acid. J. Chem. Technol. Biotechnol., 2009, 84, 1576-1581.
[44] Huh Y.S., Jun Y.S., Hong Y.K., Song H., Lee S.Y., Hong W.H. Effective purification of
succinic acid from fermentation broth produced by Mannheimia succiniciproducens.
Process Biochemistry 41 (2006) 14611465.
[45] Song H, Lee S. Y., Production of succinic acid by bacterial fermentation. Enzyme and
Microbial Technology 39 (2006) 352361.
[46] Kurzrock T, Weuster-Botz D., Recovery of succinic acid from fermentation broth.
Biotechnol Lett (2010) 32:331339.
[47] Patel MA (2006) Medium and long-term opportunities and risks of the
biotechnological production of bulk chemicals from renewable resourcesthe potential of
white biotechnology. The BREW Project (http:www.chem.uu.nl/brew/)
[48] Delhomme C, Weuster-Botz D, Kuhn F (2009) Succinic acid from renewable
resources as a C4 building-block chemicala review of the catalytic possibilities in
aqueous media. Green Chem 11:1326
[49] Wilke T, Vorlop K-D (2004) Industrial bioconversion of renewable resources as an
alternative to conventional chemistry. Appl Microbiol Biotechnol 66:131142
[50] Guettler MV, Rumler D, Jain MK. Actinobacillus succinogenes sp. nov., a novel
succinic-acid-producing strain from the bovine rumen. Int J Syst Bacteriol 1999;49:207
16.
64
[51] Urbance SE, Pometto AL, DiSpirito AA, Denli Y. Evaluation of succinic acid
continuous and repeat-batch biofilm fermentation by Actinobacillus succinogenes using
plastic composite support bioreactors. Appl Microbiol Biotechnol 2004;65:66470.
[52] Lee PC, Lee WG, Kwon S, Lee SY, Chang HN. Succinic acid production by
Anaerobiospirillum succiniciproducens: effects of the H2/CO2 supply and glucose
concentration. Enzyme Microb Technol 1999;24:54954.
[53] Samuelov NS, Lamed R, Lowe S, Zeikus JG. Influence of CO 2 HCO 3 levels and pH
on
growth,
succinate
production
and
enzyme
activities
of
Anaerobiospirillum
succiniciproducens. Appl Environ Microbiol 1991;57:30139.

[54] Lee PC, Lee SY, Hong SH, Chang HN. Isolation and characterization of new succinic
acid producing bacterium, Mannheimia succiniciproducens MBEL 55E, from bovine
rumen. Appl Microbiol Biotechnol 2002;58:6638.
[55] Hong SH, Lee SY. Metabolic flux analysis for succinic acid production of by
recombinant Escherichia coli with amplified malic enzyme activity. Biotechnol Bioeng
2001;74:8995.
[56] Lin H, San K-Y, Bennett GN. Effect of Sorghum vulgare phosphoenolpyruvate
carboxylase and Lactococcus lactis pyruvate carboxylase coexpression on succinate
production in mutant strain of Escherichia coli. Appl Microbiol Biotechnol 2005;67:51523.
[57] Bechthold I, Bretz K, Kabasci S, Kopitzky R, Springer A (2008) Succinic acid: a new
platform chemical for biobased polymers from renewable resources. Chem Eng Technol
5:647654.
[58] Okino SH, Noburyu R, Suda M, Jojima T, Inui M, Yukawa H (2008) An efficient
succinic acid production process in a metabolically engineered Corynebacterium
glutamicum strain. Appl Microbiol Biotechnol 81:459464.
[59] Huh YS, Hong YK, Hong WI, Chang HN. Selective extraction of acetic acid from the
fermentation broth produced by Mannheimia succiniciproducens. Biotechnol Lett
2004;26:15814.
65
[60] Kim BS, Hong YK, Hong WH. Effect of salts on the extraction characteristics of
succinic acid by predispersed solvent extraction. Biotechnol Bioprocess Eng 2004;9:20711.
[61] Durnin, G., Clomburg, J., Yeates, Z., Alvarez, P.J.J., Zygourakis, K., Campbell, P.,
Gonzalez, R., 2009. Understanding and harnessing the microaerobic metabolism of
glycerol in Escherichia coli. Biotech.Bioeng.103,148161.
[62] Zhang, Y., Huang, Z., Du, C., Li, Y., Cao, Z., 2009. Introduction of an NADH
regeneration system into Klebsiella oxytoca leads to an enhanced oxidative and reductive
metabolism of glycerol. Metab. Eng. 11,101106.
[63] Blankschien M.D., Clomburg J.M., Gonzalez R., Metabolic engineering of Escherichia
coli for the production of succinate from glycerol. Metabolic Engineering 12 (2010) 409
419.
[64] Gonzalez, R., Murarka, A., Dharmadi, Y., Yazdani, S.S., 2008. A new model for the
anaerobic fermentation of glycerol in entericbacteria: trunk and auxiliary pathways in
Escherichia coli. Metab.Eng.10,234245.
[65] Tran, Q.H., Bongaerts, J., Vlad, D., Unden, G., 1997. Requirement for the protonpumping NADH dehydrogenase I of Escherichia coli in respiration of NADH to fumarate
and its bioenergetic implications. Eur. J. Biochem. 244,155160.
[66] Unden, G., D unnwald, P., 2008. The aerobic and anaerobic respiratory chain of
Escherichia coli and Salmonella enterica, enzymes and energetics. In: Niedhardt, F.C.
(Ed.),EcoSalEscherichia coli and Salmonella, Cellular and Molecular Biology.
ASMPress, Washington,D.C.
[67] Sauer, U., Eikmanns, B.J., 2005. The PEP-pyruvate-oxaloacetate node as the switch
point for carbon flux distribution in bacteria. FEMS Microbiol. Rev. 29, 765794.
[68] Hsu, S., Yang, S., 1991. Propionic acid fermentation of lactose by Propionibacterium
acidipropionici: effects of pH. Biotechnol. Bioeng. 38, 571578.
[69] Roberto, M.C., Mayra, T., 2002. Production of propionate by fed-batch fermentation
of Propionibacterium acidipropionici using mixed feed of lactate and glucose. Biotechnol.
Lett. 24, 427431.
66
[70] Coral, J., Karp, S.G., Vandenberghe, L.P.S., Parada, J.L., Pandey, A., Soccol, C.R.,
2008. Batch fermentation model of propionic acid production by Propionibacterium
acidipropionici in different carbon sources. Appl. Biochem. Biotech. 151, 333 341.
[71] Goswami, V., Srivastava, A.K., 2000. Fed-batch propionic acid production by
Propionibacterium acidipropionici. Biochem. Eng. J. 4, 121128.
[72] Suwannakham, S., Yang, S.T., 2005. Enhanced propionic acid fermentation by
Propionibacterium acidipropionici mutant obtained by adaptation in a fibrousbed
bioreactor. Biotechnol. Bioeng. 91, 325337.
[73] Zhang, A., Yang, S., 2009a. Propionic acid production from glycerol by metabolically
engineered Propionibacterium acidipropionici. Process Biochem. 44, 13461351.
[74] Zhang, A., Yang, S.T., 2009b. Engineering Propionibacterium acidipropionici for
enhanced propionic acid tolerance and fermentation. Biotechnol. Bioeng. 104, 766773.
[75] Gu, Z., Glatz, B., Glatz, C., 1998. Effects of propionic acid on propionibacteria
fermentation. Enzyme Microb. Technol. 22, 1318.
[76] Goswami, V., Srivastava, A.K., 2001. Propionic acid production in an in situ cell
retention bioreactor. Appl. Microbiol. Biot. 56, 676680.
[77] Lewis, V., Yang, S., 1992. A novel extractive fermentation process for propionic acid
production from whey lactose. Biotechnol. Progr. 8, 104110
[78] Ozadali, F., Glatz, B., Glatz, C., 1996. Fed-batch fermentation with and without online
extraction for propionic and acetic acid production by Propionibacterium acidipropionici.
Appl. Microbiol. Biot. 44, 710716.
[79] Woskow, S., Glatz, B., 1991. Propionic acid production by a propionic acid-tolerant
strain of Propionibacterium acidipropionici in batch and semicontinuous fermentation.
Appl. Environ. Microb. 57, 28212828.
[80] Suwannakham, S., Huang, Y., Yang, S.T., 2006. Construction and characterization of
ack knock-out mutants of Propionibacterium acidipropionici for enhanced propionic acid
fermentation. Biotechnol. Bioeng. 94, 383395.
67
[81] Ito, T., Nakashimada, Y., Senba, K., Matsui, T., Nishio, N., 2005. Hydrogen and
ethanol
production
from
glycerol-containing
wastes
discharged
after
biodiesel
manufacturing process. J. Biosci. Bioeng. 100, 260265.

[82] Babuchowski, A., Hammond, E., Glatz, B., 1993. Survey of propionibacteria for ability
to produce propionic and acetic acids. J. Food Protect. 56, 493496.
[83] Carrondo, M., Crespo, J., Moura, M., 1988. Production of propionic acid using a
xylose utilizing Propionibacterium. Appl. Biochem. Biotech. 17, 295312
[84] Lewis, V., Yang, S., 1992b. Propionic acid fermentation by Propionibacterium
acidipropionici: effect of growth substrate. Appl. Microbiol. Biot. 37, 437442.
[85] Quesada-Chanto, A., Afschar, A., Wagner, F., 1994. Optimization of a
Propionibacterium acidipropionici continuous culture utilizing sucrose. Appl. Microbiol.
Biot. 42, 1621.
[86] Himmi, E.H., Bories, A., Boussaid, A., Hassani, L., 2000. Propionic acid fermentation
of glycerol and glucose by Propionibacterium acidipropionici and Propionibacterium
freudenreichii ssp. Shermanii. Appl. Microbiol. Biot. 53, 435440.
[87] Zhang, A., Yang, S., 2009a. Propionic acid production from glycerol by metabolically
engineered Propionibacterium acidipropionici. Process Biochem. 44, 13461351.
7. Study Cases of Chemical Conversion of Glycerol

Three different technological schemes to transform the glycerol obtained as by-product in
biodiesel industry to added-value products are here designed, simulated, and
economically assessed. Dehydration, steam gasification, and hydrogenolysis were the
analyzed processes where acrolein, hydrogen, and 1,2-propanediol are their respective
products. For dehydration and gasification processes a glycerol conversion of 100% was
reached, and the respective molar yields to acrolein and hydrogen were 85.2 % and 78.2
%. Also, these two processes were heat integrated. 175 and 67 W/(feeding kg) were
recovered for dehydration and gasification respectively. Economic results showed that the
three processes are economically viable, and the highest economical return was obtained
for 1,2-propanediol.
7.1 Generalities
Both dehydration and hydrogenolysis reactors were simulated based on a stoichiometric
approach in which acrolein, hydroxyacetone, acetaldehyde, formaldehyde, and water
were
considered
as
the
dehydration
products,
meanwhile
1,2-propanediol,
hydroxyacetone, ethylene glycol, and methane were considered as hydrogenolysis

products. On the other hand, the gasification reactor was simulated as an Rgibbs module
and carbon monoxide, carbon dioxide, hydrogen, methane, and water were considered as
the reaction products.
All processes were integrated in a basic level for a better economic performance. Thus,
the dehydration process was heat integrated, meanwhile the gasification process was
heat and mass integrated and finally the hydrogenolysis process was mass integrated.
70
In all cases the non-random two liquid (NRTL) thermodynamic model was utilized to
calculate the activity coefficients of the liquid phases and Redlich-Kwong (RK) equation of
state was used to model the vapor phase.
Process engineering looks into the design of high-performance processes that meet
mainly two kinds of criteria: high conversion levels and low production costs. Thus
comparing opportunities to convert the glycerol by-product into added-value products is
the core of this analysis. Also, simulations of the technological schemes were used to
generate their respective mass and energy balance sheets, which are the basic input for
the techno-economic analysis.
7.2 Acrolein Production

Acrolein is used as raw material to treat fiber and to produce acrylic acid and medicines,
even more it has been used as a growth control agent of microbes in feed process lines
due to its antimicrobial activity.
Although commercial manufacturing of acrolein has been based on the petrochemicalpropylene oxidation process, this compound can also be produced by homogeneous
catalytic dehydration of glycerol in presence of zinc sulfate. In the last case, for glycerol
dehydration at 360 C and 25 MPa, a maximal selectivity of 75 mol % at a conversion
level of 50% was reached [1].On the other hand, both higher selectivity and conversion
were obtained using heteropolyacid catalysts supported on silica with presence of
titanium, aluminium, and zirconium oxides. Thus, the H 3 PW 12 O 40 catalyst supported on
ZrO 2 was able to produce acrolein at a selectivity of 70 % [2]. Also, complete conversion
of glycerol with selectivities ranging from 75 to 86 % was reported at temperatures
between 275 -325 C on these heteropolyacid catalysts [3-4]. Even more, silicotungstic
acid [5] and Nb 2 O 5 [6] supported on activated carbon have also been reported to produce
acrolein from glycerol at selectivity levels near to 50%.
In addition to heterogeneous catalysis, acrolein can also be produced by glycerol

conversion on hot-compressed water (HCW) with H 2 SO 4 as catalyst. In this sense, it was
reported that yield to acrolein can be improved by increasing either the operational
7. Study Cases of Chemical Conversion
71
pressure or the concentration of glycerol or H2SO4. Using this conversion way, selectivity
values up to 80% can be obtained [7].
The highly exothermic glycerol dehydration to acrolein is carried out by an acid catalyzed
process as shown in Figure 7.1. An aqueous glycerol stream at 10 wt % is heated in two
stages; in the first one the heat produced during the dehydration reaction is recovered in
the Heat Exchanger I, meanwhile using the Heater I the reaction temperature is reached.
Thus dehydrogenation reaction takes place at 275 C and 1 bar.
DC-1
DC-2
Con-1
R-1
HE-1
Diluted
Glycerol
HE-2
H-1
Vapor
Reactives
Distillate 1
Bottoms 1
Products
Distillate 2
Bottoms 2
Condensate
Figure 7.1. Simplified flowsheet for acrolein production by glycerol dehydration.

HE-1: Heat exchanger I; H-1: Heater; R-1: Dehydration reactor; Cond-1: Condenser; HE2: Heat exchanger II; DC-1: Distillation column I; DC-2: Distillation column II.
Equations (7.1) to (7.3) describe the reactive system for catalytic glycerol dehydration [4,
8], in which acrolein (C 3 H 4 O), hydroxyacetone (C 3 H 6 O 2 ), acetaldehyde (C 2 H 4 O), and
formaldehyde (CH 2 O) are the main reaction products. The normalized yields for each
reaction are 85%, 8% and 7%, respectively.
C 3 H 8 O 3 C 3 H 4 O + 2H 2 O (Equation 7.1)
C 3 H 8 O 3 C 3 H 6 O 2 + H 2 O (Equation 7.2)
C 3 H 8 O 3 C 2 H 4 O + CH 2 O + H 2 O (Equation 7.3)
After the dehydration reaction, products stream is cooled in the Heat Exchanger I, and
thus this stream is thermally integrated with the fed diluted glycerol stream. Then, a share
of water is condensed and the resulting mixture is cooled to 80 C in the Heat Exchanger
II. Thus, the downstream process continues with a distillation column where both
remaining water and hydroxyacetone are retired by the bottoms stream. Finally, to purify
the acrolein stream from 92 to 98.5 wt %, a second distillation column could be used.
72
Thus, a mixture of acetaldehyde and formaldehyde is obtained by the distillated stream,

but the condenser should be operated with a special coolant liquid since the distillated
stream is obtained to -10 C.
During the heating of the fresh feed glycerol stream in the dehydration process, 175
W/(feeding kg) were recovered from the effluent reactor stream by mean of the Heat
Exchanger I. Also, in the dehydration reactor not only glycerol was completely converted
but also a yield to acrolein of 85.25% mol was achieved. Then, following the downstream
process line, acrolein at 92.2 wt % was obtained in the distillated stream from the
Distillation Column I and also 99.4 % of the produced acrolein was recovered.
Additionally, in order to obtain a higher purity of acrolein, a further distillation column was
analyzed. Thus, an acrolein steam at 98.5 wt % was obtained, but to reach the operation
conditions a special coolant is required since the condenser must to operate at -10 C. In
this way, when the second distillation column was used, the 98.7 % of the produced
acrolein was recovered, this operational requirements surely increase the production
costs. On the other hand, the most important energy consumptions were obtained the
Heater I and the reboilers of both distillation columns, with net heat duties of: 591.1, 74.4,
and 5.02 W/(feeding kg), respectively. The main simulations results for the dehydration
process are shown in Table 7.1.
Table 7.1. Simulation results for dehydration process from glycerol
Temp. (K)
Pressure (atm)
Comp. (wt%)
Glycerol
Water
Acrolein
Hydroxyacetone
Acetaldehyde
Formaldehyde
Total Flow Rate
(Kg/h)
Diluted Reactives Products

Glycerol
298,1
548,1
548,1
1
1
1
Stream
Condensate Vapor
372,6
1
352,7
1
Distillate
1
309,4
1
Bottoms
1
373,1
1
Distillate
2
262,5
1
Bottoms
2
325,4
1
0,1
0,9
0
0
0
0
0,1
0,9
0
0
0
0
0
0,936
0,052
0,007
0,003
0,002
0
0,978
0,002
0,019
0
0
0
0,927
0,063
0,004
0,004
0,003
0
0,006
0,922
0
0,034
0,038
0
0,994
0
0,004
0,001
0
0
0
0,087
0
0,371
0,542
0
0,006
0,985
0
0,009
0
100
100
100
17,75
82,25
5,59
76,66
0,39
5,20
73
7.3 Hydrogen production

Hydrogen is currently derived from nonrenewable natural gas and petroleum, but it could
be produced from renewable resources such as biomass or its derivates [9]. Many
applications in fields such as electricity generation, fuel cells, and automotive fuels have
been found for hydrogen since it can be used in mobile and stationary applications.
Besides, due to its high energy efficiency, sustainable, and nonpolluting character,
hydrogen is considered as an obvious alternative to hydrocarbon fuels such as gasoline.
Thus, it is expected that hydrogen plays a key role in the worlds energy future by
replacing fossil fuels and storage energy [10].
Non-catalytic processes such as pyrolysis and steam gasification are technologies able to
produce added-value products such as hydrogen and syn gas from glycerol. Pyrolysis is a
thermal cracking process of organic liquids or solids at high temperature performed in
oxygen absence; meanwhile steam gasification is carried out in presence of oxygen and
produces fuel gases with higher hydrogen content than pyrolytic process.
Pyrolysis produces liquid fuels at low temperatures (400 to 600 C), but when this process
is carried out at high temperatures (> 750 |C) gaseous products are obtained. Moreover,
gasification is performed in presence of oxygen (i.e., air, pure oxygen, or steam) and a
mixture of carbon monoxide and hydrogen is also produced [11]. In the case of steam
gasification of glycerol at 600 - 700 C, a yield of 92.3 mol % to syn-gas with a H 2 /CO
molar ratio of 2/1 was reported [12]. Meanwhile glycerol pyrolysis over carbonaceous
catalysts at 800 C produces synthesis gas up to 81 vol % [13].
On the other hand, crude glycerol has been analyzed as raw material to produce
hydrogen. For instance, yields ranging from 77 to 95 wt % were reported for catalytic
steam reforming of crude glycerol on commercial Ni [14]. Besides, higher yields have
been reached by steam gasification from crude glycerol such as 97 % to syn-gas and
65.7 % to H 2 [15].
Hydrogen is produced by supercritical water gasification (SCWG), with glycerol as carbon
source. The simplified flowsheet for hydrogen production from glycerol is shown in Figure
7.2. A mixture containing diluted glycerol at 25 wt % is heated in two stages, and an
intermediate compression process is required. Thus, the first heat exchanger produces
74
overheat vapor and then the reaction pressure is reached by mean of the compressor.
During the compression operation a heat excess is produced, this is used to heat the
fresh diluted glycerol stream in the Heat Exchanger I. Thus, compressed stream and fed
glycerol stream are thermally integrated. Then, by mean of the Hater I the reaction
temperature is achieved, and low heating requirement are needed. The resulting mixture
is fed to the gasification reactor at 600 C and 300 bar (i.e. supercritical conditions).
HE-1
Diluted
Glycerol
Comp-1
R-1
Overheat
Vapor
H-1
Reactives
Products 1
Vapor 1
HE-2
Vapor 2
Products 2
Recycle
Fresh
Water
Sep-2
Sep-1
Condensate 1
M-1
Condensate 2
Figure 7.2. Simplified flowsheet for hydrogen production by gasification.

HE-1: Heat exchanger I; Comp-1: Compressor: H-1: Heater I, R-1: Gasification reactor;
HE-2: Heat exchanger II; Sep-1: High-Pressure (HP) gas-liquid separator; Sep-2: LowPressure (LP) gas-liquid separator; M-1: Mixer.
Molar distribution of reaction products after gasification has been reported as follows [16]:
hydrogen 29.2 %, carbon dioxide 36.1 %, carbon monoxide 0.9 % and methane 33.8 %.
Then, the reaction stoichiometry can be expressed as shows the equation (7.4).
10C 3 H 8 O 3 + H 2 O 13H 4 + 15CO 2 + CO + 14CH 4 (Equation 7.4)

Reaction products are cooled in the Heat Exchanger II and a two-phase stream (gas and
liquid phases) is obtained. This mixture is fed to the high-pressure (HP) gas-liquid
separator which operates at temperatures between 25-100 C, and at 300 bar. Then the
liquid phase, obtained by bottoms, is further transferred to the low-pressure (LP) gasliquid separator which operates at 20 C and 1 bar. The HP separator produces a H 2 -rich
gas stream, while the LP separator produces a CO 2 -CH 4 -rich gas. The liquid product from
LP separator, which is mostly water, is mixed with fresh water and fed to the Heat
Exchanger II as cooling fluid, and thus an overheat-vapor stream is obtained. Finally, the
CO 2 -CH 4 -rich gas stream could be burned to generate process heat.
75
For the dehydration process, the feed stream to the gasification process was also heat
integrated. Thus, the heat excess obtained during the isentropic compression operation
(i.e., 67 W/(feeding kg)) was recovered in the Heat Exchanger I. Besides, the gasification
reactor was able to reach a conversion of 100 % at a yield of 78.2 %. In addition to the
heat integration of the fresh feed glycerol stream, the products reaction stream was also
heat integrated and 784 W/(kg of reaction products) were recovered. Thus, an overheat
vapor stream was obtained. In order to purify the hydrogen produced in the gasification
reactor, two gas-liquid separation units were used. The first one is the High-Pressure (HP)
gas-liquid separator in which H 2 at 90.9 mol % was obtained, meanwhile in the LowPressure (LP) gas-liquid separator the water was retired and a stream containing a
mixture CO 2 -CH 4 was obtained. Also, this stream containing a mixture CO 2 -CH 4 could be
used to generate process heat. The net heat duties in the gasification process were
represented by the Compressor, Heater I, and Gasification Reactor; where the heat duty
reported by the simulator were 673, 146.3 and -75.13 W/(feeding kg), respectively. The
main simulations results for the gasification process are shown in Table 7.2.
Table 7.2. Simulation results for gasification process from glycerol

Stream
Product
Diluted
Reactives s
Glycerol
Products Vapor
Condensate Vapor Condensat Recycle
e2
298,1
873,1
873,1
308,9
308,9
308,9
293,1
293,1
293,1
300
300
300
300
300
Glycerol
0,061
0,061
Water
0,939
0,939
0,804
0,804
0,841
0,023
0,978
0,984
CO 2
0,074
0,074
0,032
0,076
0,451
0,013
0,009
CO
0,002
0,002
0,007
0,002
0,011
CH 4
0,080
0,080
0,052
0,082
0,515
0,009
0,006
H2
0,041
0,041
0,909
100
100
100
100
0,994
99,01
20,69
78,32
107,99
Temp. (K)
Pressure (atm)
Comp. (wt%)
Total Flow Rate

(Kg/h)
76
7.4 1,2-propanediol production

1,2-propanediol is produced commercially by the the hydration of propylene oxide derived
from propylene by either the chlorohydrin process or the hydroperoxide process. But in
the presence of metallic catalysts and hydrogen, glycerol can be hydrogenated to
propylene glycol, 1,3 propanediol, or ethylene glycol.
1,2-propanediol is used in unsaturated polyester resins, functional fluids (antifreeze, deicing, and heat transfer), pharmaceuticals, foods, cosmetics, liquid detergents, tobacco
humectants, flavors and fragrances, personal care, paints and animal feed [17]. The
antifreeze and deicing market is growing because of concern over the toxicity of ethylene
glycol-based products.
1,2-propanediol
is
obtained
from
glycerol
by
the
sequential
processes
of
dehydrogenation-hydrogenation via hydroxiacetone, also glycerol hydrogenolysis can be

carried out in both liquid and vapor phases. Liquid process takes place at low pressure
producing mainly 1,2-propanediol and 1,3-propanediol in presence of supported catalysts
such as Rh [18], Ru [19], or Pt [20]. On the other hand, glycerol hydrogenolysis in vapor
phase is catalyzed by Cu at high hydrogen pressure [21], but in this process lateral
reactions occur and different reaction by-products are obtained. In order to overcome this
problem a two-step process has been proposed, thus dehydrogenation is first performed
under vacuum conditions and then hydrogenation is carried out at high hydrogen pressure
[22-23].
An efficient two steps process for selective production of 1,2-propanediol from glycerol
was developed [24]. The reaction is carried out in vapor phase on a copper metallic
catalyst at ambient pressure of hydrogen. In the first step hydroxyacetone is produced by
glycerol dehydrogenation and then 1,2-propanediol is produced by hydrogenation of
hydroxyacetone. Also, due to both the high temperature required for the dehydrogenation
reaction and the improved selectivity at low temperatures for the hydrogenation reaction,
the reactor configuration has a gradient temperature ranging from 200 C (on the reactor
top) to 120 C (on the reactor bottom). Thus, a molar selectivity of 96% to 1,2-propanediol
with total glycerol conversion is achieved.
77
The simplified flowsheet for 1,2-propanediol production from glycerol is shown in Figure
7.3. Glycerol is diluted at 30 g/L and then this stream is heated up to the reaction
temperature. This stream is fed to the dehydrogenation-hydrogenation reactor, besides a
hydrogen stream at 200 C is fed in a volumetric ratio of 1/141 (glycerol/H 2 ) to the reactor.
The gradient reactor is available to convert glycerol completely to 1,2-propanediol,
hydroxyacetone, ethylene glycol, and methane, as is shown by the equation system (7.5)
to (7.7).
Purge
D-1
R-1
M-1
Reactives
Glycerol
Water
M-2
Fresh
H2
HE-1
H-1
DC-1
DC-1
Sep-1
Distillate 1
Products
Distillate 2
H-2
Condensate
Reactive H2
Bottoms 1
Bottoms 2
Recycled H2
Figure 7.3. Simplified flowsheet for 1,2-propanediol production by hydrogenolysis.

M-1: Mixer I; H-1: Heater I; R-1: Hydrogenolysis reactor; HE-1: Heat exchanger; Sep-1:
Gas-lquid separator; D-1: Divisor; DC-1: Distillation column I; DC-2: Distillation column II;
M-2: Mixer II; H-2: Heater III.
C 3 H 8 O 3 C 3 H 6 O 2 + H 2 O (Equation 7.5)
C 3 H 6 O 2 + H 2 C 3 H 8 O 2 (Equation 7.6)
C 3 H 6 O 2 + 2H 2 C 2 H 6 O 2 + CH 4 (Equation 7.7)
1,2-propanodiol purification process starts with a flash operation at 30 C and 50 bar.
Then, the gas stream containing mainly H 2 is recycled and mixed with fresh H 2 . The
resultant stream of H 2 must be heated up to reaction temperature and then fed to the
reactor. The liquid stream obtained from the flash operation is purified using two
distillation columns. In the first one, most of the water quantity is retired meanwhile in the
second distillation column the remaining water and the non-converted hydroxyacetone are
retired.
78
During the simulation of the hydrogenolysis process, the fresh feed glycerol stream was
heated up to 200 C. Then, a hydrogen stream at the reaction temperature was fed to the
reactor at a volumetric ratio of 1/141 (glycerol/H 2 ) and glycerol is completely converted in
the hydrogenolysis reactor. The purification process for 1,2-propanediol requires an
evaporation process at 30 C and 50 bar, in which a gaseous stream containing mainly
H 2 (99.9 mol %) was recycled and mixed with fresh H 2 . The liquid stream obtained by the
bottom stream from the evaporation process was purified by mean of two distillation
columns. Thus, 99.96 % of water was discarded using the Distillation Column I, and both
the remaining water and no-converted hydroxyacetone were obtained by the distillated
stream in the Distillation Column II. In this way, 1,2-propanediol at 99 wt % was achieved.
The main simulation results for the hydrogenolysis process are shown in Table 7.3.
Table 7.3. Simulation results for hydrogenolysis process from glycerol

Stream
Temp. (K)
Reactives
Products
Condensate Bottoms 1
Bottoms 2
Recycled H 2
Reactive H 2
200
200
30
183.6
186.7
30
200
50
50
Pressure (bar) 1
Comp. (wt%)
Glycerol
0,3
1,2-PD
0,020
0,240
0,969
0,99
Hydroxyacetone
0,001
0,007
0,029
0,01
Ethylene glycol
0,001
0,001
Water
0,7
0,069
0,753
0,001
0,008
0,007
H2
0,911
0,992
0,992
33333
408099
33398
8263
7997
355966
374765
Total Flow Rate

(Kg/h)
7.5 Economic assessment

Operational and capital costs were disaggregated by raw material, utilities, operation
labor, maintenance, operating charges, plant overhead, general and administration costs,
and depreciation cost, as shows the Table 7.4 for each glycerol conversion process [2532]. Besides, all costs were normalized and their shares are shown in Table 7.5.
79
Table 7.4. Production costs for glycerol conversion to added-value

Production costs
Acrolein at
Acrolein at
Hydrogen
1,2-propanediol
(US$/L of product)
92 wt %
98.5 wt %
Raw materials
0,2927
0,2920
4,777E-05
0,1203
Utilities
0,3067
1,2006
3,731E-05
0,0813
Operating labor
0,0033
0,0042
1,129E-05
0,0073
Maintenance
0,0116
0,0123
1,031E-05
0,0066
Operating charges
0,0008
0,0011
2,823E-06
0,0081
Plant Overhead
0,0069
0,0082
1,080E-05
0,0063
General and
0,1499
0,2105
9,625E-06
0,0191
capital
0,0555
0,0646
1,069E-4
0,0192
Total costs
0,8274
1,7935
2,368E-4
0,2682
Sale Price
1,110
1,779
2,498E-4
0,4200
Administrative
Depreciation of
Table 7.5. Percentage of Production costs for glycerol conversion to added-value

Production costs
Acrolein
Acrolein
Hydrogen 1,2-propanediol
(US$/L of product)
at 92%
at 98.5%
Raw materials
35.38
16.28
20.17
44.85
Utilities
37.07
66.94
15.75
30.31
Operating labor
0.40
0.23
4.77
2.72
Maintenance
1.40
0.69
4.35
2.46
Operating charges
0.10
0.06
1.19
3.02
Plant Overhead
0.83
0.46
4.56
2.35
General and Administration
18.12
11.74
4.06
7.12
Depreciation of capital
6.71
3.60
45.14
7.16
Although glycerol at technical grade was considered as raw material for the three
technological schemes, some differences in the raw material costs can be observed due
to the differences among yield, selectivity, and products density (see Table 7.4). For
80
instance, because of the dehydration process had the lowest selectivity; the highest raw
material cost was obtained for acrolein. Also, due to hydrogen is only product here
obtained in gas phase not only the lowest raw material cost but also the lowest production
cost in US$/L were obtained for the gasification process. Then, in order to compare the
three technological schemes, it was necessary to include the commercial sale price for
each product as shown Table 4. Even more, the shares of each item allow identifying the
main economical resource consumers, as shown Table 7.5.
Two qualities of acrolein are observed in Table 7.4, there are 92 and 98.5 wt %. These
assessment were performed because of the acrolein production process at 98.5 wt %
requires a powerful coolant system which implies high operational costs; and thus its total
production cost is higher than the commercial sale price. On the other hand, since the
service cost to produce acrolein at 92 wt % is only the 25 % required to obtain acrolein of
high purity, the total production cost for this process is lower than its commercial sale
price. During the acrolein production at 92 wt %, most of the production costs are
represented by raw materials and services which totaling 72 % of the total production
cost. Meanwhile for acrolein production at 98.5 wt %, only the services contributing the 67
% of total production cost. Thus, the production process of acrolein at high purity is not
economically viable.
Although in most of the chemical process the raw material cost represents near to 50 % of
the total production cost, in the case of hydrogen production from glycerol the sum of both
raw material and services costs were almost 36 % of the total production cost. But, for this
process the main investment is represented by the process units since the equipment
depreciation is 45.14% of the total production cost. The high depreciation cost occurs
because extreme operational conditions (i.e., high temperatures and pressures) are
required during this process.
Finally for 1,2-propanediol production the raw material and services costs represent the
most share of the total production cost, which add 75%. And then, the equipment
depreciation cost is 16.7 % of the total production cost. These values are typical for a
chemical process.
81
By comparing the ratio of the commercial sale price respect to the obtained total
production cost (i.e., sale/production costs), a ratio of 1.055 was found for hydrogen
production, followed by the acrolein process with a ratio of 1.34, and the highest value
obtained was for 1,2-propanediol production at a ratio of 1.57. Thus, the production of 1,2propanediol could generate the highest economical return for glycerol conversion into
added-value products among the analyzed processes.
7.6 Conclusions
Acrolein, hydrogen, and 1,2-propanediol, are three of the most commercially important
products obtained from glycerol, due to their applications, established market, and sale
prices. Here the technological schemes to produce these compounds were designed,
simulated, and economically assessed. Thus, simulation results showed that all the
processes are technologically feasible reaching high purity of product. Also, acrolein
production was found to be viable at a purity of 92 wt %, but do not at a purity of 98.5 wt
%. Finally, both hydrogen and 1,2-propanediol production processes are also
economically viable, where the last one generates the highest profit margin.
References
[1] Ott, L., Bicker, M., Vogel, H., 2006. Catalytic dehydration of glycerol in sub- and
supercritical water: a new chemical process for acrolein production. Green Chem. 8, 214220.
[2] Chai, S.-H., Wang, H.-P., Liang, Y., Xu, B.-Q., 2008. Sustainable production of
acrolein: Preparation and characterization of zirconia-supported 12-tungstophosphoric
acid catalyst for gas-phase dehydration of glycerol. Appl. Catal., A 353, 213-222.
[3] Atia, H., Armbruster, U., Martin, A., 2008. Dehydration of glycerol in gas phase using
heteropolyacid catalysts as active compounds. J. Catal. 258, 71-82.
[4] Tsukuda, E., Sato, S., Takahashi, R., Sodesawa, T., 2007. Production of acrolein from
glycerol over silica-supported heteropoly acids. Catal. Commun. 8, 13491353.
82
[5] Lili, N., Yunjie, D., Weimiao, C., Leifeng, G., Ronghe, L., Yuan, L., Qin, X., 2008.
Glycerol dehydration to acrolein over activated carbon- supported silicotungstic acids.
Chin. J. Catal. 29, 212-214.
[6] Chai, S.-H., Wang, H.-P., Liang, Y., Xu, B.-Q., 2007. Sustainable production of
acrolein: Gas-phase dehydration of glycerol over Nb 2 O 5 catalyst. J. Catal. 250, 342-349.
[7] Watanabe, M., Iida, T., Aizawa, Y., Aida, T. M., Inomata, H., 2007. Acrolein synthesis
from glycerol in hot-compressed water. Bioresour. Technol. 98, 12851290.
[8] Pathak, K.D., 2005. Catalytic conversion of glycerol to value-added liquid chemicals.
Thesis for the degree of Master of Science, University of Saskatchewan, Canada.
[9] Cortright, R.D., Davda, R.R., Dumesic, J.A., 2002. Hydrogen from catalytic reforming
of biomass-derived hydrocarbons in liquid water. Nature 418, 964-966.
[10] Demirbas, A., 2002. Fuel properties of hydrogen, liquefied petroleum gas, and
compressed natural gas for transportation. Energ. Source. 24, 601610.
Franco, C., Pinto, F., Gulyurtlu, I., Cabrita, I., 2003. The study of reactions influencing
biomass steam gasification process. Fuel 82, 835-842.
[11] Franco, C., Pinto, F., Gulyurtlu, I., Cabrita, I., 2003. The study of reactions influencing
biomass steam gasification process. Fuel 82, 835-842.
[12] Chaudhari, S.T., Bakhshi, N.N., 2002. Steam gasification of chars and bio-oil, In:
Report to bioenergy development program renewable energy branch: Energy, mines, and
resources. Canada, Ottawa, 396-436.
[13] Fernndez, Y., Arenillas, A., Dez, M.A., Pis, J.J., Menndez, J.A., 2009. Pyrolysis of
glycerol over activated carbons for syn-gas production. J. Anal. Appl. Pyrolysis 84, 145150.
[14] Garcia, L., French, R., Czernik, S., Chornet, E., 2000. Catalytic steam reforming of
bio-oils for the production of hydrogen: effects of catalyst composition. Appl. Catal., A
201, 225239.
[15] Valliyappan, T., Bakhshi, N.N., Dalai, A.K., 2008. Pyrolysis of glycerol for the
production of hydrogen or syn gas. Bioresour. Technol. 99, 44764483.
83
[16] Mozaffarian, M., Deurwaarder, E.P., Kersten, S.R.A, 2004. Project: green gas (SNG)
production by supercritical gasification of biomass, pp. 71, 2004.
[17] Dasari, M.A., Kiatsimkul, P.-P., Sutterlin, W.R., Suppes, G.J., 2005. Low-Pressure
hydrogenolysis of glycerol to propylene glycol. Appl. Catal., A. 281, 225-231.
[18] Chaminand, J., Djakovitch, L., Gallezot, P., Marion, P., Pinel, C., Rosie, C., 2004.
Glycerol hydrogenolysis on heterogeneous catalysts. Green Chem. 6, 359-361.
[19] Maris, E.P., Davis, R.J., 2007. Hydrogenolysis of glycerol over carbon-supported Ru
and Pt catalysts. J. Catal. 249, 328-337.
[20] Kurosaka, T., Maruyama, H., Naribayashi, I., Sasaki, Y., 2008. Production of 1,3propanediol by hydrogenolysis of glycerol catalyzed by Pt/WO 3 /ZrO 2 . Catal. Commun. 9,
1360-1363.
[21] Wang, S., Liu, H., 2007. Selective hydrogenolysis of glycerol to propylene glycol on
CuZnO catalysts . Catal. Lett. 117, 62-67.
[22] Chiu, C.-W., Tekeei, A., Ronco, J.M., Banks, M.-L., Suppes, G.J., 2008a. Reducing
byproduct formation during conversion of glycerol to propylene glycol. Ind. Eng. Chem.
Res. 47, 6878-6884.
[23] Chiu, C.-W., Tekeei, A., Sutterlin, W.R., Ronco, J.M., Suppes, G.J., 2008b. Lowpressure packed-bed gas phase conversion of glycerol to acetol. AIChE J. 54, 2456-2463.
[24] Akiyama, M., Sato, S., Takahashi, R., Inui, K., Yokota, M., 2009. Dehydration
hydrogenation of glycerol into 1,2-propanediol at ambient hydrogen pressure. Appl.
Catal., A 371, 60-66.
[25] Cardona, C.A, Snchez, O.J., 2006. Energy consumption analysis of integrated
flowsheets for production of fuel ethanol from lignocellulosic biomass. Energy 31, 24472459.
[26] Cardona, C.A., Posada, J.A., Quintero, J.A., 2010. Use of agroindustrial subproducts
and wastes: Glycerin and Lignocellulosics, first ed. Artes Grficas Tizn, Manizales. (In
Spanish).
84
[27] Gutirrez, L.F., Snchez, O.J., Cardona, C.A., 2009. Process integration possibilities
for biodiesel production from palm oil using ethanol obtained from lignocellulosic residues
of oil palm industry. Bioresour. Technol. 100, 1227-1237.
[28] Posada, J.A., Cardona, C.A., 2010. Design and analysis of fuel ethanol production
from raw glycerol. Energy, doi:10.1016/j.energy.2010.07.036.
[29] Posada, J.A., Cardona, C.A., 2010b. Validation of glycerin refining obtained as a byProduct of biodiesel production. Ingeniera y Universidad. 14, 2-27. (In Spanish).
[30] Posada, J.A., Naranjo, J.M., Lpez, J.A., Higuita, J.C., Cardona, C.A., 2010a. Design
and analysis of poly-3-hydroxybutyrate production processes from crude glycerol.
Process Biochem., doi:10.1016/j.procbio.2010. 09.003..
[31] Posada, J.A., Cardona, C.A., Rincn, L.E., 2010b. Sustainable biodiesel production
from palm using in situ produced glycerol and biomass for raw bioethanol. In 32nd
symposium on biotechnology for fuels and chemicals. Clearwater Beach, Florida.
[32] Quintero, J.A., Montoya, M.I., Snchez, O.J., Giraldo, O.H., Cardona, C.A., 2008.
Fuel ethanol production from sugarcane and corn: Comparative analysis for a Colombian
case. Energy 33, 385399.
8. Study Cases of Biochemical Conversion of

Glycerol
This chapter presents the process design, simulation, and economical assessment of six
different possibilities for glycerol transformation by fermentation. For the production of 1,3propanediol, a kinetic model was used allowing optimizing the fermentation stage by three
different approaches. For ethanol, poly-3-hydroxybutirate, lactic acid, succinic acid, and
propionic acid production, a yield approach was used in all cases. Thus, several
scenarios were analyzed in each case depending on the glycerol fermentation stage or on
the downstream process.
8.1 1,3-Propanediol production

Although 1,3-propanediol could be biologically produced from glycerol by several bacterial
strains such as: Klebsiella pneumoniae, Citrobacter freundii, Enterobacter agglomerans,
Clostridium butyricum, and Clostridium acetobutylicum [1-2]; the K. pneumoniae and C.
butyricum strains are the most promising bacterial because of their high yield,
productivity, and resistance to both substrate and product inhibition. Among these two
bacteria, K. pneumoniae DSM-2026 has been presented as one of the most appropriate
bacterial strain for glycerol fermentation to 1,3-propanediol [3]. First of all, here the
fermentation process of glycerol to 1,3-propanediol by K. pneumoniae is analyzed in one
and two continuous stages.
The material balances for continuous glycerol fermentation in one single stage are solved
using two independent variables namely the glycerol concentration in the feed stream and
the dilution rate. Since, acetic acid and ethanol are also produced during glycerol
fermentation to 1,3-propanediol by K. pneumoniae, the material balances in a dynamic
state needed to be solved for biomass, substrate, and all the obtained products as shown
in equations (8.1) to (8.5).
86
dX iOut
= Di ( X iIn X iOut ) + i X iOut
dt
(8.1)
dC
Out
G ,i
dt
= Di ( C GIn,i C GOut,i ) q G ,i X iOut
Out
dC PD
,i
dt
dC
Out
HAc , i
dt
dC
Out
EtOH ,i
dt
(8.2)
In
Out
Out
= Di ( C PD
,i C PD ,i ) + q PD ,i X i
(8.3)
In
Out
Out
= Di ( C HAc
,i C HAc ,i ) + q HAc ,i X i
In
Out
Out
= Di ( C EtOH
,i C EtOH ,i ) + q EtOH ,i X i
(8.4)
(8.5)
Where: X, C G , C PD , C HAc , and C EtOH , are the concentrations for biomass (g/L), glycerol
(mol/L), 1,3-propanediol (mol/L), acetic acid (mol/L), and ethanol (mol/L), respectively. D
is the dilution rate (i.e., ratio between volumetric flow and reactor volume) (h-1), is the
specific rate of cellular growth (h-1), q G is the specific rate of glycerol consumption, and
q PD , q HAc , and q EtOH are the generation rates of each product (h-1). Subscript i, indicates
the fermentation stage for a multistage system. In the i fermentation stage, In and Out
superscripts indicate the in and the out conditions respectively.
The kinetic model of glycerol fermentation by K. pneumoniae has been previously

explained [4-6]. Specific rates of cell growth, substrate consumption, and products
formation are given in the equations (8.6) to (8.11).
i = max
C G ,i
C
1 G,i
C G ,i + K S
CG
q G ,i = m G +
i
m
G
C ,i
1 PD
C PD
+ q Gm
C ,i
1 HAc
C HAc
(8.6)
C G ,i
C G ,i + K S
m
m
+ q PD
q PD ,i = m PD + i * YPD
(8.7)
C G ,i
C G ,i + K PD
m
m
q HAc ,i = m HAc + i * YHAc
+ q HAc
(8.8)
C G ,i
C G ,i + K HAc
q EtOH ,i = qG ,i Y(mEtOH / G ),i
Y(mEtOH / G ),i =
,i
1 EtOH
C EtOH
b1
b2
+
c1 + Di CG ,i c 2 + Di CG ,i
(8.9)
(8.10)
(8.11)
8. Study Cases of Biochemical Conversion
87
Equation (8.6) describes the specific rate of cell growth which represents a kinetic model
*
*
*
with inhibition by both substrate and products. The kinetic parameters C G , C PD , C HAc and
*
C EtOH
are the critical concentrations (i.e., the concentration where biological activity is
stopped). The required parameters to solve the kinetic model (valid at 37 C and at
neutral pH [4, 6]) are: Maximum Specific Growth Rate, max =0.67 h-1 and the Monod
Saturation Constant, Ks=2.8e-4 gmol/L.
Constants for specific rate of substrate consumption and product formation are: m G = 2.20
e-3, m PD =-2.69e-3, m HAc =-9.7e-4, Ym Gly = 8.2, Ym PD =6.769e-2, Ym HAc =3.307e-2, qm Gly
=2.858e-2, qm PD =2.659e-2, qm HAc =5.74e-3, K* Gly =1.143e-2, K* PD =1.55e-2, K* HAc =
8.571e-2. Also, b 1 , b 2 , c 1, and c 2 constants in equation (8.11) are: 2.5e-5, 5.18e-3, 6e-5,
and 5.045e-2 mol/(L*h), respectively.
The critical concentrations required in equation (8.6) were taken from [2]. These
concentrations have an average global deviation of 8.6% for 29 stable states of glycerol
fermentation by K. pneumoniae and C. butyricum [5-6]. The critical concentrations for
glycerol, 1,3-propanediol, acetic acid, and ethanol are: 2.012, 0.8975, 0.7798, and 0.3975
mol/L, respectively.
On the other hand, glycerol fermentation to 1,3-propanediol by K. pneumoniae presents a

metabolic overflow of products causing dynamic phenomena of non-lineal behavior such
as multiplicity of steady states, hysteresis, and oscillations [5]. Thus, the operational
conditions that take to multiplicity of steady states were determined.
In order to obtain the best performance in the first fermentation stage, the volumetric
productivity was optimized using the Levenverg-Marquardt method [7]. Volumetric
productivity is one of the most important functions to be optimized from an operative point
of view, since it implies a high production in a small reactive volume on a short period of
time. The volumetric productivity is shown in equation (8.12), where Pr 1 is the volumetric
productivity in (mol/(L*h)), C PD1 is the outlet 1,3-propanediol concentration in (mol/L),and
D 1 is the dilution rate in (h-1). The first fermentation stage in this equation is indicated by
the subscript 1.
88
Pr1 = C PD1D1
(8.12)
In this case, when multiplicity occurs, the steady states with the highest concentration of
1,3-propanediol were considered. This is to ensure that the steady states with the higher
volumetric productivities are evaluated. Regardless of the inhibition phenomenon caused
by substrate or products, fermentation systems could be successfully performed in two
continuous stages reaching simultaneously a high productivity and a high product
concentration in the first and second fermentation stages respectively [4, 8].
To assess the glycerol fermentation process in two continuous stages, three optimization
models were analyzed. The first optimization model is a sequential procedure in which the
outlet stream from the first fermentation stage (operated at optimal conditions) is directly
fed on the second one. The volumetric productivity on the second fermentation stage was
calculated as a function of both the dilution rate and the achieved change on the 1,3propanediol concentration as shown in equation (8.13). Also, the maximum outlet
concentration of 1,3-propanediol on the second fermentation stage was determined by the
Levenverg-Marquardt method.
Pr2 = D2 ( C PD 2 C PD1 )
(8.13)
Although in the second optimization model the objective function is the volumetric
productivity in the second fermentation stage (see equation 8.13), only the optimal dilution
rate in the first fermentation stage was kept unchanged. Thus, the volumetric productivity
was calculated as a function of both the dilution rate on the second stage and the feed
glycerol concentration on the first stage.
Finally, in the third optimization model the productivity of both fermentation stages were
simultaneously considered as a function of the dilution rate in both stages and the feed
concentration of glycerol in the first fermentation stage. The objective function in this
model is the product of productivities between both fermentation stages (P r3 ), as shown in
equation (8.14).
Pr3 =Pr1 Pr2
(8.14)
89
Figure 8.1 shows the multiple steady states, hysteresis loops, and the wash out line for
the continuous glycerol fermentation. Multiplicity of steady states and hysteresis loops
were studied considering the dilution rate as a parameter from a low glycerol
concentration up to the wash out conditions. In the hysteresis loops when the feed
glycerol concentration increases, low 1,3-propanediol yields are obtained. Moreover,
when the feed glycerol concentration decreases, high 1,3-propanediol yields are acquired.
The latter condition corresponds to the upper curves in the hysteresis loops. The "wash
out" line indicates the extreme operational conditions where the dilution rate equals to the
cellular growth rate (
i = Di ).
Thus, volumetric productivity was calculated as a function of both the feed glycerol
concentration and the dilution rate, using a polygon mesh (partition) from low feed
glycerol concentration up to the wash out line (see Figure 8.2.a.). The conditions that
generate the higher 1,3-propanediol concentration were selected at the multiplicity of
steady states region. As a consequence, a discontinuity between A and B was obtained
and the higher volumetric productivity values were located at the right side of these
points.
Figure 8.1. Hysteresis loops and multiple steady states. Concentrations of: a) Biomass
(g/L). b) Residual Glycerol (mol/L). c) 1,3-Propanediol (mol/L). d) Acetic Acid (mol/L) and
e) Ethanol (mol/L). Vertical lines indicate the limits of the multiple steady states region.
Dotted lines show the "wash out" conditions for each dilution rate.
90
Figure 8.2.a) 1,3-propanediol volumetric productivity, (the column in the right side gives
the scale). b) Region of multiplicity of steady states, optimal productivity for each dilution
rate, global optimal productivity, and wash-out line.
For each dilution rate exists a feed glycerol concentration that generates a maximum
volumetric productivity as obtained by the polygon mesh distribution shown in Figure 2.a.
In order to obtain the global optimum for volumetric productivity both independent
variables (i.e., feed glycerol concentrations and dilution rate) must be simultaneously
considered. Since the highest volumetric productivity is close the steady states region,
this area must be considered when selecting the initial estimated to apply in the
optimization method.
In order to find the conditions for feed glycerol concentration and the dilution rate that
generates the highest volumetric productivity, the Levenverg-Marquardt optimization
method was employed [7]. Since volumetric productivity is a non-continuous function, the
initial estimated for both the feed glycerol concentration and the dilution rate must be
higher than the conditions obtained in the A point.
The multiple steady states region for glycerol fermentation was reported by Xiu et al. [26],
but the used critical concentration parameters have a smaller fitting than the ones used in
this work. The optimal conditions for glycerol fermentation in one continuous stage are as
follows: 0.2821 h-1 for the dilution rate and 0.6882 mol/L for the feed glycerol
concentration, with a volumetric productivity of 0.1076 mol/(L*h).
91
Additionally, the obtained outlet concentration of 1,3-propanediol is 0.3811 mol/L. This

optimal volumetric productivity is outside the multiple steady states region as shown in
Figure 8.2.b. Since this volumetric productivity is very close to the multiple steady states
region, minimum requirements in the automatic control of the equipments are
recommended. Additionally, Figure 8.2.b. shows the wash out conditions and the optimal
volumetric productivity for each dilution rate.
The kinetic model given for the fermentation system by equations (8.1) to (8.12) is equally
applicable for simulation of a fermentation process with two continuous stages. Thus,
three models to optimize the second fermentation stage were used.
In the first model, optimal conditions for the first fermentation stage were used to calculate
the volumetric productivity on the second fermentation stage, but this function increases
proportionally to the dilution rate, contrary to the behavior shown by the concentration of
1,3-propanediol (see Figure 8.3). The optimal concentration of 1,3-propanediol was
0.4126 mol/L at a dilution rate of 1.9850 h-1, with a productivity of 0.0625 mol/(L*h).
Figure 8.3. 1,3-Propanediol productivity and concentration in the second fermentation

stage.
92
In the second optimization model the dilution rate for the first stage was kept from the first
model and the productivity in the second fermentation stage was optimized as a function
of the feed glycerol concentration. The reached productivity is 0.1128 mol/(L*h), at a
dilution rate on the second stage of 0.79 h-1, with a feed glycerol concentration on the first
stage of 0.8817 mol/L. The outlet concentration of 1,3-propanediol from the first and
second stages are 0.3405, and 0.4833 mol/L, respectively. Since each dilution rate has its
own optimal productivity, it was necessary to calculate the global optimal productivity
using the Levenverg-Marquardt method (see Figure 8.4). Optimal productivities for each
dilution rate in the second fermentation stage are represented by the discontinuous curve.
Also, the P point indicates the global optimal productivity in the second stage.
Figure 8.4. Volumetric productivity in the second fermentation stage using the optimal
dilution rate obtained by the model 1 for the first fermentation stage, (the column in the
right side gives the scale).
Finally, in the third optimization model the productivities of both fermentation stages were
simultaneously optimized considering the two dilution rates and the feed glycerol
concentration as independent variables. The obtained results using the optimal dilution
rate in the first fermentation stage are shown in Figure 8.5. The optimum product of
productivities for each dilution rate in the second fermentation stage is represented by the
discontinuous curve.
93
Figure 8.5. Product of productivities of both fermentation stages using the optimal dilution
rate obtained by the model 1 for the first fermentation stage, (the column in the right side
gives the scale).
The Q point indicates the global optimum for the product of productivities. This point
corresponds to a dilution rate of 0.2821 h-1 and 3.08 h-1 in the first and second stages
respectively, and a feed glycerol concentration of 0.7362 mol/L in the first stage. The
reached product of productivities was 0.0116 (mol/(L*h))2 where the outlet concentration
of 1,3-propanediol was 0.4124 mol/L and the global molar yield was 0.5602 1,3-PD/ Gly.
Table 8.1 shows the results of the three different models used to optimize the
fermentation of glycerol in two stages. The highest global yield and volumetric productivity
in the first fermentation stage were generated using the sequential optimization model. On
the other hand, the volumetric productivity in the second fermentation stage was
optimized using the combined optimization model under the optimal dilution rate in the
first fermentation stage. But, when the dilution rate in the first fermentation stage was
decreased at 0.25 h-1, the best final concentration of 1,3-propanediol was obtained as
shown in Table 8.1.
94
Table 8.1. Results summary for each optimization model.

Optimization CS 0 a
D1
D2c
CPD 1 d
CPD 2 e Y PD/S f
Pr 1 g
Pr 2 h
Pr 3 i
Model
Sequential.
0,688 0,282 1,985 0,381
0,413
0,599
0,107
0,063
6,72e-3
Combined
0,882 0,282 0,790 0,341
0,483
0,548
0,096
0,113
1,09e-2
Combined
0,932 0,250 0,940 0,396
0,512
0,549
0,099
0,109
1,08e-2
Combined
0,852 0,300 0,720 0,311
0,469
0,551
0,093
0,114
1,06e-2
Simultaneous 0,736 0,282 3,080 0,377
0,412
0,560
0,106
0,109
1,15e-2
Feed glycerol concentration in the first stage in (mol/L),

-1
fermentation stage in (h ),
Dilution rate in the first
Dilution rate in the second fermentation stage in (h-1),
propanediol concentration in the first fermentation stage in (mol/L),

concentration in the second fermentation stage in (mol/L),
1,3-
1,3-propanediol
Global fermentation yield,
Volumetric productivity in the first fermentation stage in (mol/(L*h)),
Volumetric
productivity in the second fermentation stage in (mol/(L*h)), Product of productivities in

(mol/(L*h))2.
Also, when the dilution rate in the first fermentation stage was increased to 0.30 h-1, the
highest volumetric productivity in the second stage was obtained as shown in Table 8.1.
Then, the volumetric productivity in the second fermentation stage can be optimized only
at a specific dilution rate in the first fermentation stage.
Using the simultaneous optimization model a high volumetric productivity in both

fermentation stages and the highest product of productivities were obtained. Also, the
obtained value for the optimal dilution rate in the first stage was the same using the
sequential optimization model. Thus, the use of a sequential optimization model allowed
obtaining the highest global yield for 1,3-propanediol (0.599) and the maximum volumetric
productivity in the first fermentation stage (0.1075 mol/(L*h)), whereas the highest 1,3propanediol outlet concentration (0.512 mol/L) was observed when the combined
optimization model was employed. Meanwhile, using the simultaneous optimization model
showed both: high volumetric productivities in the two fermentation stages and the highest
product of productivities (0.01157 (mol/(L*h))2). In this way, for the fermentation of
glycerol in two continuous stages, three different operational configurations are available
depending on the desired process objective namely global yield, 1,3-propanediol outlet
concentration, or high simultaneous productivity.
95
On the other hand, the main problem for designing the downstream process for 1,3propanediol recovery and purification from the fermentation broth is the high hydrophilicity
and high boiling point of 1,3-propanediol. The purification scheme proposed here is based
on integrated reaction-separation units to carry out the recovery of 1,3-propanediol. The
first integrated stage is a reactor-extraction process where the hydrophilic nature of 1,3propanediol is changed by the acetylation reaction with iso-butyl aldehyde, which
produces 2-iso-propyl-1,3-dioxane as shown in Figure 8.6.
H3C
HO
OH
H3C
H3C
O
HC
H3C
CH2
CH2
H2O
CH2
Figure 8.6. Acetylation reaction of 1,3-propanediol with iso-butyl aldehyde to 2-iso-propyl1,3-dioxane
The 2-iso-propyl-1,3-dioxane has a hydrophobic character which is dragged into the

organic phase containing mainly iso-butyl aldehyde. This aldehyde acts as both reagent
and solvent for the reactive-extraction process [9-11]. Subsequently, the 1,3-propanediol
is recovered by reactive distillation of 2-iso-propyl-1,3-dioxane and water though out the
reverse reaction of cyclical acetylation. Thus, 1,3-propanediol at high purity is obtained by
bottom while the iso-butyl aldehyde is recovered by distillated and it is able to be reused
in the downstream process. Then, the aldehyde is not consumed in the purification
process.
Based on the fermentation results obtained previously for the fermentation stage, three
scenarios are selected in order to perform the process design and analysis for glycerol
conversion to 1,3-propanodiol.
The first scenario considers conditions of optimal volumetric productivity in the first
fermentation stage and optimal final concentration of 1,3-propanediol according to the
sequential model presents in Table 8.1. The second scenario considers conditions of the
highest final concentration of 1,3-propanediol and the highest productivity in the second
fermentation stage according to the second combined model presented in Table 8.1. And
finally, the third scenario considers the optimal global productivity having into account
both fermentation stages according to simultaneous model presented in Table 8.1.
96
Calculated results of the two-stage fermentation process for each scenario are shown in
Table 8.2, where the column named Feed indicates the fed stream to the first
fermentation stage, Products 1 corresponds to the fermentation products stream from the
first fermentation stage, and Products 2 is the fermentation products stream from the
second fermentation stage.
The normalized stoichiometry for the fermentative reactions are shown in Table 8.3
according to fermentation results obtained for each scenario (Scen. in Table 8.3) and
each fermentation tank (Ferm. in Table 8.3). Also, the molecular formula used for K.
pneumoniae is CH 1.75 O 0.46 N 0.23 .
Table 8.2. Fermentation results for the three considered scenarios
Concentration mmol/L
Feed
Products 1
Products 2
Scenario 1
688.2
77.6
8.9
13PD
0.0
381.1
419.7
AcAc
0.0
109.6
122.5
EtOH
0.0
43.4
51.3
Biomass (g/L)
0.0
2.7741
3.1788
932.0
330.1
153.6
13PD
0.0
395.1
511.4
AcAc
0.0
118.2
149
EtOH
0.0
23.6
35.1
Biomass (g/L)
0.0
2.4924
3.1489
736.2
147.8
92.1
13PD
0.0
376.8
412.2
AcAc
0.0
111.7
121.7
EtOH
0.0
33.4
37.3
Biomass (g/L)
0.0
2.6267
2.8725
Glycerol
Scenario 2
Glycerol
Scenario 3
Glycerol
97
Table 8.3. stoichiometric reactions for each scenario and each fermentation stage
Reactions
Glycerol
Residual Gly 13PD
AcAc
EtOH
Biomass
Molecular Weight
92.09
92.09
76.09
60.05
46.07
23.94
Scen 1, Ferm 1
1.0
0.1067
0.5238 0.1506 0.0597
0.1593
Scen 1, Ferm 2
1.0
0.1045
0.4530 0.1514 0.0927
0.1984
Scen 2, Ferm 1
1.0
0.3399
0.4069 0.1217 0.0243
0.1072
Scen 2, Ferm 2
1.0
0.4523
0.3424 0.0907 0.0339
0.0807
Scen 3, Ferm 1
1.0
0.1896
0.4834 0.1433 0.0429
0.1408
Scen 3, Ferm 2
1.0
0.4832
0.3071 0.0868 0.0338
0.0891
The simplified flowsheet for 1,3-propanediol production from raw glycerol is shown in
Figure 8.7. Cell mass contained in the fermentation is withdrawn throughout a
centrifugation process. Then, the clarified fermentation broth is mixed with iso-butyl
aldehyde in a weight ratio of 5/1 (iso-butyl aldehyde /1,3-propanediol). The reactiveextraction process takes place at 10 C since better distribution coefficients are obtained
at lower temperatures. From the reaction extraction unit two product streams are
obtained.
Aqueous stream should be purified in order to recover significant amounts of both isobutyl aldehyde and 2-iso-propyl-1,3-dioxane. This stream is subjected to two distillation
stages, in the first one the heterogeneous azeotrope iso-butyl aldehyde-water is obtained
by distillated, and then iso-butyl aldehyde is obtained at 97.3 wt % by decantation. In the
second distillation column the homogeneous azeotrope composed by 2-iso-propyl-1,3dioxane and water is obtained at the top of the distillation column. Then, the obtained
azeotrope is mixed with the organic stream obtained during the reactive extraction
process which contains mainly iso-butyl aldehyde and 2-iso-propyl-1,3-dioxane. The
resulting mixture is distilled and the heterogeneous azeotrope iso-butyl aldehyde-water is
once again obtained as the top product, and thus a mixture containing 2-iso-propyl-1,3dioxane and water is obtained. This stream is directly fed to the reactive distillation
column as follows.
98
Waste
DC-1
Water 1
Metanol
E-1
C-1
R-1
E-2
Water
Waste RII-1
Water 2
Dec-1
Raw
Glycerol
Water
F-1
F-2
M-1
Glycerol
(98wt%)
Glycerol
(85wt%)
Solids
Aqueous
glycerol
Organic
Phase
Adsorbate
Diluted
Glycerol
Dec-3
Dec-2
IBuAld
Distillated-6
Waste
water
Distillated-5
DC-5
RDC
Waste
water
Distillated-4
DC-4
Cen-1
Organic
Phase
M-2
Aqueous
Phase
M-2
Bottoms-4
Fresh
IBuAld
Distillated-2
Water
Bottoms-5
RE-1
DC-2
DC-3
Distillated-3
13PD
Fermentation
Broth
P-3
Bottoms-3
Solids
Bottoms-2
Figure 8.7. Simplified flowsheet for 1,3-propanediol production from raw glycerol. E:
evaporator, R: reactor, C: centrifuge, Dec: Decanter, DC: distillation column, M: Mixer, F:
fermentator, RE: Reactor extractor, RDC: Reactive distillation column.
In order to determine both the operational viability and the best configuration in the
reactive-distillation tower (localization of the reaction zone), the Static Analysis is applied
[12-16]. This methodology is the main tool for the qualitative study of the reactive
distillation process which requires minimum initial information. Also it is based on both the
thermodynamic topological behavior of reactive system and on the selection of stable
state limits of highest conversion.
Static Analysis was developed by Serafimov et al [12] and has been sufficiently illustrated
for Pisarenko et al [13] and validated in multiple reactive systems [14-16]. Some
considerations should be made to carry out this analysis: (i) the reaction takes place
under equilibrium conditions and (ii) the reactive distillation column operates to total both
reflux and efficiency. In other words
/)( to conditions are considered. T
he main
operation parameters are: the flow ratio of distillated to bottom (P/W) and the volume and
localization of the reaction zone.
99
To carry out the thermodynamic topological analysis of the reactive system their singular
points are characterized as shown Table 8.4 and the corresponding quaternary residue
map curves is obtained as shown in Figure 8.8.
Table 8.4. Singular Points ** - Acetilation System of 1,3-PD* with 2iP13DO*

Component
Type
Temperature
X1
X2
Azeotrope_H 2 O-iBuAld*
Heterogeneous
61,35 C
0,2079
0,7921
iBuAld*
Homogeneous
64,10 C
---
Azeotrope_H 2 O-2iP13DO*
Heterogeneous
92,87 C
0,7449
0,2551
H2O
Homogeneous
100,00 C
---
2iP13DO*
Homogeneous
138,12 C
---
13PD*
Homogeneous
214,40 C
---
* iBuAld: iso-Butyraldehyde; 2iP13DO: 2-iso-Propil-1,3-Dioxano; 13PD: 1,3-Propanediol; H2O: Water.

** Singular Points at 1Atm.
Figure 8.8. Residue map curves for the reactive system.
Thus, only one distillation region is obtained with a bunch of residue curves starting from
the azeotrope of minimum boiling point (iso-butyl aldehyde-water) and ending in corner of
1,3-propanediol. By direct separation (formulated distilled) three distillation subregions are
obtained while in the case of indirect separation (bottoms formulated) two distillation
100
subregiones are founded. Thus, ten different possibilities for the feeding ratio of 2-isopropyl-1,3-dioxane to water to the reactive distillation column were analyzed (results no
shown). Then, the feeding ratio that generates the highest products distribution (P/W) at a
total conversion of 2-iso-propyl-1,3-dioxane (see Figure 8.9) corresponds to the 2-isopropyl-1,3-dioxane/water molar mixture of 0.3776/0.6224, as is shown in Figure 8.10.
Also, based on both the chemical equilibrium and the residue curve maps, it was
determined that the reactive zone must be localized in the stripping section of the reactive
distillation column. Also, the product obtained in the distillated stream is the
heterogeneous azeotrope iso-butyl aldehyde-water.
Then, in order to verify it was possible to obtain the conditions and the trajectory predicted
by the Static Analysis method, simulations at both / (stages/ reflux ) and finite condition
were carried out. Also, it was found that a self-extractive phenomenon (which can be
understood as a non-lineal variation in the relative volatility of a system with the change of
concentrations in a multicomponent mixture, see Figure 8.11) affects strongly the reactive
distillation column performance. The analysis of the isovolatility curves allowed
determining that a redistribution of the feeding streams lead to a high conversion keeping
the configuration obtained by the Static Analysis method.
Figure 8.9. Direct separation with fed 0.377645/0.6223552iP13DO/Water
101
Figure 8.10. P/W ratio, Direct Separation (XF: 0.377645/0.622355-2iP13DO/water)
Figure 8.11. iso-Volatility curve (Wateriso-Butyraldehyde2-iso-Propil-1,3-Dioxane)
In this case, the water required as reactive to carry out the hydrolysis reaction of 2-isopropyl-1,3-dioxane is fed in five different stages to the reactive distillation column. For
instance, in the Scenario 1 the reactive distillation column has 45 stages and the water
stream is fed to the stages: 9, 15, 22, 29, 36, and 43. And the respective mass flows are:
7.56, 11.46, 15.36, 19.27, and 23.17 kg/h.
102
In this way high conversion levels of 2-iso-propyl-1,3-dioxane are obtained. But, because
of the 1,3-propanediol purity ranges 83.3 and 93.9 wt %, a final distillation process is
required in order to achieve a higher purity of 1,3-propanediol. A summary of the main
simulation results for each scenario is given in Table 8.5.
Table 8.5. Summary of the main simulation results for 1,3-propanediol production from
glycerol
Scenario1
Dilgly
13PD2
Organic2
Aqueous1 IP13DO3
13PD3
13PD4
310
310
283.2
283.1
404.2
457.9
486.8
9115.8
8900.1
920.0
9270.3
453. 8
272.6
252.7
WATER
8536.2
8536.2
14.28
8582.9
0.752
GLYCE-01
578.9
6.45
0.273
6.18
0.273
0.273
0.273
KPNEUMON
27.16
1,3-P-01
273.67
1.633
10.95
1.63
251.1
250.83
ACETI-01
62.95
0.387
59.07
1.08
1.08
0.980
ETHAN-01
20.15
1.335
17.48
3.74
0.003
ISOBU-01
491.32
557.88
0.449
0.183
2IP13DOX
410.77
35.83
446.60
19.24
0.656
Temperature K
Mass Flow
kg/hr
Scenario2
Dilgly
13PD2
Organic2
Aqueous1 IP13DO3
13PD3
13PD4
310
310
283.2
283.1
409.1
444.4
487.7
6766.1
6594.4
925.9
6847.5
423.5
247.9
245.0
WATER
6186.5
6186.5
15.51
6227.5
2.74
0.007
GLYCE-01
578.9
89.0
5.95
83.04
5.95
5.95
5.95
KPNEUMON
20.26
1,3-P-01
250.30
1.70
7.54
1.703
238.4
238.4
ACETI-01
57.58
0.473
52.84
1.021
0.747
0.686
ETHAN-01
10.37
0.560
8.50
1.001
ISOBU-01
515.1
442.4
1.515
1 E-03
2IP13DOX
386.6
25.73
412.3
0.017
0.002
Temperature K
Mass Flow
kg/hr
103
Scenario3
Dilgly
13PD2
Organic2
Aqueous1 IP13DO3
13PD3
13PD4
310
310
283.2
283.1
411.4
424.7
487
8531.1
8338.9
872.37
8687.6
427.6
253.6
248.4
WATER
7951.5
7951.5
13.77
7995.5
5.15
0.027
GLYCE-01
578.9
53.03
2.277
50.7
2.28
2.28
2.28
KPNEUMON
23.73
1,3-P-01
259.07
1.535
10.20
1.53
245.6
245.6
ACETI-01
60.30
0.374
56.55
0.7
0.59
0.507
ETHAN-01
14.28
0.573
12.37
ISOBU-01
465.07
527.9
0.001
2IP13DOX
388.77
34.32
423.1
0.011
Temperature K
Mass Flow
kg/hr
The final production of 1,3-propanediol is mainly related to the fermentation yield of both
fermentation stages. Thus, while the decreasing order for the final concentration of 1,3propanediol after the second fermentation stage was Scenario 2 > Scenario 1 > Scenario
3 (see Table 8.2) and the decreasing order for the fermentative yield of glycerol to 1,3propanediol was Scenario 1 > Scenario 3 > Scenario 2 (see Table 8.3), the decreasing
order for the actual production of 1,3-propanediol was Scenario 1 > Scenario 3 > Scenario
2 (see Table 8.5).
Otherwise, high recovery percentages were achieved for iso-butyl aldehyde, indicating
that low requirements of fresh reactive are required. Higher differences are noticed when
the whole technological scheme is analyzed. The maximum global molar yield from
glycerol to 1,3-propanediol was obtained for the Scenario I, while the minimum was
obtained for the Scenario 2. The relative difference between these two scenarios was
5.21 %, which was close to the relative difference for the fermentation yield obtained for
the same both scenarios, 11.14 %. Thus, it can be stated that the technological
performance of 1,3-propanediol production from raw glycerol depends mostly on the
global conversion of substrate to the main product during the fermentation stage. See
Table 8.6.
104
Table 8.6. Data representing the behavior of the downstream process

Scenario 1
Scenario 2
Scenario 3
Extraction efficiency (%)
96.00
96.99
96.06
Distribution coefficient
204.09
203.39
204.91
Loading (Z)
0.177
0.182
0.177
Global 13PD recovery (%)
91.66
95.26
94.80
IBuAld recovery (%)
98.66
99.48
99.54
0.5244
0.4984
0.5134
Reactive-Extraction
Downstream Process
Global Process
Global process yield from Glycerol to Lactic acid (%)
8.2 Ethanol production

Ethanol can be produced from sugarcane [17], corn starch [17], sugar [18], molasses [19],
cassava [20], wheat [21] or lignocellulosic biomass [22-25]. Glycerol fermentation by
Escherichia coli produces a mixture containing predominantly ethanol, acetate, and
succinate, also low amounts of formiate could be produced [26]. Succinate and acetate
are competitive by-products which could eventually decrease the ethanol yield as was
shown in Figure 6.2. Thus, glycerol can be converted into ethanol and either hydrogen or
formiate. The resulting mixture can be easily purified due to the significant
physicochemical differences among its compounds.
The analysis here performed is based on the results presented by Yazdani and Gonzalez,
about glycerol conversion to ethanol by Escherichia coli SY04 (pZSKLMgldA) [27]. Their
experimental study used two approaches: (i) ethanol and H 2 co-production, and (ii)
ethanol and formiate co-production. It was found that the maximum theoretical yield in
both cases was 1 mol of ethanol plus 1 mol of either formiate or hydrogen per each mol of
consumed glycerol. As additional information to perform the simulation, the average
molecular formula for E. coli of CH 1.9 O 0.5 N 0.2 was used [28].
Three different possibilities for ethanol production from glycerol were considered. The first
and second possibilities use crude glycerol (88 wt %) in a fermentation stage at a dilution
105
of 10 g/L and 20 g/L, respectively. Meanwhile, the third possibility considered pure
glycerol (98 wt %) at 10 g/L. The flowsheet of these three simulated bioprocess for fuel
ethanol production from glycerol using E. coli is shown in Figure 8.12.
In all cases the flowsheet is the same, but the operational conditions are different.
Obtained glycerol from the purification process (88 wt % or 98 wt %) was cooled at 37 C
and diluted (10 g/L or 20 g/L) in fresh water at 37 C. Then the glycerol fermentation
process was carried out by E. coli SY04 (pZSKLMgldA) [27] and a mixture of ethanol,
formiate, and cells was produced. Cells were withdrawn by centrifugation and an aqueous
stream of ethanol and formiate was obtained. This stream was distilled and ethanol
concentrated in two distillation columns with 40 and 30 stages respectively. Then, an
ethanol stream between 93 wt % and 94 wt % of purity was obtained (concentration near
to ethanol-water azeotrope 95.6 wt %). Finally, ethanol was dehydrated in a molecular
sieve and fuel ethanol was obtained at 99.5 wt %. The main results of this simulation are
shown in Table 8.7.
Water
Broth
Glycerol
Diluted
Glycerol
Solids
Distillate
1
Distillate
2
Water
waste 1
Ethanol
Adsorbate
Water
waste 2
Figure 8.12. Simplified flowsheet of fuel ethanol production from glycerol at 88 wt % and
98 wt %. 1. Mixed tank, 2. Fermentation tank, 3. Centrifuge, 4. First distillation column, 5.
Second distillation column, 6. Molecular sieves.
In general terms the ethanol production has been described as a process composed of
four main stages named: raw material conditioning, fermentation, separation and
dehydration, and waste treatment. A comparison among the ethanol production from
traditional feedstocks (i.e., sugar cane and corn) versus raw glycerol as raw material was
performed. Figure 8.13 shows the required stages for ethanol production from these three
feedstocks.
106
Table 8.7. Simulation results for fuel ethanol production from glycerol
STREAM
Diluted
Broth
Distillate
glycerol
Ethanol
From Crude Glycerol at 10 g/L

Temperature (C)
Mass Flow (kg/hr)
37
37
57633.551 57624.871
77.9
77.9
292
273.072
Mass Fraction:
Water
0.99
0.9899
0.06
0.005
Glycerol
0.01
0.0002
E. coli
0.0004
Ethanol
0.0048
0.94
0.995
Formiate
0.0046
77.9
77.9
313
290.508
From Crude Glycerol at 20 g/L

Temperature (C)
Mass Flow (kg/hr)
37
37
28896.25 28881.1230
Mass Fraction:
Water
0.98
0.9801
0.067
0.005
Glycerol
0.02
0.0000
E. coli
0.0006
Ethanol
0.0103
0.933
0.995
Formiate
0.0089
77.9
77.9
317
293.383
From Pure Glycerol at 10 g/L

Temperature (C)
Mass Flow (kg/hr)
37
37
57530.195 57538.901
Mass Fraction:
Water
0.99
0.9896
0.07
0.005
Glycerol
0.01
0.0000
E. coli
0.0004
Ethanol
0.0053
0.93
0.995
Formiate
0.0047
107
Sugar cane
Corn
Co-generation
Grinding
Grinding
Evaporation
Steam
Clarification
Cooking
Neutralization
CO2
Fermentation
Enzyme
Liquefaction
Washing
Centrifugation
Enzyme
Saccharification
Distillation
DIstillation train
CO2
Fermentation
Fermentation
Dehydration by
adsorption
DIstillation train
Centrifugation
Evaporation train
DIstillation train
Combustion gases
Crude Glycerol
Bagasse
Ethanol
Dehydration by
adsorption
Whole
Stillage
Evaporation train
Ethanol
Concentrated
stillage
Whole
Stillage
Centrifugation
Thin
Biomass
Dehydration by
adsorption
Stillage
Drying
Ethanol
Distillers dried
grains
Figure 8.13. Stages for ethanol production from sugar cane, corn, and crude glycerol.
Although simulations were carried out for ethanol production from sugar cane and corn
according to the flowsheets shown in Figure 8.14, the most relevant results were obtained
from the economic assessments and then they are discussed in the Section 8.8.2.
On the other hand, based on the possibility of transforming both raw glycerol and biomass
to ethanol, a sustainable production of biodiesel from oil palm was here proposed. Thus,
sustainable biodiesel production can be performed using only oil palm as a single
feedstock. Palm oil extraction produces mainly two lignocellulosic residues: empty fruit
bunches (EFB), produced in the highest amount; and palm press fiber (PPF) resulting
from press cake separation. They have an important lignocellulosic content and low
moisture, thus both residues can be used as feedstock for bioethanol production [24].
Besides, glycerol is the main by-product of biodiesel production and it can also be
transformed to ethanol. Thus, both oil extraction residues (EFB and PPF) and raw
glycerol are used as feedstock to produce the ethanol required to carry out the
transesterification reaction with palm oil.
108
Figure 8.14. Simplified flowsheet for ethanol production from: (A) sugar Cane: 1.
Washing tank. 2. Mill. 3. Clarifier. 4. Rotary Filter. 5. Fermentator. 6. Centrifuge. 7.
Absorption column. 8. Concentration column. 9. Rectifying column. 10. Molecular sieves.
11. Evaporator. 12. Boiler. 13. Turbo-generator. (B) Corn: 1. Washing tank. 2. Mill. 3.
Liquefaction reactor. 4. SSF Reactor. 5. Absorption column. 6. Concentration column. 7.
Rectification column. 8. Molecular sieves. 9. First evaporation train. 10. Centrifuge. 11.
Second evaporation train. 12. Air dryer.
109
The first step is the overall extraction process of palm oil from fresh fruit brunches (FFB).
The whole extraction process includes: Pretreatment stage, where FFB are first cooked
using saturated steam in order to prepare fruit to a subsequent remotion from brunches,
and then they are digested in a cylindrical vertical tank at 100C, obtaining a separation of
pulps from nuts. Extraction stage, mashed fruits are passed through a screw pressing,
where crude oil is splited up from cake. Refining stage, is made, first decanting crude with
hot water at 90C, obtaining a decanter cake from additional ,oil is recover. Clarified oil
contains 1% of water, for that reason, must be dried under vacuum conditions before be
stored in oil tanks. Also, obtained press cake is treated, in order to obtain Palm Press
Fiber (PPF) and nuts, these vegetable wastes are used to extract palm kernel oil (PKO)
and palm kernel cake (PKC).
Then, EFB and PPF obtained in the oil extraction process is used in bioethanol production
from lignocellulosic biomass, composed up to 75% of cellulose and hemicellulose. The
overall process usually includes five main steps: biomass pretreatment, cellulose
hydrolysis, fermentation of hexoses, separation and effluent treatment. In first step,
feedstock is pretreated, because composition of this biomass, containing up to 75% of
cellulose and hemicellulose, it should be broken down into fermentable sugars able to be
converted into ethanol and other products [23]. Among available pretreatment methods, in
this work is used a diluted acid pretreatment with sulphuric acid 1-10% at 121 C [29], in
order to hydrolyze hemicellulose, producing Hexose and Pentose. In a previous work
Cardona et al [18], showed a very promising integrated configuration for bioethanol
production from an energy viewpoint [18, 30] known as simultaneous saccharification and
co-fermentation (SSCF). In this configuration the hydrolysis of cellulose, the fermentation
of glucose released, and the fermentation of pentoses present in the feed stream is
simultaneously accomplished in a same single unit, using a genetically modified
Zymomonas mobilis, Culture broth exiting SSCF bioreactor has an ethanol concentration
of about 6% weight. This stream is concentrated up to 92% in two distillation columns.
The dehydration of ethanol is made by adsorption with molecular sieves. Stillage obtained
from the bottoms of concentration column is evaporated to reduce its volume and
diminishing the costs of its further treatment and the lignin is separated using
centrifugation.
110
Otherwise, biodiesel is produced from refined palm oil obtained in extraction section. This
vegetable oil is composed by a mixture of triglycerides, being major compounds:
Tripalmitin, Triolein and Trilinolein. They are transeterified, reacting with ethanol using
potassium hydroxide as catalyst. This process is carried out with an integration approach
named multistage reactor-extractor. This process combines the chemical reaction and
liquidliquid extraction, achieving high selectivity, conversion, productivity, and purity [31].
In this way two main streams are obtained: biodiesel-enriched liquid phase (65% of ethyl
esters), continuously removed from the reactor-extractor and sent to a separation unit
where ethanol is recovered, and glycerol-enriched liquid phase (44% of glycerol) [32].
Finally, an additional amount of ethanol can be produced from raw glycerol as it was
above described.
Simulations are based on Colombian palm industry conditions, reported by Gutierrez et al.
[24] with an average installed processing capacity of crude palm oil of 122 tonnes per day
of FFB. And the lignocellulosic residues are 28.06 tonnes per day of EFB, and 17.98
tonnes per day of PPF. Thus, the total crude palm oil is 21.76 tonnes per day. To carry
out the simulation of integrated biodiesel production process some particularities of each
stage must be considered, which are described as follows: Extraction process considers
the composition data reported by Abdul Aziz et al. [33, 34], and Wan Zahari and Alimon
[35], for both lignocellulosic residues, EFB and PPF, obtained during palm oil extraction,
also the yield extraction was based on the reported data by Prasertsan and Prasertsan
[36], for processing of FFB to crude palm oil.
Ethanol production process from lignocellulosic biomass was analyzed in a previous work
[30], and a brief description of the main processing units is given: pretreatment of
lignocellulosic biomass, enzymatic hydrolysis, and co-fermentation processes were
simulated based on a stoichiometric approach. Thus, lignocellulosic biomass was
converted into glucose and pentoses, which after were converted into cell biomass,
ethanol, and fermentation by-products.
Biodiesel production process from palm oil was previously analyzed by Gutirrez et al.
[24] in an integrated raw-ethanol production process from lignocellulosic biomass, where
a kinetic approach and a multi-stage reactorextractor were used. This kinetic model
considers a serial reactive system which transforms triglycerides and ethanol into an ethyl
111
ester (biodiesel) molecule and either diglycerides, monoglycerides, or glycerol. Figure

8.15 shows jointly the ethanol production process from lignocellulosic biomass, the
biodiesel production process from palm oil, and the ethanol production process from
glycerol.
Here, the process integration is made at different levels to increase efficiency and
productivity. First level is given for two individual processes: i) Integration reactionreaction for the process of producing ethanol from biomass feedstock EFB and PPF. In
this process the reaction of hydrolysis of cellulose is carried out simultaneously with the
fermentation of pentoses and hexoses in the SSCF process. ii) Integration reactionseparation of the process of producing biodiesel from palm oil, which uses a multistage
extractor reactor. Second level of integration takes place between the processes of
biodiesel and ethanol production using lignocellulosic biomass and glycerol. Second level
uses a totally integrated configuration, using oil palm residues EFB and PPF, which are
proposed as raw materials for ethanol production. They enter the pretreatment reactor
where react with dilute acid at high pressure. Then, the pretreated lignocellulosic biomass
undergoes the transformations described in Bioethanol Production from lignocellulosic
biomass Section obtaining dehydrated ethanol with purity greater than 99.5% by weight.
This stream of ethanol, is mixed with an incoming one form (Bioethanol from glycerol)
section, where process crude glycerol is first purified, finally, the crude glycerol is first
refined, and then converted to ethanol by mean of an Escherichia coli strain in a
fermentative process [37]. Final ethanol mixture with a purity of 99.5 % weight, along with
crude oil, is fed to a multi-stage reactorextractor (Biodiesel from palm oil where
transesterification reaction is continuously accomplished by reactive extraction process
using KOH. Also, main input data and operation conditions used in the simulation process
are shown in Table 8.8.
Thus, sustainable biodiesel production from oil palm was simulated considering jointly
four processes named: palm oil extraction and refining, biodiesel production, and ethanol
production from two feedstocks, lignocellulosic residues and raw glycerol. The
corresponding simulation results for the main process streams are shown in the Table
8.9. Due to extraction process is not showed in the Figure 8.15 the main feed streams are
EFB, PPF, and palm oil; and the other feed streams are service fluids and catalytic
112
agents. In this sense, the main product streams are biodiesel and ethanol, and also some
waste water streams are obtained.
Figure 8.15. Flowsheet for the integrated process of combined biodiesel and bioethanol
production. Syrup (concentrated sugars in water). (1) Pretreatment reactor, (2) Washing,
(3) Ionic exchange, (4) Simultaneous saccharification and co-fermentation, (5)
Concentration column, (6) Rectification column, (7) Molecular sieves, (8) Evaporation
train, (10) Centrifuge, (11) Multi-stage reactorextractor, (12) Distillation column for
biodiesel purification, (13) Distillation column for glycerol purification, (14) Neutralization
tank, (15) Centrifuge, (16) First distillation column, (17) Washing tank, (18) Evaporation
column, (19) Second distillation column, (20) Mixed tank, (21) Fermentation tank, (22)
Centrifuge, (23) Third distillation column, (24) Fourth distillation column, (25) Molecular
sieves.
113
Table 8.8. Main input data and operation conditions used in the simulation process.
Feature
Description
FFB: Cellulose 18,38; Hemicellulose 12,52; Lignin 9,05; Others 9,05;
Raw materials Moisture 56,84.

composition
EFB: Cellulose 15,47; Hemicellulose 11,73; Lignin 7,14; Ash 0,67;

Moisture 65,00.
PPF: Cellulose 24,00; Hemicellulose 14,40; Lignin 12,60; Ash 3,00; Oil
3,48; Others 2,52; Moisture 40,00.
Ethanol
production
from
Lignocellulosic biomass pretreatment: H 2 SO 4 diluted at 190C and

12.2 atm by 10 min. Hemicellulosic conversion 75%.
Simultaneous saccharification and co-fermentation: T. reesei
lignocellulosi cellulases and recombinant Z. mobillis at 30C by 144 h. Cellulose and

c biomass
cellobiose conversion are 80% and 100% respectively. Ethanol and

biomass yield are 92% and 2,7% of theoretical.
Ethanol distillation: Two distillation columns at 1,77 atm, where the
final ethanol concentration is 92,3 wt %.
Ethanol dehydration: Molecular sieves at 116 C by 10 min at 1,7 atm.
Final ethanol concentration: 99,5 wt %.
Multi-stage reactorextractor: 5 counter-current stages at 60C with a
Biodiesel
production
residence time of 6 h. Liquid phase equilibrium was considered.

Biodiesel purification: Ethanol recovery by distillation from both light
from palm oil (biodiesel) and heavy (glycerol) phases.
Glycerol fermentation: E. coli SY04 (pZSKLMgldA) is used in a

Ethanol
fermentation broth of 20 g/L of glycerol (previously purified until 88 wt %)
production
at 37 C and pH 7.
from crude
Ethanol distillation: Two distillation columns at atmospheric pressure,
glycerol
where the final ethanol concentration is 94,1 wt %.

Ethanol dehydration: Molecular sieves at 116 C by 10 min at 1,7 atm.
Final ethanol concentration: 99,5 wt %.
114
Table 8.9. Main process streams for ethanol production from lignocellulosic biomass
Recycle
Lignocell
Biomass
Stream
Rectific.
BioEtOH Biodiese
Broth water for
Column
From
washing
Distillate
Biomass
Refined
BioEtOH
Glycerol
From
Glycerol
T (C)
20
30
77,4
93,4
25
73,7
244,9
P (bar)
1,793
1,793
1000
0,2
0,3
Mass flow (Kg/h)
1913,1
4185
667,3
310,8
230,7
947,8
95,8
Cellulose (%)
18,38
1,52
0,04
--
--
--
--
Hemicellulose (%)
12,52
1,31
0,03
--
--
--
--
Lignin (%)
9,05
3,72
0,09
--
--
--
--
Glucose (%)
--
0,56
0,5
--
--
--
--
Xylose (%)
--
0,67
0,89
--
--
--
--
Water (%)
56,84
80,47
97,78
8,5
0,5
0,002
10,5
0,5
Triolein (%)
--
--
--
--
--
0,6
--
--
Diolein (%)
--
--
--
--
--
0,2
1,2
--
Monoolein (%)
--
--
--
--
--
0,02
0,298
--
Ethanol (%)
--
5,59
0,01
91,5
99,5
1,32
0,002
99,5
Ethyloleate (%)
--
--
0,01
--
--
98,9
--
--
Glycerol (%)
--
--
0,02
--
--
0,02
88
--
This configuration considers two simultaneous processes: (i) saccharification and

fermentation, where the cellulose hydrolysis produces glucose, which is assimilated by
the microorganisms and converted into ethanol, and thus the inhibitory effect of glucose
over cellulases are reduced. And (ii) reactive extraction where reaction and separation are
integrated in only one processing unit and then conversion, yield and productivity are
improved related to conventional processes because of the continuous products
extraction. In this way, a high product concentration is obtained in both cases.
A high ethanol/palm oil ratio is used inside the multistage reactor-extractor since the
excess of ethanol leads to a better conversion of feedstock during biodiesel production,
as it was showed by Gutirrez et al [24], because of the ethanol fed to the reactorextractor is a mixture of the ethanol obtained from both production processes
(lignocellulosic biomass and glycerol) and the recycled ethanol during biodiesel
77,9
47,11
115
purification. This mixture ensures a high ethanol flow inside the multi-stage reactor
extractor. Thus, a 99,9 % of triolein conversion is reached with a final ethyloleate purity of
98,4 wt %.
Biodiesel production from oil palm and ethanol production from lignocellulosic biomass
have been individually described and analyzed in many times. An important integration
approaches was recently performed by Gutirrez et al [24], they considered different
process configurations for heat and mass integration, and results were discussed based
only on data obtained from process simulation. But the integration of these two processes
leaves an unsolved problem, which is the production of low cost glycerol; since its sales
do not represent a significant income for the integrated biodiesel production. In this way,
the integration of a biorefinery that uses crude glycerol as feedstock to produce more rawethanol was analyzed. Thus, it is possible to have the oil palm as single raw material to
produce biodiesel. Glycerol conversion to ethanol was 99,8 % and the yield was 99 % of
theoretical.
8.3 PHB production

Glycerol purification, glycerol fermentation (cell growth and PHB accumulation), mass cell
pretreatment, PHB isolation, and PHB purification are the five stages needed for the
process of PHB production from raw glycerol. The purification process of raw glycerol was
described in the Chapter 4. This process includes a methanol recovering which decreases
the purification costs in 37.5% [38].C. necator JMP 134 can synthesize PHB up to 70 wt
% of the cell dry mass from various carbon substrates [39]. The fermentation process is
carried out in two stages, in the first stage cell growth occurs and in the second stage
PHB is synthesized. Air and pure oxygen are fed at the first fermentation tank where the
fermentation broth is saturated between 15 or 20 DOC %, thus PHB accumulation takes
place inside the cell mass [40]. The detailed conditions for the fermentation process are
shown in Table 8.10
After fermentation, the next step is PHB isolation and purification. PHB must be extracted
from the cell cytoplasm. Cell membrane is broken and PHB is dissolved and separated
from the residual biomass. The separation step can be divided in three parts:
pretreatment, extraction, and purification. In the pretreatment step cell disruption is
116
carried out easily and some alternatives for this step are: heat, alkaline or salt
pretreatment and freezing [41].
Table 8.10. Process conditions for glycerol fermentation.

Feature
Description
Glycerol 88 wt%
Feedstock
Pumped
Sterilization
Heat exchange
Glycerol 98 wt%
Feed concentration: 170.8 g/L
249 g/L
Feed flow rate: 1000 kg/h
1000 kg/h
Outlet Pressure: 25 atm
25 atm
Net Work required: 8.1 KJ/Kg
7.9 KJ/Kg
Temperature: 139 C
Heat duty: 443.96 KJ/Kg
429.31 KJ/Kg
402.29 KJ/Kg
Required area: 63.62 m2
51.34 m2
Temperature: 35 C
pH: 7
Cell mass growth
Residence time: 21 h.
Aeration: 0.6 vol/(vol*min)
Cell mass concentration: 4.91 wt % or 50.4g/L 4.24 wt % or 44.4 g/L
PHB concentration: 0.44 wt % or 4.5 g/L
0.70 wt % or 7.3 g/L
Temperature: 35 C
pH: 7
Residence time: 22.5 h.
PHB accumulation Cell mass concentration: 7.1 wt % or 73.4 g/L
8.7 wt % or 91.5 g/L
PHB concentration: 2.7 wt % or 27.8 g/L
5.5 wt % or 57.1 g/L
Flow rate: 929.05 kg/h
893.173 kg/h
Some of the different extraction methods to separate PHBs from the cell residual
material are: solvent extraction, digestion, mechanical cell disruption, supercritical fluids
extraction, cell fragility and spontaneous liberation. In Table 8.11 advantages and
disadvantages of the most commonly used PHB extraction methods are listed. Solvent
extraction modifies the cell membrane permeability and the PHB is then dissolved [41].
117
Some used solvents are: chlorinated hydrocarbon (e.g. chloroform), cyclic carbonates
(e.g., propylene and ethylene carbonates), halogenated solvents (e.g., chloroethanes and
chloropropanes), non-halogenated solvents (e.g., chain (410 carbons) alcohols, esters,
amides, and ketones (both cyclic and acyclic compounds)). Digestion can be chemically
or enzymatically performed. Chemical digestion uses different chemical agents to destroy
lipids, carbohydrates, proteins and enzymes. According to the chemical agent used, the
chemical digestion could be: digestion by surfactants (e.g. anionic sodium dodecyl sulfate
(SDS) and synthetic palmitoyl carnitine), by sodium hypochlorite, by sodium hypochlorite
and chloroform, surfactant-hypochlorite digestion, surfactant-chelate digestion, and
selective dissolution of non-PHA cell mass by protons.
The enzymatic digestion uses enzymes to degrade the cell membrane. Some varieties of
proteolytic enzymes have high activities on protein dissolutions and slight effects on PHB
degradation. Enzymatic digestion can be complemented by other extraction methods.
Mechanical cell disruption has been widely used to recover intracellular proteins by
different ways [42-43] such as: bead mill disruption, high pressure homogenization,
disruption by ultrasonication, centrifugation, and chemical treatment. Supercritical fluids
have unique physicochemical properties such as high densities and low viscosities that
make them suitable as extraction solvents. Due to its low toxicity and reactivity, moderate
critical temperature and pressure (31C and 73 atm), availability, low cost, and
nonflammability CO 2 is the most used fluid [44]. This extraction method can also be
combined with NaOH or salt (NaCl) pretreatments to get higher disruption levels [41]. The
cell fragility method takes advantage of the cell fragility shown by some bacteria after
large amounts of PHB accumulation. Other extraction methods use air such as: air
classification and dissolved-air flotation. Finally, purification methods involve a hydrogen
peroxide treatment combined with action of enzymes or chelating agents [41].
Besides, based on the available methods for PHB extraction (see Table 8.11) three PHB
production processes from either raw glycerol (88 wt %) or pure glycerol (98 wt %) were
designed.
118
Table 8.11. PHB Extraction Methods (Adapted from Jacquel et al [25])

Extraction
Methods
Advantages
Disadvantages
Results (wt %)
Break PHA granules

morphology.
Hazards connected
with halogenated
solvents. High
price/Low recovery
Low purity/Water
waste treatment
needed
Degradation of the
polymer
Degradation of the
polymer
Surfactant: High
cell density
digestion by
SDS)Purity >95%;
release rate >90%
Purity: 99%;
Recovery: 94%
High quantity of
solvent needed
Purity: >97%;
Recovery: 91%
Solvent extraction
Elimination of
Endotoxine/high
purity No polymer
degradation
Digestion by
surfactants
Treatment of high
cell densities No
polymer
degradation
Digestion by NaOCl
High purity
Digestion by NaOCl
and chloroform
Low polymer
degradation high
purity
Digestion by NaOCl
and surfactants
Limited
degradation/low
operating cost
Surfactant-EDTA
disodium salt.
Purity: 98%;
Recovery: 86.6%
Digestion by
chelate and
surfactants
High purity/low
environmental
pollution
Large volume of
wastewater Low
degradation of the
polymer
Purity: 98.7%;
Recovery: 93.3%
High recovery and

high purity low
operating costs
Purity: 98.7 wt%

Recovery: 95.4%
Selective
dissolution of
NPCM (Non PHB
cell mas) by
protons
Enzymatic
digestion
Good recovery
Bead mill disruption
No chemicals
used
High pressure
homogenization
No chemicals
used
Supercritical CO2
Using cell fragility
Low cost, low

toxicity
Use of weak
Purity: 99.5%;
Recovery> 90%
High cost of
enzymes
Require several
passes
Poor disruption rate
for low biomass
levels
Low micronization
Purity: 92.6 wt%

Recovery: 90%
Low recovery
Recovery: 89%
Purity: 99%
Yield: 98%
Purity: 95%
119
extracting
conditions
Air classification
High purity
Dissolved air
flotation
No chemicals
used
Spontaneous
liberation
No extracting
chemicals needed
Recovery: 96%
Low recovery
Require several
consecutive flotation
steps
Low recovery (80%
cells secretes PHB
granules
spontaneously
Yield: 90%
Purity: 97%
Purity: 86%
Yield: 80%
The flowsheets for PHB production are shown in Figure 8.16. The fermentation process
begins with a sterilization of diluted glycerol. Pure glycerol was diluted at 249 g/L and raw
glycerol at 170.8 g/L based on the total glycerol consumption by C. necator [40]. Glycerol
conversion takes place in two continuous fermentation stages for cell growth and PHB
accumulation, with operation times of 21 and 22.5 h, respectively. Total glycerol
consumption is considered on the second fermentation stage. The sterilization and
fermentation stages are common for the three PHB production processes. Table 8.12
shows the first downstream process (see Figure 8.16) and it is based on a variation of the
BIOPOL flowsheet [45-46]. The first step is a thermal treatment at 85 C, and then a
digestion process using the pancreatin enzyme Burkholdeira sp. PTU9 and NaOCl is
carried out [47]. This pretreatment causes an appropriate cell disruption releasing the
PHB to the fermentation broth. The digestion product containing between 7 to 9 wt % of
biomass is filtrated and the residual cell mass is withdrawn. The mixture containing the resuspended PHB at 5.5 5.7 wt % is treated with a hydrogen peroxide solution. Then,
using a flash process the majority of the water content is retired. Finally, PHB at 99.9 wt
% is obtained by spray drying.
Figure 8.16 shows the second downstream process and the process conditions are given
in Table 8.13. After passing through the high pressure homogeniser, the depressurized
stream is centrifuged and the solid product is heated and mixed with diethyl succinate
(DES) in a 1/20 ratio of biomass/solvent. The solvent extraction process takes place by
modification of the cell membrane permeability and PHB dissolution [41]. Residual cell
mass is withdrawn by centrifugation and a mixture of PHB-water is gelled by cooling and
the DES is recovered. Finally PHB at 99.9 wt % is obtained by spray drying.
120
Figure 8.16 also shows the third downstream process for PHB production, which is
described in Table 8.14. This downstream process uses an alkaline pretreatment with a
NaOH solution. Then, a digestion process is carried out using NaOCl and sodium dodecyl
sulfate (SDS) as detergent. The disrupted cells are centrifuged and PHB is washed with
H 2 O 2 . The obtained mixture is subjected to an evaporation process and most of the water
1
GLYCEROL
(88 wt % or
98 wt %)
Downstream
Process I
O2
12
WASTE
WATER 1
Downstream
Process II
10
11
AIR
ENZYME
H2O2
NaOCl
WASTE
WATER 2
SOLIDS
PHB
STEAM
14
11
13
10 STEAM
DES
9
STEAM
DES
12
12
WASTE
WATER 2
PHB
Downstream
Process III
GASES
11
SOLIDS 2
10
WASTE
WATER 1
STEAM
H2O2
WASTE
WATER 2
SDS
NaOCl
NaOH
SOLIDS
PHB
STEAM
Figure 8.16. Flowsheets for PHB production from glycerol (88 or 98 wt %). Fermentation
stage: 1. Pump; 2. Sterilizer; 3. Heat exchanger I; 4. Fermenter I; 5. Fermenter II.
Downstream Process I: 6. Heater; 7. Digestor; 8. Centrifuge; 9. Washer tank; 10. Heat
exchanger II; 11. Evaporator; 12. Spray drier. Downstream Process II: 6. Homogenizer;
7. Centrifuge I; 8. Heat exchanger II; 9. Heat exchanger III; 10. Extractor; 11. Centrifuge
II; 12. Heat exchanger IV; 13. Decanter; 14. Spray drier. Downstream Process III: 6.
Alkaline tank; 7. Digester; 8. Centrifuge; 9. Washer tank; 10. Heat exchanger II; 11.
Evaporator; 12. Spray drier.
Fermentation
broth
Fermentation
stage
content is discarded. Finally, PHB at 99.9 wt % is obtained by spray drying.
121
Table 8.12. Process conditions for PHB recovery: Downstream Process I

Feature
Feedstock
Heat Pretreatment
Chemical + enzymatic
Digestion
Centrifugation
H 2 O 2 -Water washing
Heat Exchanging
Water evaporation
Spray Drying
Description
Fermentation broth from glycerol at : 88 wt %
Temperature: 85 C
Residence time: 15 min
Temperature: 50 C
NaOCl at 30 wt %
Ratio NaOCl/cell mass: 1/2
Enzyme: Burkholdeira sp. PTU9
Residence time: 1 h.
pH: 9
Enzyme concentration: 2 wt %
Residence time: 20 min.
Retired products: mass cell, mainly.
Concentration: 1.2 v/v %
Heat duty: 2.305 MJ/Kg
PHB purity: 37.7 wt %
Product Purity: 99.9 wt %
98 wt %
114 KJ/Kg
2.227 MJ/Kg
2.95 m2
53.0 wt %
1.02 MJ/Kg
99.9 wt %
48.25 kg/h
Table 8.13. Process conditions for PHB recovery: Downstream Process II

Feature
Feedstock
Pumped
Description
Pressure outlet: 70 Mpa
Net work required: 239.73 KJ/Kg
Pressure: 70 Mpa
High
pressure Temperature: 110 C
Residence time: 45 min
homogenizer
Heat Duty: 218.40 KJ/Kg
Centrifugation 1
Recovered products: solids at 62.5 wt %
Heat Exchanging 2
Solvent to heat: Diethyl-succinate
Heat Exchanging 3
98 wt %
249.68 KJ/Kg
250.02 KJ/Kg
65 wt %
0.829 MJ/Kg
0.45 m2
0.158 MJ/Kg
0.43 m2
122
Solvent extraction
Centrifugation 1
Heat Exchanging 4
Decantation
Spray Drying
Temperature: 110 C
Pressure : 1 atm
Mass ratio of PHB/solvent: 1/20
Extracted products: cell mass
Temperature: 25 C
PHB purity: 38 wt %
0.216 MJ/Kg
18 m2
41.7 wt %
1.09 MJ/Kg
99.9 wt %
48.74 kg/h
Table 8.14. Process conditions for PHB recovery: Process III

Feature
Feedstock
Description
Temperature: 35 C
Concentration: 3 M
Alkaline pretreatment
Ratio: 0,4 (Kg of NaOH)/(Kg of mass cell)
Temperature: 55 C
NaOCl at 30 wt %
Chemical + surfactant Ratio NaOCl/cell mass: 1/3
Surfactant: Anionic sodium dodecyl sulfate (SDS)
Digestion
Heat Duty: 109.20 KJ/Kg
Centrifugation 1
Retired products: mass cell
H 2 O 2 -Water washing
Concentration: 1.2 v/v %
Heat Exchanging 4
Water evaporation
PHB purity: 25.2 wt %
Spray Drying
Feed flow rate: 25.13 kg/h
98 wt %
125.01 KJ/Kg
2.12 MJ/Kg
6.3 m2
37.2 wt %
2.14 MJ/Kg
99.9 wt %
48.46 kg/h
123
PHB production from crude glycerol starts with the glycerol purification process. Glycerol
content in the feedstock is 60.05 wt % and the rest is mainly methanol, which is recovered
at 99.9 wt % of purity using an evaporation process. The process continues with
impurities treatment and water evaporation and then the stream containing 80.5 wt % of
glycerol is distilled. Two different operation conditions were used for the molar distillated
ratio: 0.11 and 0.40, to obtain glycerol at 88 and 98 wt %, respectively.
The glycerol fermentation process can be carried out by two ways, using glycerol at 88 or
98 wt %. Each way requires different glycerol concentrations in the fermentation media,
which are 170.8 and 249 g/L, for 88 and 98 wt % of glycerol respectively. These
differences account for the impurities of glycerol at 88 wt % which affect the metabolic
process of C. necator. The diluted glycerol stream is sterilized at 139 C and 25 atm, in
both cases. Then, temperature and pressure are fitted to operation conditions (i.e., 35 C
and 1 atm). The first fermentation stage is called Cell mass growth, where air and oxygen
are fed to reach the stress conditions. This process is carried out for 21 h at pH 7; then if
glycerol at 88 wt % is used, 50.4 g/L of cell mass and 4.5 g/L of PHB are obtained. When
glycerol at 98 wt % is used, 44.4 g/L of cell mass and 7.3 g/L of PHB are obtained. In the
second fermentation stage the operation conditions are kept equal to the first fermentation
stage, where the residence time is 22.5 h and PHB accumulation occurs. Thus, 27.8 g/L
(for glycerol at 98 wt %) and 57.1 g/L (for glycerol at 98 wt %) of PHB are obtained in the
fermentation outlet stream. The fermentation broth is mainly a mixture of incorporated
PHB in the cell mass and water. To recover the PHB from this broth, three different
downstream processes were considered, and each one was evaluated with glycerol at 88
and 98 wt % (see Figure 8.17).
In the Downstream Process I a heat pretreatment is carried out, which denatures the
genetic material and proteins, and destabilizes the outer membrane of the bacterial cells.
The cell mass is then disrupted and PHB is released using for 1 hour a combined
digestion involving Burkholdeira sp. PTU9 enzyme (2 wt %) and a sodium hypochlorite
solution (30 wt %) in a 1/2 mass ratio of NaOCl/cell mass. Furthermore, solids are
removed by centrifugation, followed by water washing and a peroxide hydrogen
treatment. The resulting mixture is heated and near 90 % of water is evaporated. When
the fermentation process is carried out with glycerol at 88 or 98 wt %, a stream with 37.7
or 53.0 wt % of PHB is obtained respectively. Finally, this steam is spray dried until 99.9
124
wt % of PHB is obtained. Energetically wise the most efficient process is the one that
uses glycerol at 98 wt % since the other one requires evaporating higher water quantities
in the spray drying process (see Table 8.12). The total energy consumptions were 4.57
MJ/Kg and 3.36 MJ/Kg when glycerol at 88 or 98 wt % was used respectively.
Figure 8.17. Scheme for the simulation procedure to synthesize PHB from crude glycerol.
Raw Glycerol: 60 wt %
Glycerol Purification Process
Crude Glycerol:
88
Pure Glycerol:
t%
98
Fermentation Process
in Two Stages
t%
Fermentation Process
in Two Stages
Downstream
Process III
Downstream
Process II
Downstream
Process I
Downstream
Process III
Downstream
Process II
Downstream
Process I
PHB 99.9 wt %
125
The pretreatment step in the Downstream Process II is carried out in a high pressure
homogenizer at 70 Mpa, and 110 C for 45 min and then the water excess is extracted by
centrifugation requiring 218.40 KJ/Kg processed. The products stream and the stream
containing the solvent Diethyl-succinate (DES) are mixed at 110 C, with a mass ratio
PHB/solvent of 1/20. Solvent extraction takes place and the disrupted cell mass is
retrieved by centrifugation. The resulting mixture is decanted at 25 C and the recovered
DES is recicled in the extraction process. When fermentation is performed with glycerol at
88 or 98 wt %, a 38.0 or 41.7 wt % of PHB purity is achieved respectively. Finally, this
PHB stream is spray dried up to 99.9 wt % of purity. Using glycerol at 98 wt % implies a
higher energy consumption (2.79 MJ/Kg) than that for glycerol at 88 wt % (2.77 MJ/Kg).
These energy consumptions were calculated as a sum of the main energy consumer units
from Table 8.13.
The Downstream Process III differs from the Downstream Process I in the pretreatment
and digestion steps. Pretreatment is carried out in alkaline media at 35 C using a solution
of NaOH (3 M) in a NaOH/cell mass ratio of 0.4. Then, the combined digestion process
takes place at 55 C for 20 min. This process involves anionic sodium dodecyl sulfate as
surfactant and sodium hypochlorite (30 wt %) with a mass ratio NaOCl/cell mass of 1/3.
After water evaporation, PHB at 25.2 or 37.2 wt % of purity are obtained when the
fermentation process is carried out with glycerol at 88 or 98 wt %, respectively. Then
spray drying is carried out and PHB is purified up to 99.9 wt %. Nevertheless, glycerol at
98 wt % takes lower energy consumption than glycerol at 88 wt % (i.e., 4.38 MJ/Kg and
4.98 MJ/Kg, respectively).
8.4 D-Lactic acid production

In order to analyze the production process of D-lactic acid from raw glycerol, three main
stages have been distinguished: (i) glycerol purification, (ii) glycerol fermentation, and (iii)
D-lactic acid recovery and purification.
Although lactic acid bacteria have been used for D-lactic acid production from
carbohydrate rich feedstocks, it has also been reported the use of alternative biocatalysts
which are mainly engineered Escherichia coli strains able to produce D- or L-lactic acid
[48-52]. But only a few papers have been published on the use of glycerol as carbon
126
source for D-lactic acid production [53-54]. For instance, Hong et al. [54] compared eight
bacterial strains for lactic acid production from glycerol. Thus, the strain named AC-521
and a member of E. coli, showed the best performance for a fed-batch fermentation
process. On the other hand, Mazumdar et al. [53] engineered several E. coli strains by
overexpressing pathways involved in the conversion of glycerol to lactic acid and blocking
those leading to the synthesis of by-products as it was above described. In all cases they
used a minimal medium supplemented with sodium selenite, Na 2 HPO 4 , (NH 4 ) 2 SO 4 ,
NH 4 Cl, and 20 (or 40 or 60) g/l of pure (or crude) glycerol.
Conventional purification of lactic acid from the fermentation broth could be performed by
two routes: (i) crystallizing and acidifying the previously clarified and concentrated (32 wt
%) fermented liquor; or (ii) crystallizing, filtering, dissolving, and subsequently acidifying
the previously precipitated calcium lactate. But, these conventional routes generate huge
quantities of calcium sulphate cake which is difficult to dispose of [55]. The main
impurities contained in the clarified fermentation broth are residual substrate, color, and
other organic acids. Thus, in order to recover and purify the lactic acid, and also to
remove these impurities from the fermentation broth, several processes have been
proposed. The most remarkable purification processes are: adsorption, electrodialysis,
reactive extraction, and reactive distillation. Here, a brief description of each one is given
and some of their advantages and disadvantages are also discussed.
Recovery of lactic acid from a fermentation broth by adsorption requires special

characteristics of extractants and solid sorbents such as: high adsorption capacity and
selectivity, regenerability, and in some cases biocompatibility with microorganisms. Many
carboxylic acid fermentations operate effectively at a pH > pKa of the acid product; for
example lactic acid (pKa = 3.86) fermentation is typically produced at pH 56 [55]. In this
case, agents sufficiently basic to retain a significant capacity several pH units above the
acids pKa are recommended. Different basic extractants and polymeric sorbents have
been investigated for the extraction and sorption of lactic acid, but the uptaking degree
depends mainly upon the agent basicity and capacity [56]. For instance, weak base
polymer adsorbents such as: IRA-35 [57], MWA- 1, and VI-15 [58], which did not show
high final purity of lactic acid from fermentation broth. Better results were reported for
other studies [59] which used adsorbents with a water-insoluble macro-reticular gel, or a
127
weak basic anionic exchange resin with a tertiary amine, or a pyridine functional group, or
a strongly basic anionic exchange resin with quaternary functional groups.
Ion exchange technique has also been studied for the recovery of lactic acid from a
fermentation broth by several ionic exchanger resins such as: poly(4-vinylpyridine) resin
(PVP) [60], IRA-420 [61], IRA-400 [62-63], and IRA-92 [64]. Using IRA-92 (weakly basic
exchanger) and under optimal conditions of the fermentation broth (pH 6.0), lactic acid
was recovered with a yield, purity, and specific productivity of 0.826, 96.2%, and 1.16 g
L.A./(g-resin day), respectively [64]. Otherwise, when IRA-400 (a strong anionic exchange
resin) was used by different authors in an fluidized bed column, 0.18 g L.A./(g-resin) and
0.126 g L.A./(g-resin) [65] were recovered. Similar results have also been reported for
lactic acid recovering by ionic exchange resins, such as: 0.1 [66], 0.18 [67], and 0.2 [68] g
L.A./(g-resin).
By mean of a simulated moving bed (SMB) chromatography process (a continuous

separation process consisting of a circle of chromatographic columns) with a PVP resin,
high product purity (99.9 wt %) and high purification yield (>93%) were achieved [69]. On
the other hand, the PVPs adsorption capacity was found to decrease about 14 % each
time after base regeneration [70].
An advantage for adsorption on ion exchange resin is its possibility to couple it with the
fermentation process. But in the same way, adsorption and ion exchange technologies
require: (i) regeneration of the ion exchange resin, (ii) adjustment the pH of the fed stream
in order to increase the sorption efficiency, (iii) large amounts of extra-chemicals, and (iv)
treatment and disposal of large quantities of salts and effluents [55]. Besides, the
decreasing on the adsorption capacity for the ion exchange resins has also been reported
[55]. Additionally, because of the low adsorption selectivity of some ion exchange resins,
a further purification by esterification is necessary.
Electrodialysis has been recognized as a promissory technology for lactic acid recovery
from the fermentation broth since the product can be continuously removed maintaining
constant the pH of the medium [71]. Recovery of lactic acid is performed from lactate salts
in a two steps process, a conventional electrodialysis to concentrate and purify the
product, and a bipolar electrodialysis to convert the of lactate salts into lactic acids [55]. In
128
situ lactate recovery electrodialysis has been used with free and immobilized cells in order
to reduce the product inhibition. But, although the final amount of product was increased
in the medium containing immobilized cells, problems related to deposition and fouling of
bacteria on the membranes were found [55]. On the other hand, complete technological
schemes have also been suggested around electrodialysis process for lactic acid
purification. Bailly et al. [72-73] proposed a process in which a conventional
electrodialysis is utilized prior to an electrodialysis stage which uses bipolar membranes
to increase the concentration of organic acid salts. The same configuration of tow-stage
electrodialysis was reported by Habova et al. [74] as an efficient technique for lactate ions
recovering. The same configuration of two-stage electrodialysis was reported by Habova
et al [74] as an efficient technique for lactate ions recovering, while Li et al. [75] combined
both conventional electrodialysis and bipolar membrane electrodialysis in one laboratory
scale bioreactor. These processes let to have a good pH control, lead to reduce the
generation of troublesome salts, and seem to be economically and environmentally
attractive. But, exploitation of electrodialysis with bipolar membranes will require two
previous stages, named: micro-filtration and monopolar electrodialysis. Despite the
several studies performed in order to improve the eletrodialysis fermentation method,
commercialization of this process has not been reported [55].
Other widely studied alternative to recover lactic acid from a fermentation broth is the
reactive extraction. This process uses the reaction between extractants which are in the
organic phase and the extracted materials which are in the aqueous phase. Then, the
complexes formed during the reactions are solubilized in the organic phase. The most
used extractans for the reactive extraction of carboxylic acids are hydrocarbon,
phosphorous, and aliphatic amine [76]. Thus, three categories of extractions are
recognized: (i) extraction by solvation with carbon-bonded oxygen-bearing extractants, (ii)
salvation with phosphorous-bonded oxygen-bearing extractants, and (iii) proton transfer
or ion-pairing formation with high molecular weight aliphatic amines and their salts [77].
But, although only the last two extractants have been mainly used in the recovery of
carboxylic acids, the best extractabilities have been noticed for aliphatic amines. This
attribute is due to the behavior of the acid proton during the transfer from an aqueous
phase to an organic solution. That is, meanwhile the measures of extractability in systems
containing oxygen-bearing extractans are given by both the acid strength in the aqueous
solution and the hydrogen bond in the organic solution; in the case of using aliphatic
129
amines, the extracted compounds are more stable ammonium salts. The reactive
extraction process of carboxylic acids with tertiary amine extractants is composed of three
sequential steps: dissociation of carboxylic acid, proton transfer to the amine, and
recombination of ammonium salt [77]. The following reaction describes the overall
process, but its stoichiometry varies with several factors, such as: the property and
concentration of amine, acid, and diluent.
R 3 N + HA- R 3 NHA
(8.15)
A successful reactive extractive process depends mainly on a high distribution coefficient

for the lactic acid (K d ), and also the extractant should have both low water solubility and
low distribution coefficient for the impurities. The distribution coefficient is defined, as ratio
of the lactic acid concentration in the solvent phase to lactic acid concentration in the
aqueous phase, as shown in equation (8.16).
Kd =
Concentration of LA in the in the organic phase

Concentration of LA in the in the aqueous phase
(8.16)
Other two important parameters used to evaluate the performance of the reactive
extraction process are the extraction efficiency (E) and the loading (Z), as shown in
equations (8.17) and (8.18), respectively.
E (%) =
Z=
(Initial concentration of LA - Reffinate concentration of LA)

100
Initial concentration of LA
Concentration of LA in the organic phase

Inital concentration of amine
(8.17)
(8.18)
Although long chain aliphatic amines are effective extractants of carboxylic acid from
dilute aqueous solutions, these extractants must always be used dissolved in organic
diluents due to their physical properties such as viscosity, density, and corrosivity [78].
Solvents containing functional groups which interact strongly with complex are called as
Active Diluent (e.g., 1-octanol), while the solvents with low interaction level with complex
130
are called Inert Diluent (e.g. n-hexane). Thus, nature of the organic diluents affects not
only the basicity of amines but also the behavior of the extraction process [78].
Extractants, mainly ternary amines, including tri-n-octylamine (TOA), tripropylamine
(TPA), tributyl amine (TBA), trilauryl amine, tri-n-butyl phosphate (TBP), triisooctylamine,
Alamine 336 have been reported for lactic acid recovery from aqueous medium; besides
inert and active diluents such as: hexane, heptane, xylene, chloroform, chlorobenzene,
chlorobutane,
octanol,
decanol,
dodecanol,
oleyl
alcohol,
tributyl
phosphate,
methylisobutylketone, and methylene chloride-n-hexane, have also been used for this
process [55].
Recovery of lactic acid by reactive extraction is performed through the formation of an

acid-amine complex according to equation (8.15). A second step of regeneration is
required in order to reverse this reaction and to recover both the acid product phase and
the extractant phase available to be recycled. Regeneration could be carried out through
backextraction into an aqueous phase [78] by two approaches, swing either temperature
of diluent composition which leads to changes in the equilibrium relationship. Moreover,
recovering of lactic acid from a loaded solvent phase can also be performed using
solutions of NaOH and HCl [79-80]. One of the most suitable techniques for the
regeneration process is the so-called temperature-swing regeneration [78], where the
extracted stream is mixed with a fresh aqueous stream at a higher temperature to
produce an acid-laden aqueous product and an acid-free organic phase.
Lactic acid can also be obtained by a sequential process containing esterification of crude
lactic acid, distillation of ester, and hydrolysis of ester by reactive distillation in order to
obtain the preceding alcohol and lactic acid. Among the different alcohols analyzed for the
esterification process, methanol appears to be the most suitable one because of the
relatively low boiling of both methanol and methyl lactate. This implies lower energy
consumption for heating is the subsequent processes (i.e., distillation of ester and
reactive distillation). A typical configuration analyzed for lactic acid purification through
reactive batch distillation includes two columns reactants separation from the product and
two reboilers for esterification reaction and hydrolysis reaction [81-83]. In most of the
studies performed on lactic acid purification by reactive distillation, the cation exchange
resin Dowex 50W has been used for both esterification reaction and hydrolysis reaction
[81-84]. The main drawbacks for implementing this technology at industrial scale are not
131
only the low conversion levels on the esterification reaction (around 50%) but also the
high energy requirements to evaporate a mixture of produced ester and the water present
in the fermentation broth.
Joglekar et al. [55] drew four possible routes for lactic acid purification from a fermentation
broth considering both different fermentation modes and downstream processes as
shown in Table 8.15. Also, the purification costs were estimated, for an assumed
production of 1000 Tons of lactic acid (100 wt %) per year, based on reported costs and
on prices of raw materials and utilities for India. According to the authors, purification cost
of Route 2 was not calculated since the data available on expanded bed ion exchange
adsorption technology is not enough for estimating the costs.
Table 8.15. Downstream processes for lactic acid recovery from a fermentation broth
Route Fermentation
Downstream process
Cost
mode
1
Continuous
(USD$/LA kg)
Reactive extraction, re-extraction, esterification, and
hydrolysis by reactive distillation
Continuous
Adsorption/desorption
using
1.59
methanol
as
eluent,
esterification, and hydrolysis by reactive distillation
xxx
Addition of lime, precipitation of calcium lactate,

3
Batch
dissolution in methanol, acidification to separate calcium

sulphate, esterification, and hydrolysis by Reactive
distillation.
Batch
1.40
Addition of ammonium hydroxide, micro filtration,

monopolar
electrodialysis,
bipolar
electrodialysis,
esterification with reactive distillation and hydrolysis.
1.74
Here, based on the above reviewed literature, a technological scheme for lactic acid
production is proposed, simulated, and economically assessed as follows. Based on the
results reported by Mazumdar et al. [53], five fermentative scenarios were identified to be
analyzed for the D-lactic acid production from raw glycerol. The scenarios differ on: strain,
substrate concentration, substrate purity, fermentation time, and fermentation stages. In
this way, different values of yield, glycerol consumption, and productivity are reported.
Detailed information for the fermentation stage of each scenario is given in Table 8.16.
132
Table 8.16. Base information for the glycerol fermentation to D-lactic acid
Scenario
Strain
Glycerol
Glycerol
Glycerol
Molar
Conc.
purity for
consumption
yield to
(g/L)
fermentation
(%)
D-LA
Details
Main by product:
Ethanol.
1
LA01(pZSKLMgldA)
20
Pure
100
0.820
Fermentation time: 36 h
Main by product:
Succinic acid.
LA02(pZSglpKglpD)
20
Pure
100
0.812
LA02dld(pZSglpKglpD)
40
Pure
100
0.833
40
Crude
100
0.859
Two fermentation
stages: 48 h and 36 h,
60
Crude
90
0.934
each one.
The downstream process for D-lactic acid recovery and purification from the fermentation
broth is based on a reactive-extractive process because of its good process
characteristics, such as: low toxicity, low cost, low boiling point, extraction yield, and
recovery yield. Tri-n-octylamine and dichloromethane are used as extractant and active
diluent, respectively. A mixture of tri-n-octylamine diluted in dichloromethane at 0.6 M was
considered for this process and the used weight ratio of fermentation broth to organic
media was 2/1. During the reactive extraction process, a complex molecule is formed
according to equation (8.15) (see Figure 8.18). The back-extraction process is carried out
by a combined effect of changing the extractant concentration (Regeneration Swing
Concentration Process) and changing the temperature profile (Regeneration Swing
Temperature Process), which is reached by a distillation process under vacuum
conditions in order to obtain a highly pure D-lactic acid. Also, distillated water is fed in a
molar ratio of 1/1 respect to the formed complex.
The simplified flowsheet for D-lactic acid production is shown in Figure 8.19. The main
differences among the five scenarios are determined by the fermentation stage, which
leads to different: flows, equipment sizes, and operational conditions. The fermentation
product is a mixture containing mainly organic acids and cell mass, where the cell mass is
133
withdrawn by centrifugation. The clarified broth is mixed with three organic streams,
where two of those streams correspond to recycled tri-n-octylamine and dichloromethane.
The third one is a mixture of fresh both extractant and diluent because of their lost during
the purification process. Then, the reactive extraction process is performed at 20 C with
a residence time of 1.5 h and with a mixture of 0.6 M of tri-n-octylamine dissolved in
dichloromethane. Thus, the complex is produced and extracted to the organic phase,
which is distillated in order to remove the remaining water. The distillation product is the
heterogeneous azeotrope of dichloromethane-water. This azeotrope is also obtained from
the distillation of the aqueous phase obtained during the reactive extractive process,
which contains a significant amount of dichloromethane. Then, these two streams are
mixed and treated by decantation in order to recover dichloromethane at 99.7 wt %. The
bottom stream obtained after distillation of the organic phase from the reactive distillation
process is also distillated and the remaining dichloromethane is recovered. This stream is
mixed with the decantation product containing dichloromethane at 99.7 wt %, and the final
obtained purity was 99.8 wt %. The new bottom stream, containing mainly the complex, is
mixed with distillated water and then the back-extraction process takes place by
distillation. The distilled product is tri-n-octylamine at 99.1 wt % while the bottom product
is a mixture of D-lactic acid (85.5 wt %) and water. This last stream is finally purified by
vacuum distillation and D-lactic acid at 99.9 wt % is obtained.
The fermentation processes were simulated using a yielding approach where glycerol is
consumed and products including cell mass are formed according to Table 8.17. The
molecular formula used for E. coli strains was CH 1.9 O 0.5 N 0.2 [85-86].
Figure 8.18. Complex formed during the reactive extraction process of D-lactic acid
134
Waste
Water 1
Metanol
E-1
RII-1
E-2
Water
C-1
R-1
Water
DC-1
Waste
Water 2
Dec-1
Raw
Glycerol
Organic
Phase
Solids
Aqueous
Phase
Adsorbate
Distillated-2
DC-2
Diluted
Glycerol
Fresh
TOA
DCE
Cen-1
RE-1
DC-5
Distillated-5
Organic
Phase
M-1
Glycerol
(85 wt %)
Aqueous
glycerol
Dec-2
Glycerol
(98 wt %)
DC-3
F-1
Fermentation
Broth
Distillated-3
Bottoms-2
M-2
Solids
M-3
DC-4
Lactic Acid
Aqueous
Phase
DC-6
Distillated-4
Distillated-6
Bottoms-3
Bottoms-6
Bottoms-4
Figure 8.19. Simplified flowsheet for D-lactic acid production from raw glycerol. E:
fermentator, RE: Reactor extractor.
Table 8.17. Stoichiometry for glycerol fermentation to D-Lactic Acid by Engineered E. coli.
Scenario Glycerol Residual Ac Ac Succ Ac EtOH D-Lac Ac For Ac CO 2
Biomass
Glycerol
1
-1
0.021
0.024
0.82
0.045
11.5
-1
0.044
0.008
0.812
0.044
6.5
-1
0.048
0.0076
0.833
0.048
3.4
-1
0.045
0.008
0.859
0.006
0.045
4.5
-1
0.1
0.8406
0.0414
0.0414 0.00855
D-lactic acid production process starts with the purification of raw glycerol up to the
required purity according to each scenario as it was showed in Table 8.16. Detailed
information about the glycerol purification process was previously reported [87-88]. A
summary of the main simulation results for each scenario is given in Table 8.18. The final
production of D-lactic acid is directly related not only to the fermentation yield but also to
135
the substrate consumption. In other words, while the decreasing order respect to the yield
was: Scenario 5 > Scenario 4 > Scenario 3 > Scenario 1 > Scenario 2, the decreasing
order respect to the D-Lactic acid production was: Scenario 4 > Scenario 5 > Scenario 3 >
Scenario 1 > Scenario 2. This change in the order between the Scenarios 5 and 4 occurs
due to the incomplete consumption of glycerol during the fermentation in the Scenario 5.
Table 8.18. Summary of the main simulation results for D-lactic acid production process
Dilut gly Lactac1
Scenario 1
Lacacid4 Lacacid6
Destwater
Lactac9
Lactacp
Mass Flow (kg/hr)
28575.1
28560.0
13959.4
3073.5
83.96
487.3
419.5
Water
27995.5
27995.5
68.2
83.96
67.5
0.165
578.9
Formic Acid
Ethanol
6.95
0.855
0.001
0.001
Acetic Acid
7.9
0.612
0.612
0.417
0.004
Succ Acid
Lact Acid
464.3
0.273
0.273
419.3
419.3
Ecoli
72.2
Tri-n-amine
986.1
986.0
Dichlmethan
Complex
0
0
0
0
10813.9
2085.2
1.394
2085.2
0
0
0.125
0
0
0
Lacacid4
Lacacid6
Destwater
Lactac9
Lactacp
Glycerol
Scenario 2
Dilut gly Lactac1
Mass Flow (kg/hr)
28575.1
28531.5
13954.7
3068.9
84.6
484.1
416.3
Water
27995.5
27995.5
68.0
84.633
67.97
0.172
578.9
Formic Acid
Ethanol
16.6
1.28
1.28
0.87
0.87
Acetic Acid
5.9
0.08
0.08
0.079
0.079
Succ Acid
459.8
0.27
0.27
415.1
415.1
Lact Acid
40.8
Ecoli
1002.4
1002.4
Tri-n-amine
10813.5
Dichlmethan
2064.8
2064.8
Lacacid6
Destwater
Lactac9
Lactacp
Glycerol
Scenario 3
Dilut gly Lactac1
Lacacid4
136
Mass Flow (kg/hr)
14359.1
14310.3
14231.1
3081.2
86.89
498.6
421.58
Water
13779.5
13779.5
69.92
86.89
70.18
578.9
Formic Acid
Ethanol
18.1
2.702
2.702
1.85
Acetic Acid
5.64
0.161
0.161
0.157
0.157
Succ Acid
471.7
0.586
0.586
426.4
421.4
Lact Acid
21.35
Ecoli
959.5
959.5
Tri-n-amine
11073.0
Dichlmethan
2118.2
2118.2
Lacacid4
Lacacid6
Destwater
Lactac9
Lactacp
Glycerol
Scenario 4
Dilut gly Lactac1
Mass Flow (kg/hr)
14402.8
14375.6
14243.9
3095.2
89.87
516.36
441.7
Water
13821.2
13821.2
70.13
89.87
73.09
0.188
580.6
Formic Acid
1.741
0.03
Ethanol
17.04
2.535
2.535
1.759
0.019
Acetic Acid
5.956
0.17
0.17
0.166
0.166
Succ Acid
487.8
0.605
0.605
441.3
441.3
Lact Acid
28.33
Ecoli
901.1
901.1
Tri-n-amine
11071.9
Dichlmethan
2190.8
2190.8
Scenario 5
Glycerol
Dilut gly Lactac1
Lacacid4
Lacacid6
Destwater
Lactac9
Lactacp
Mass Flow (kg/hr)
9622.8
9640.9
14320.4
3087.3
88.01
505.9
432.5
Water
9041.2
9041.2
70.94
88.01
71.28
0.114
Glycerol
580.6
56.32
0.066
0.066
0.066
0.066
Formic Acid
Ethanol
15.67
3.3
3.3
2.274
0.012
Acetic Acid
6.365
0.274
0.274
0.267
0.267
Succ Acid
477.4
0.902
0.902
432.0
432.0
Lact Acid
31.48
Ecoli
938.8
938.8
Tri-n-amine
11155.0
Dichlmethan
2143.9
2143.9
137
Otherwise, for the reactive extraction process no high differences were noticed in terms of
Distribution coefficient or Loading as is shown in Table 8.19. Global recovery of D-lactic
acid was around 90 % respect to its production during the fermentation process. Also,
high recovery percentages were achieved for both tri-n-octylamine and dichloromethane,
indicating that low requirements of fresh both extractant and diluent are required. Higher
differences are noticed when the whole technological scheme is analyzed. The maximum
global molar yield from glycerol to D-lactic acid was obtained for the Scenario 4, while the
minimum was obtained for the Scenario 2. The relative difference between these two
scenarios was 5.93 %, which was close to the relative difference for the fermentation yield
obtained for the same both scenarios, 5.47 %. Thus, it can be stated that the
technological performance of D-lactic acid production from raw glycerol depends mostly
on the global conversion of substrate to the main product during the fermentation stage.
Table 8.19. Data representing the behavior of the downstream process for D-lactic acid
production
Scenario 1
Scenario 2
Scenario 3
Scenario 4
Scenario 5
92
92
92
92
92
11.59
11.58
11.68
11.68
11.78
Loading (Z)
0.63
0.62
0.64
0.66
0.65
Global lactic acid recovery (%)
90.32
90.3
89.36
90.48
90.51
Tri-n-octylamine recovery (%)
99.998
99.992
99.998
99.998
99.998
Dichloromethane recovery (%)
99.98
99.57
99.99
99.99
99.93
from 71.39
70.68
71.75
75.14
73.55
Reaction-Extraction Process
Downstream Process
Global Process
Global
process
yield
Glycerol to Lactic acid (%)
138
8.5 Succinic acid production

In general terms, the downstream process for succinic acid purification and recovery from
the fermentation broth starts with a cell separation by centrifugation or microfiltration, and
in some cases an additional ultrafiltration process is used in order to separate residual cell
mass, proteins, and other fermentation supernatants. For succinic acid recovery and
purification different alternatives have been proposed, such as: precipitation with
ammonia or calcium hydroxide, electrodialysis, reactive extraction, and sorption/ion
exchange. Here, the advantages and disadvantages of these alternatives are briefly
discussed.
Industrially the most used method for recovery of carboxylic acids from a fermentation
broth is the precipitation process with calcium hydroxide or calcium oxide, especially for
the cases of lactic and citric acids. Addition of calcium hydroxide or calcium oxide to the
clarified fermentation broth leads to calcium salt formation of succinic acid, which are
filtered off and treated with concentrated sulfuric acid. This last addition generates
calcium sulfate (CaSO 4 ), gypsum, in an equimolar amount. Then, free succinic acid is
purified by active carbon or ion exchange, and finally the product is further concentrated
and crystallized by evaporation. From a commercial view of point, this purification way
cannot be used because high amount of calcium sulfate are by-produced as a waste a
adequate disposal is required [89-90]. Additionally, the precipitation process requires high
consumption of calcium hydroxide, calcium oxide, and sulfuric acid which cannot be
regenerated or recycled causing high process costs. That is why precipitation with
calcium hydroxide or calcium oxide has been reported as unlikely process for large-scale
production of bio-succinic acid [91]. Precipitation with ammonia has also been reported for
succinic acid recovery at laboratory scale [92-93]. During this process the produced
diammonium succinate must be later treated with sulfate ions, or ammonium bisulfate, or
sulfuric acid at a low pH in order produce both succinic acid precipitate and ammonium
sulfate. Finally succinic acid is obtained after the dissolution in methanol and recrystallisation processes while ammonium sulfate can be cracked thermally in order to
produce ammonia and ammonium bisulfate. Although precipitation with ammonia reduces
the amount of waste production, low selectivities have been reported for this purification
alternative [93].
139
Other alternative for succinic acid purification is the electrodialysis process. Membranes
are charged either with positive or negative groups and selectively allow to cations or
anions passing through the membranes, thus succinate anions are able to passage
through positively charged membranes while sodium cations are repelled. Glassner et al.
[94] reported a total purification yield of 60% for a desalting electrodialysis combined with
a water-splitting electrodialysis. This process requires both a set of chelating ionexchange columns to replace the divalent cations of the succinate salt with sodium ions
and a bipolar membrane water-splitting dialysis to obtain succinic acid from succinate.
Then, after electrodialysis and ion exchange process, evaporation of water and
crystallization of the succinic acid are required [94-96]. Electrodialysis is known as an
expensive alternative not only by the high energy consumption but also by the materials
cost. Besides to the low yield, some other problems such us: low selectivity [97], handle
on binary ions [95], and fouling [98] have also been reported.
On the other hand, since liquid-liquid extraction has shown low distribution coefficients for
carboxylic acids recovery from the fermentation broth [99-100], reactive extraction
appears as a better option to increase yield and selectivity to organic acids from an
aqueous phase [101]. Mixtures of amines (reactive components) dissolved in non-water
miscible organic solvents have been widely studied for carboxylic acids recovery [102103]. Amine reacts with the succinic acid thought out a proton transfer or ion pair
formation mechanism depending on the type of amine and the organic solvent [104].
Long-chain aliphatic primary, secondary, and tertiary amines have been proposed for
succinic acid extraction [105-111], but in these cases only the undissociated acid can be
extracted. Otherwise, although quaternary amines can extract the dissociated and the
undissociated succinic acid, its regeneration is difficult by back extraction. Thus, ternary
amines have been the most used for carboxylic acids extraction from an aqueous solution
[112] using solvents such as: octanol, xylene, heptane, kerosene, methylenchloride or
nitrobenzene [105-108, 111, 113]. Mixtures of amines such us: tripropylamine/
trioctylamine or trialkylamines have also been reported [108, 114]. On the hand, different
operational alternatives for the reactive extraction process of succinic acid with amines
have been proposed. For instance, Huh et al. [115] studied the removal of by-products
and impurities present in the fermentation broth such as: acetic acid, pyruvic acid, and
salts, by mean a pre-treatment step of reactive extraction with trioctylamine. Then the
aqueous succinic acid is purified up to 99.8 wt % by an evaporative crystallizer with a
140
purification yield of 71.3 %. Finally, Kurzrock and Weuster-Botz [101] stated that if the
recycling of the costly amines is done efficiently, it is very likely that optimized reactive
extraction processes may be applied in the near future for industrial production of biosuccinic acid.
In general terms, the final step of the succinic acid purification process is an ionic
exchanger unit in which the residual cations and anions are removed. Different kinds of
exchange resins have been investigated in order to produce succinic acid at high purity
from either the fermentation broth or succinate. Some examples are the alkaline
anionexchange resin (NERCB 04) [116] and the H-type strongly acidic cation-exchange
resins [117]. On the other hand, mesoporouses silicas (SBA-15) functionalized with
primary, secondary and tertiary amino-functional silanes were reported to be able for for
the isolation of pyruvic and succinic acid from fermentation broth [118]. Ion-exchange
must be only considered as an additional purification step for succinic acid recovery
because of its low both selectivity and yield [119].
In this case, three different types of strains are here analyzed for the succinic acid
production from raw glycerol. The first one is the Escherichia coli recently reported by
Blankschien et al. [120], the second strain is the Mannheimia sp. Pasteurellaceae
reported by Scholten and Dagele [121], and the last one is the Anaerobiospirillum
succiniciproducens reported by Lee et al. [122]. In the case of Mannheimia sp.
Pasteurellaceae
three
scenarios
are
studied
while
for
Escherichia
coli
and
Anaerobiospirillum succiniciproducens one scenario is analyzed in each case, as shown

in Table 8.20. Although it can be observed that three different qualities of glycerol are
considered for the fermentation stage (i.e., 76, 90, and 98 wt %), a unique raw glycerol is
considered as feedstock. A typical composition of a raw glycerol stream obtained from a
biodiesel production process is: methanol 32.59 wt %, glycerol 60.05 wt %, NaOCH 3 2.62
wt %, fats 1.94 wt %, and ash 2.8 wt % [123]. This stream must be purified up to the
specified concentration established in Table 1 for the fermentation stage. The purification
process previously studied in the Chapter 4. As result of the purification process, a
glycerol stream at 80 wt % is obtained. Thus, the glycerol concentration required for the
Scenario 4 is obtained. Finally, in order to purify the glycerol stream up to 90 or 98 wt %,
a distillation process is carried out.
141
Table 8.20. Base information for the glycerol fermentation to succinic acid
Scenario
Strain
1
2
Escherichia coli
Mannheimia sp.
Pasteurellaceae
Mannheimia sp.
Pasteurellaceae
Mannheimia sp.
Pasteurellaceae
Anaerobiospirillum
succiniciproducens
3
4
5
Glycerol
Glycerol
Glycerol
purity for consumption
Conc.
(%)
fermentation
(g/L)
20
98 wt %
96
9.6
98 wt %
55.2
Molar
yield to
S.A.
0.522
0.287
8.3
90 wt %
75.9
0.542
9.1
80 wt %
71.4
0.453
6.5
98 wt %
58.5
0.344
Then, the fermentation process can be performed, but due to these scenarios differ on:
strain, substrate concentration, substrate purity, and fermentation time, different
composition profiles are obtained in the fermentation broth according to the fermentation
stoichiometry reported for each case and shown in Table 8.21. The fermentation broth is
then clarified by a centrifugation process where the cell mass is withdrawn.
Table 8.21. Stoichiometry for glycerol fermentation to succinic acid by Engineered E. coli.
Scenario
1
2
3
4
5
Purity
of Gly
(wt %)
99
99
90
76
99
Glycerol
92.09
-1
-1
-1
-1
-1
Res
Gly
92.09
0.040
0.448
0.241
0.286
0.415
Succinic
acid
118.09
0.544
0.520
0.714
0.634
0.588
Acetic
acid
60.05
0.064
0.048
0.055
0.051
0.020
Lactic
acid
90.08
0.000
0.000
0.000
0.011
x
Formic
acid
Biomass
46.03
x
0.066
0.083
0.044
0.072
0.044
0.066
0.044
x
0.032
Recovery and purification of succinic acid from the clarified fermentation broth is based on
the downstram process recently reported by Huh et al. [124]. During the so-called
complex process, the by-produced acids are removed selectively from the fermentation
broth by reactive extraction, followed by a vacuum distillation step where the volatile
impurities are removed. Then a crystallization process is performed and succinic acid is
concentrated in the order of five to six folds. Finally, another crystallization process is
carrying out at pH 2.0 and 4 C in order to obtain succinic acid highly purified.
142
The pretreatment step of the reactive extraction process selectively removes

contaminated organic acids from the dilute fermentation broth using tri-n-octylamine
(TOA) and 1-octanol. Since TOA only extracts the undissociated form of carboxylic acids
[110, 124], the selective removal of specific acids from the fermentation broth is made
possible by using different degrees of dissociation of each acid with the pH. Although the
distribution coefficient values decreased with the increase of pH for succinic acid, pyruvic
acid, and acetic acid, the distribution coefficient of succinic acid at pH values between 4.0
and 5.0 is close to 0. Thus, the increase of the dissociated acid concentration leads to the
reduction of succinic acid extraction. In this, case a multi-stage reactive extractive process
was analyzed, but no significant improvements were noticed after each stage. Besides,
the author recommended a one stage reactive extraction process in order to do economic
the removal of succinic acid from the fermentation broth. Then, by mean a vacuum
distillation process volatile impurities such as acetic acid and formic acid are effectively
removed, and thus the pretreated fermentation broth is concentrated five- to six-fold by a
crystallization process at low temperature (4 C) and in an acid media (pH 2.0). Table
8.22 shows the calculated removal efficiency of carboxylic acids from the fermentation
broth based on the data reported by Huh et al. [124]. Thus, applying this complex process
all by-produced carboxylic acids are effectively withdrawn with a succinic acid lost of 27%.
Table 8.22. Removal efficiency (%) of the carboxylic acids from the fermentation broth.
Fermentation product
Reactive
Vaccum distillation Cristallization
extraction
Succinic acid
0.45
0.00
26.58
Maleic acid
87.50
0.00
100.00
Acetic acid
44.44
90.00
100.00
Pyruvic acid
27.14
11.76
99.78
Fumaric acid
87.50
0.00
100.00
On the other hand, due to the fermentation products here considered are: succinic acid,
acetic acid, and formic acid according to Table 8.21, it is required to generate the complex
molecules as the corresponding reaction products of TOA with of carboxylic acids.
Complex I (see Figure 8.20.a) is the reaction product of succinic acid with TOA, while the
Complex II (see Figure 8.20.b) and Complex III (see Figure 8.20.c) are the reaction
products of formic acid and acetic acid respectively.
143
The simplified flowsheet for succinic acid production is shown in Figure 8.21. As it was
observed for the D-lactic acid production processes, the main differences among the five
scenarios for the succinic acid production processes are determined by the fermentation
stage. Thus different flows, equipment sizes, and operational conditions are obtained for
each scenario. Glycerol is first purified up to the required quality according to Table 8.20
and then the fermentation occurs under the stoichiomentry presented in the Table 8.21.
The fermentation product contains a mixture of cell mass and organic acids such as
succinic acid, acetic acid, lactic acid, and formic acid. The biomass produced during the
fermentation stage is withdrawn by centrifugation and the obtained clarified fermentation
broth is mixed with both the fresh and the recycled streams containing TOA and octanol.
Then, the resulting mixture is subjected to the reactive extraction process.
During the reactive extractive process the complex molecules are produced from each
carboxylic acid according to the Figure 8.20, and then most of the succinic acid complex
is contained in the aqueous phase while the rest of the impurities are extracted to the
organic phase. This organic phase is subjected to a back extraction process by mean a
distillation column in which some water is withdraw and most important the used TOA is
recovered. This stream is again distilled and most of the carboxylic acids are discarded
while a mixture of octanol and TOA containing low quantities of succinic acid and acetic
acid is recycled in order to be used in the reactive extractive process. On the other hand,
the aqueous phase containing most of the succinic acid is first distilled not only water but
also acetic acid and and a few amount of octanol are discarded by distillated. The
bottoms product is then subjected to a crystallization process and succinic acid at high
purity is obtained.
The main simulation results for each scenario are shown in Table 8.23 and significant
differences for the final production of succinic acid can be observed among the five
analyzed scenarios. For instance based on Table 8.21, the final production of succinic
acid depends mainly on the succinic acid yield (increasing order: Scenario 2 < Scenario 1
< Scenario 5 < Scenario 4 < Scenario 3) and also on the glycerol consumption (increasing
order: Scenario 2 < Scenario 5 < Scenario 4 < Scenario 3 < Scenario 1). This higher
dependence on the succinic acid yield can be explained because of the increasing order
of succinic acid production (Scenario 2 < Scenario 1 < Scenario 5 < Scenario 3 <
Scenario 4) was most similar to the succinic acid yield than the glycerol consumption.
144
Figure 8.20. Reaction complexes of succinic acid, formic acid and acetic acid with TOA.
Waste
Water 1
Metanol
E-1
C-1
R-1
E-2
Water
Raw
Glycerol
Solids
DC-4
Distillated-4
Waste
Water 3
Succinic
acid
DC-3
Cry-1
Glycerol
(98 wt %)
M-1
Glycerol
(85 wt %)
Aqueous
glycerol
Distillated-2
DC-2
RII-1
Waste
Water 2
Dec-1
Organic
Phase
Water
DC-1
Adsorbate
Diluted
Glycerol
RE-1
Fresh
TOA
Octanol
Cen-1
Distillated-3
Fermentation
Broth
Bottoms-4
Bottoms-3
Bottoms-2
M-2
Solids
Aqueous
Phase
Figure 8.21. Simplified flowsheet for succinic acid production from raw glycerol. E:
fermentator, RE: Reactor extractor, Cry: Crystallizator
F-1
145
The change between the Scenarios 3 and 4 occurs due to the difference in the glycerol
consumption is higher than the succinic acid yield. For the reactive extractive process was
noticed that most of the fermentation by-products such as formic acid and acetic acid
were selectively removed from the clarified broth with extraction efficiency around 88 %
for formic acid and 90 % for acetic acid, while the losses for succinic acid were lower than
1.4 % as shown in Table 8.24. The good performance of the downstream process is
stated because of the high recovery levels not only for succinic acid (> 98 %) but also for
both tri-n-octylamine and 1-octanol.
Table 8.23. Summary of the main simulation results for succinic acid production process
Mass Flow
kg/hr
Water
Glyce-01
Cell mass
Succini
Formi-01
Aceti-01
Tri-N-01
1-oct-01
Complex
Complex3
Mass Flow
kg/hr
Water
Glyce-01
Cell mass
Succini
Formi-01
Aceti-01
Tri-N-01
1-oct-01
Complex
Complex2
Complex3
Dilutgly
Succaci1
28575.1
27995.5
578.9
0
0
0
0
X
X
X
X
28447.3
27995.5
23.16
10.25
403.8
0
24.16
X
X
X
X
Dilutgly
Succaci1
59382.1
58802.5
578.9
X
X
X
X
X
X
X
X
X
59490.7
58802.5
259.3
6.83
386.0
24.01
18.12
X
X
X
X
X
Scenario 1
Complex2
Aqueous2
29668.5
27813.1
27992.7
27659.3
23.15
23.1
X
X
403.4
398.2
0
0
2.32
0.097
1.99
1.91
1368.8
2.83
1.61
149.1
X
Scenario 2
Complex2
Aqueous2
62220.1
58796.6
259.3
X
385.6
3.00
1.81
3.86
2737.5
1.54
181.5
111.8
58525.4
58134.5
258.6
X
380.9
2.89
1.74
3.71
5.62
0
0
0
Extract2
Concent1
Succaci2
1855.5
333.4
0.072
X
5.18
0
1.74
0.08
1365.9
1.61
149.1
3448.4
3298.1
23.1
X
398.2
0
X
1.91
trace
X
X
123.6
0.003
X
X
397.6
0
0.811
Extract2
Concent1
Succaci2
3694.7
662.2
0.759
X
4.7
0.115
0.069
0.147
2731.9
1.54
181.5
111.8
7297.5
6915.4
258.6
X
380.9
0.033
1.31
3.71
0
0
0
0
117.2
0.007
0
X
379.7
0
0
0
0
0
0
0
X
X
X
146
Mass Flow
kg/hr
Water
Glyce-01
Cell mass
Succini
Formi-01
Aceti-01
Tri-N-01
1-oct-01
Complex
Complex2
Complex3
Mass Flow
kg/hr
Water
Glyce-01
Cell mass
Succini
Formi-01
Aceti-01
Tri-N-01
1-oct-01
Complex
Complex2
Complex3
Mass Flow
kg/hr
Water
Glyce-01
Cell mass
Succini
Aceti-01
Tri-N-01
1-oct-01
Complex
Complex3
Dilutgly
Succaci1
68527.7
67945.3
578.9
X
X
X
X
X
X
X
X
X
68659.9
67945.3
139.5
6.83
530.0
20.83
20.76
X
X
X
X
X
Dilutgly
Succaci1
61264.8
60675.0
579.3
0
0
0
0
X
X
X
X
X
61370.3
60675.0
165.7
6.84
471.0
19.10
29.09
X
X
X
X
X
Dilutgly
Succaci1
87662.1
87082.5
578.9
0
0
0
X
X
X
X
87767.5
87082.5
240.2
4.97
436.5
7.55
X
X
X
X
Scenario 3
Complex2
Aqueous2
61970.6
58546.1
58867.9
58248.9
120.9
120.5
X
X
458.7
453.5
2.26
2.17
1.80
1.73
26.92
25.96
2555.0
5.59
1.83
0
136.4
0
111.0
0
Scenario 4
Complex2
Aqueous2
64451.9
60269.7
60689.0
59936.9
165.7
165.1
X
X
470.5
464.2
2.39
2.29
2.91
2.79
13.42
12.86
3102.52
5.83
1.88
0
144.5
0
179.5
0
Scenario 5
Complex2
Aqueous2
87961.0
87073.8
240.2
X
436.0
0.755
2.43
456.2
1.74
46.58
87355.2
86965.4
240.1
X
435.4
0.752
2.42
7.82
0
0
Extract2
Concent1
Succaci2
3424.4
619.1
0.33
X
5.23
0.081
0.064
0.961
2549.4
1.83
136.4
111.0
7237.9
6948.8
120.5
X
453.5
0.025
1.3
25.96
0
0
0
0
141.3
0.007
0
X
452.9
0
0
0
0
0
0
0
Extract2
Concent1
Succaci2
4182.2
752.1
0.534
X
6.29
0.100
0.122
0.562
3096.7
1.88
144.5
179.5
7467.8
7143.8
165.1
X
464.2
0.026
2.088
12.86
0
0
0
0
143.9
0.007
0
X
463.5
0
0
0
0
0
0
0
Extract2
Concent1
Succaci2
605.8
108.4
0.077
X
0.603
0.003
0.011
448.4
1.74
46.58
10757.5
10375.8
240.1
X
435.4
0.563
2.42
0
0
0
138.6
0.01
0
X
434.8
0
0
0
0
0
147
Finally, the maximum global yield from glycerol to succinic acid was obtained for the
Scenario 4, while the minimum was obtained for the Scenario 2 as shown in Table 8.24.
This behavior agrees with the order of succinic acid production above described. The
relative difference between these two scenarios was 18.24 %, which indicates that the
fermentation process has a high influence on the final production of succinic acid.
Table 8.24. Data representing the behavior of the downstream process for succinic acid
production
Scenario 1 Scenario 2 Scenario 3 Scenario 4 Scenario 5
Extraction efficiency of SA (%)
1.28
1.22
1.14
1.34
0.14
Extraction efficiency of FA (%)
NA
87.98
87.95
88.02
NA
Extraction efficiency of AA (%)
90.40
90.38
90.36
90.42
90.04
Distribution coefficient for SA
0.168
0.168
0.170
0.168
0.172
Distribution coefficient for FA
NA
99.863
107.627
91.304
NA
Distribution coefficient for AA
121.648
128.240
138.222
117.236
1120.455
Loading for SA (Z)
0.118
0.054
0.064
0.063
0.041
Loading for FA (Z)
NA
0.618
0.498
0.528
NA
Loading for AA(Z)
0.980
0.367
0.465
0.590
0.915
Downstream Process
Global SA recovery (%)
98.62
98.38
98.76
98.56
99.76
97.62
98.15
88.84
95.20
91.49
1-octanol recovery (%)
99.66
93.30
95.98
95.99
94.59
0.603
0.565
Global Process
Global process yield from
0.517
0.493
Glycerol to SA (%)
S.A. succinic acid; F.A.: formic acid; A.A.: acetic acid
0.589
148
8.6 Propionic acid production

Propionic acid is a naturally occurring carboxylic acid which, in the pure state, is a
colorless, corrosive liquid with an unpleasant odor. Propionic acid is used in the
manufacture of herbicides, chemical intermediates, artificial fruit flavors, pharmaceuticals,
cellulose acetate propionate, and preservatives for food, animal feed, and grain.
As it was performed for D-lactic acid and succinic acid production, the whole technological
scheme for propionic acid production was divided in three main stages. These stages are:
(i) glycerol purification, (ii) glycerol fermentation, and (iii) propionic acid recovery and
purification. The glycerol purification has been widely discussed throughout this document
(see Chapter 4). On the other hand, for the glycerol fermentation to propionic acid two
strains were identified as the most promissory bacteria available from literature. The first
one is a commercial Propionibacterium acidipropionici which consumes pure glycerol
[125], and the second one is the engineered Propionibacterium acidipropionici ACK-Tet
[126], which is able to consume both pure and crude glycerol as only source of carbon
and energy.
Respect to the recovery and purification methods of organic acids several alternatives
have been evaluated. Some examples are: liquid extraction [127], reverse osmosis [128],
electrodialysis [129], liquid surfactant membrane extraction [130], anion exchange [63],
precipitation and adsorption [131], and reactive liquid-liquid extraction [132]. These
processes were above described for recovering and purifying of carboxylic acids.
Otherwise, Keshav et al [133-144] have studied widely the reactive extraction of propionic
acid from a fermentation broth, and different diluent (e.g., benzene, toluene, hexane, nheptane, n-octane, n-dodecane, ethyl acetate, butyl acetate, 1-octanol, 2-octanol, 1decanol, 1-dodecanol, petroleum ether, paraffin liquid, MIBK, oleyl alcohol, sunflower oil,
) and extractant (e.g., Tri-n-butylphosphate, tri-n-octylamine, Aliquat 336, and tri-noctylphosphine oxide)agents have been analyzed. Besides of the final extraction
performance, kinetic behavior has also been studied [135]. Based on the results reported
by Keshav et al [133-144] for the reactive extraction of propionic acid from the
fermentation broth, it was noticed that the best configuration for this process requires the
use of tri-n-octyl amine (TOA) as extractant agent while the diluent agent must be ethyl
149
acetate. The concentration that leads to the best performance is 0.686 kmol/m3 and the
extraction temperature is 305 K according to Keshav et al [136].
Here and based on the reviewed literature, a technological scheme for propionic acid
production is proposed, simulated, and economically assessed. First of all, five different
scenarios are considered for the glycerol fermentation stage. Scenario 1 uses a
commercial Propionibacterium acidipropionici strain and glycerol diluted at 20 g/L, while in
the Scenario 2 the fermentation media contains the same strain and glycerol diluted at 50
g/L. For these two scenarios the fermentation process is carry out at 30 C using pure
glycerol [125]. For Scenario 3, Scenario 4, and Scenario 5 the engineered strain
Propionibacterium acidipropionici ACK-Tet is considered and pure glycerol at 46 g/L is
required in Scenario 3. While crude glycerol at 17 g/L is used in Scenario 4, Scenario 5
considers a completely different configuration for the fermentation process. It is a fibrousbed bioreactor packed with immobilized cells and fed with pure glycerol at 41 g/L. For
these three scenarios the fermentation process is carry out at 32 C [126]. In all cases,
100% of glycerol conversion was reached and the fermentation times reported in each
case were: 120 h (Scenario 1), 150 h (Scenario 2), 280 h (Scenario 3), 160 h (Scenario
4), and 104 h (Scenario 5).
Since the analyzed scenarios differ mainly on the fermentation stage according to Table
8.25, which shows the stoichiometry of the fermentation process for each scenario,
different operational conditions, material and energy requirements, and equipment sizes
are required for each scenario. The principles of the reactive extraction process and the
corresponding back extraction process for recovering carboxylic acids from an aqueous
media were above described.
Table 8.25. Stoichiometry of the fermentation process for each scenario.

Scenario Glycerol Propionic Ac. Acetic Ac. Succinic Ac. Biomass
MW
92.09
74.08
60.05
118.09
24.73
1
0.9821
0.0925
0.0905
0.3481
1
1
0.7086
0.0785
0.0660
0.2820
2
1
0.6713
0.0368
0.0507
0.3442
3
1
0.8826
0.0537
0.0546
0.1903
4
1
0.7334
0.0414
0.0569
5
150
The simplified flowsheet for propionic acid production is shown in Figure 8.22. First, the
raw glycerol is purified up to the required quality in order to perform the fermentation
process. Then, fermentation takes place according to Table 8.25, and the fermentation
broth is clarified throughout a centrifugation process. This clarified stream is mixed with
two organic streams. These streams contain fresh and recycled mixtures of TOA and
ethyl acetate and the resulting mixture is TOA in ethyl acetate at 0.686 kmol/m3. Thus, the
reactive extraction process is carried out at 32 C. The aqueous phase, containing high
quantities of ethyl acetate, is distilled in order to recover it. Otherwise, the organic phase
is subjected to a distillation process in which the contained ethyl acetate is recovered and
mixed with this coming from the aqueous phase. Then, the resulting organic phase is
subjected to the back extraction process, and TOA is recovered by bottoms and recycled
to the reactive extraction process. Finally, the resulting stream is distilled under vaccum
conditions and propionic acid at high quality is obtained.
A summary of the main simulation results for each scenario is given in Table 8.26. In this
case and due to glycerol is completely consumed during the fermentation process in all
scenarios, the final production of propionic acid is only related to the fermentation yield.
Thus, the decreasing order for propionic acid production is Scenario 1 > Scenario 4 >
Scenario 5 > Scenario 2 > Scenario 3 (see Table 8.26). This obtained order agrees with
the fermentation yield (see Table 8.25)
Waste
Water 1
Metanol
E-1
C-1
R-1
DC-2
DC-3
RII-1
E-2
Water
Waste
Water 2
Dec-1
Raw
Glycerol
Solids
Water
DC-1
Organic
Phase
Adsorbate
Diluted
Glycerol
RE-1
Fresh
TOA
EtAc
Cen-1
Distillated-2
Distillated-3
Prop Acid
Bottoms-3
Bottoms-1
M-1
Glycerol
(85 wt %)
Aqueous
glycerol
Distillated-1
DC-1
Glycerol
(98 wt %)
Aqueous
Phase
M-2
Fermentation
Broth
Solids
F-1
151
Table 8.26. Summary of the main simulation results for prpionic acid production process
Dilutgly
303.1
Temperature (K)
Mass Flow (kg/hr)
27995.5
Water
578.9
Glycerol
0
Cell mass
0
Prop. acid
0
Suc. acid
0
Ac. acid
0
Tri-N
0
Complex
0
Ethyl
Propaci1
303.1
Dilutgly
303.1
Temperature (K)
Mass Flow (kg/hr)
11007.5
Water
578.9
Glycerol
0
Cell mass
0
Prop. acid
0
Suc. acid
0
Ac. acid
0
Tri-N
0
Complex
0
Ethyl
Propaci1
303.1
Dilutgly
305.1
Temperature (K)
Mass Flow (kg/hr)
11987.5
Water
578.9
Glycerol
0
Cell mass
0
Prop. acid
0
Suc. acid
0
Ac. acid
0
Tri-N
0
Complex
0
Ethyl
Propaci1
305.1
27995.5
0
54.05
457.3
67.18
457.3
0
0
0
11007.5
0
43.78
329.96
48.99
29.63
0
0
0
11987.5
0
53.44
312.6
37.63
13.89
0
0
0
Scenario 1
Complex2 Aqueous2
293.1
283.1
27992.7
27763.1
0
0
0
0
96.00
88.89
67.19
62.23
34.89
32.31
357.9
26.52
2075.7
0
11117.4
2754.03
Scenario 2
Complex2 Aqueous2
293.1
283.1
11006.4
10724.5
0
0
0
0
69.26
55.48
49.01
39.20
29.61
23.72
838.21
53.76
1497.6
0
11117.4
1114.0
Scenario 3
Complex2 Aqueous2
293.1
283.1
11986.3
0
0
65.63
37.67
13.87
903.6
1418.9
11117.4
11708.6
0
0
53.56
30.70
11.35
59.06
0
1203.8
Extract2
283.4
Organic5
293.1
Propacp
379.8
229.6
0
0
7.11
4.96
2.58
331.4
2075.7
8363.3
8.79
0
0
368.4
4.96
2.58
2056.3
0
0
0.018
0
0
367.4
4.96
0.54
0
0
0
Extract2
283.3
Organic5
293.1
Propacp
380
281.9
0
0
13.78
9.80
5.88
784.4
1497.6
10003.4
6.34
0
0
274.5
9.80
5.88
2029.0
0
0
0
0
0
274.0
9.80
1.20
0
0
0
Extract2
283.3
Organic5
293.1
Propacp
380
277.8
0
6.00
0
0
259.1
6.97
2.58
2023.7
0
0
0
0
0
258.3
6.97
0.540
0
0
0
12.07
6.97
2.58
844.6
1418.9
9913.6
152
Dilutgly
305.1
Temperature (K)
Mass Flow (kg/hr)
12036.6
Water
578.9
Glycerol
0
Cell mass
0
Prop. acid
0
Suc. acid
0
Ac. acid
0
Tri-N
0
Complex
0
Ethyl
Propaci1
305.1
Dilutgly
305.1
Temperature (K)
Mass Flow (kg/hr)
13492.5
Water
578.9
Glycerol
0
Cell mass
0
Prop. acid
0
Suc. acid
0
Ac. acid
0
Tri-N
0
Complex
0
Ethyl
Propaci1
305.1
12036.6
0
29.55
411.0
40.5
20.27
0
0
0
13492.5
0
53.44
341.5
42.24
15.63
0
0
0
Scenario 4
Complex2 Aqueous2
293.1
283.1
12035.4
11757.2
0
0
0
0
86.30
70.45
40.50
33.06
20.30
16.57
532.6
34.66
1865.4
0
11117.4
1213.7
Scenario 5
Complex2 Aqueous2
293.1
283.1
13491.2
0
0
71.71
42.27
15.61
794.7
1550.4
11117.4
13218.4
0
0
59.93
35.31
13.03
53.05
0
1351.4
Extract2
283.3
Organic5
293.1
Propacp
379.9
278.2
0
0
15.85
7.44
3.72
498.0
1865.4
9903.7
7.89
0
0
340.47
7.44
3.72
2047.8
0
0
0.018
0
0
339.5
7.44
0.781
0
0
0
Extract2
283.4
Organic5
293.1
Propacp
379.9
272.8
0
0
11.78
6.97
2.58
741.66
1550.4
9766.0
6.56
0
0
281.6
6.97
2.58
2029.4
0
0
0
0
0
280.8
6.97
0.54
0
0
0
Finally, the behavior of the reactive extraction process was analyzed based on its
extraction efficiency, the obtained distribution coefficient and the used loading, but no high
differences were found for the extraction efficiency while the distribution coefficient varied
from 3.8 to 15. Also, it was observed that the global recovery of propionic acid from the
fermentation broth is directly related to the extraction efficiency in the reactive extractive
process as shown in Table 8.27.
Besides of the high recovery levels for propionic acid, the extractan and diluent agents
were almost completely recovered during the downstream process. Thus, low quantities
of both agents must be fed as a fresh stream. As it could be expected, the global yield for
the downstream process agreed with the order found for the fermentation yield and the
153
propionic acid production (i.e, Scenario 1 > Scenario 4 > Scenario 5 > Scenario 2 >
Scenario 3) with a relative difference between Scenario 1 to Scenario 3 of 29.69%.
Table 8.27. Data representing the behavior of the downstream process for propionic acid
production
Scenario
Scenario
Scenario
Scenario
Scenario
80.56
83.19
82.87
82.85
82.46
8.761
3.790
14.156
12.965
15.032
Loading (Z)
0.844
0.629
0.594
0.781
0.645
Global P.A. recovery (%)
80.34
83.05
82.64
82.61
82.24
98.73
97.42
97.17
98.33
97.45
EtAc recovery (%)
99.53
99.45
98.51
98.49
98.34
0.7605
0.5672
0.5347
0.7027
0.5814
Downstream Process
Global Process
Global process yield from
Glycerol to PA (%)
PA: Propionic acid. EtAc: Ethyl acetate.
8.7 Economic assessment

8.7.1 1,3-propanediol production
For most industrial processes the cost of raw material represents near to 50 % of the total
production costs, while if raw glycerol is used for the production of 1,3-propanediol by
mean of engineered K. pneumoniae strains this value is lower between 8.2 to 9.2 % of the
total production cost as shown in Table 8.28. The values here obtained do not consider
the transportation costs, since the 1,3-propanediol production process was assumed to be
a biorefinery adjacent the biodiesel production process. Both utilities and capital costs
represent the highest production cost which combined vary between 64.46 % for the
Scenario 3 to 66.99 % for the Scenario 1. Also, it was noticed that capital costs increased
when final concentration of 1,3-propanediol increased.
154
Table 8.28. Economic results for raw glycerol conversion to 1,3-propanediol: Cost
Scenario
1
Raw materials
Utilities
Operating labor
Maintenance
Operating charges
Plant Overhead
G and A cost
Co-products sales
Gly. Purify. + ferm.
1,3-PD purification
Total
Sale price/production cost
Scenario 2 Scenario 3
Cost
0.0889
0.0892
0.0887
Share
8.61
8.19
9.20
Cost
0.2151
0.2245
0.1458
Share
20.83
20.61
15.12
Cost
0.0698
0.0746
0.0714
Share
6.76
6.85
7.40
Cost
0.0479
0.0503
0.0486
Share
4.64
4.62
5.04
Cost
0.0172
0.0187
0.0178
Share
1.66
1.71
1.85
Cost
0.0577
0.0625
0.0600
Share
5.59
5.73
6.22
Cost
0.0650
0.0712
0.0615
Share
6.29
6.54
6.38
Cost
0.4767
0.5042
0.4757
Share
46.16
46.28
49.34
Cost
-0.0055
-0.0057
-0.0054
Share
-0.53
-0.52
-0.56
Cost
0.304
0.331
0.313
Share
29.40
30.38
32.42
Cost
0.729
0.758
0.652
Share
70.60
69.62
67.58
Cost
1.033
1.089
0.964
1.710
1.622
1.832
In order to obtain more detailed information about the 1,3-propanediol production from
raw glycerol, the total production cost was divided in two processing sections: (i) Glycerol
purification plus glycerol fermentation and (ii) 1,3-propanediol recovery and purification,
as shown in Table 8.28. The first section (i.e., glycerol purification and fermentation)
represents in all cases between 29.4 to 32.4 %, while the second section (i.e., 1,3-
155
propanediol purification) is between 67.6 to 70.6 %. The 1,3-propanediol purification cost

was estimated to be between 0.652 to 0.758 USD$/kg of 1,3-propanediol. The increasing
order for the total production is: Scenario 3 < Scenario 1 < Scenario 2, which is not
related to the order found to the 1,3-propanediol production, neither its yield.
Also, the commercial sale price for 1,3-propanediol was compared to the production cost
and it was found that this ratio (i.e., sale price/production cost) is higher than the unity for
all scenarios, and for the Scenario 3 this ratio was 1.832. These results indicate that 1,3propanediol production from raw glycerol by mean of engineered K. pneumoniae could be
a profitable alternative for glycerol usage.
8.7.2
Ethanol production
The economic assessment for the purification process considers two scenarios. In the first
one, the retired methanol from raw glycerin stream is considered as a waste, and in the
second scenario the methanol is recycled to the transesterification process, which
contains 99 wt % of methanol and 1 wt % of glycerol. Therefore, in the second case
methanol is obtained as a co-product as it was shown in Chapter 4.
Raw glycerol in bioethanol production represented only 30% of the total costs as shown in
Table 8.29. Transportation costs were not considered in the economic assessment since
the purification step was assumed to be adjacent to the biodiesel production process. On
the other hand, utilities and capital costs represent the highest cost on the purification
process (i.e., between 20% and 30%). Also, the final quality of glycerol increases mainly
the utility costs. Purification costs of raw glycerol to pure glycerol at 88 wt % are 0.1574
US$/L (scenario I) and 0.0984 US$/L (scenario II) when the methanol price is considered.
Moreover, when glycerol at 98 wt % is used the Purification costs are 0.1782 US$/L
(scenario I) and 0.1124 US$/L (scenario II). Approximate costs for refining raw glycerol
have been reported to about 0.15 US$/lb or 0.26 US$/L [37], which are higher than the
purification costs obtained here, but near to the obtained purification costs in the scenario
I. Also, purification costs obtained are lower than the sale price of each product, which are
0.28 US$/L for glycerol at 88 wt %, 1.39 US$/L for glycerol at 98 wt % from vegetable oil
and 1.11 US$/L for glycerol at 98 wt % from tallow. Then a decrease in the whole fuel
ethanol production costs from glycerol can be expected due to the purification process.
156
The economic assessment carried out for the glycerol bioconversion process to fuel
ethanol does not consider the raw material cost because it is involved in the purification
costs. Table 8.29 shows the bioconversion costs (BCCs) obtained using Aspen Icarus.
The lowest BCC was obtained for crude glycerol (88 wt %) when it was diluted at 20 g/L,
since it uses a lower quantity of water than the other two processes. In this way,
equipment size and utilities are modified in each case. On the other hand, when pure
glycerol (98 wt %) is used a higher water quantity is necessary then increasing sizing and
utilities.
Table 8.29. Bioconversion costs (BCCs) for fuel ethanol production form raw glycerol
Crude
Crude
Pure
Share glycerol Share
glycerol
Share
glycerol
(10 g/l)
(%)
(20 g/l)
(%)
(10 g/l)
(%)
Utilities
0.0599
31.82
0.0503
29.41
0.0975
41.28
Operating labor
0.0188
9.97
0.0188
10.99
0.0188
7.96
0.0205
10.89
0.0154
9.02
0.0193
8.17
and administrative costs
0.0266
14.14
0.0309
18.05
0.0410
17.37
0.0625
33.18
0.0556
32.54
0.0596
25.23
0.1883
100.00
0.1710
100.00
0.2361
100.00
Item (US$/L)
Maintenance and operating

charges
Plant overhead and general
Product
(US$/L)
production
cost
Finally, global production costs (GPCs) for raw glycerol bioconversion to fuel ethanol are
obtained by adding the purification costs and the BBCs, like shown in Table 8.30. In all
cases the purification costs are near 35 % and the BCCs are near 65 %. Furthermore,
these obtained GPCs from crude glycerol are lower than those reported by Quintero et al
[17] for fuel ethanol production from corn (0.3381 US$/L), where using crude glycerol at
10 g/L and 20 g/L could represent a saving of 15 % and 20 %, respectively. The obtained
GPCs are higher than those reported by Quintero et al [17] for fuel ethanol production
from sugar cane (0.2153 US$/L). Nevertheless, these obtained GPCs are lower than the
international prices for fuel ethanol ranging from (0.4552 US$/L [145] to 0.6057 US$/L
[146]). The
157
Although a rigorous analysis of ethanol fuel market and its prices should be carried out,
the obtained results indicate that the production process of ethanol fuel from raw glycerol
using E. coli can be as profitable as those using sugar cane or corn as feedstocks.
Table 8.30. Global production costs (GPCs) for fuel ethanol production from raw glycerol.
Crude
Crude
glycerol
Share
glycerol
Share
Pure glycerol Share
(10 g/l)
(%)
(20 g/l)
(%)
(10 g/l)
(%)
0.0984
34.32
0.0984
36.53
0.1124
32.26
Costs
0.1883
65.68
0.1710
63.47
0.2361
67.74
Global Costs
0.2867
100.00 0.2694
Costs
Purification Costs
Bioconversion
100.00 0.3485
100.00
On the other hand, for the sustainable production of biodiesel from oil palm, total
production cost of ethanol from crude glycerol was estimated in 0.2694 US$/L, but this
result considers the buying of crude glycerol. This cost is lower than these reported for
fuel ethanol production from corn (0.3381 US$/L) and higher that these reported from
sugar cane (0.2153 US$/L) as showed Quintero et al [17].
Total production cost of ethanol from lignocellulosic biomass has been calculated by
McAloon et al. [147] was 0,396 US$/L, which is lower than the current international price
of fuel ethanol, reported by ICISpricing [145] as 0.4552 US$/L. On the other hand, total
production cost of biodiesel from oil palm under above described operation conditions
was estimated in 0,7971 US$/L (results are not shown), where its sale price ranged
between 0,76 0,86 US$/L (ICISpricing, 2010) [145].
Total production cost of biodiesel from oil palm in this integrated production process was
estimated in 0,8803 US$/L, which is higher than biodiesel production cost from palm oil.
This cost has been discriminated by raw material, services, operative and administrative
costs, depreciation and co-products credit, as shows the Table 8.31. Also, column 2
shows the share for each item.
158
Table 8.31. Discriminated costs for integrated biodiesel and raw-ethanol production from
oil palm.
Item
Cost (US$/L)
Share (%)
Raw materials
0,3376
35,69
Service fluids
0,2105
22,25
Labour
0,0083
0,87
Maintenance
0,0621
6,57
Operating charges
0,0301
3,18
General and
0,0750
7,92
0,2225
23,52
Co-products credit
-0,0657
-6,95
Total production cost
0,8803
100,00
administrative costs
This technology is promising from an environmental viewpoint because of petrochemical

methanol is not required and also in the case of oil palm industry an independence from
feedstock can be achieved. On the other hand, regarding crude glycerin, traditional
biodiesel industries have had only two options, the first one is its sale as low-quality
glycerin with a rather low price; the second is its refining until either, industrial or USP
grades, where the involved purification cost have been previously estimated as 0.20 and
0.27 US$/L, respectively Posada and Cardona [37]. These values agree with the
purification cost reported by Johnson and Taconi [146], which is 0.26 US$/L. Although,
biodiesel production cost by this technology indicates that the process is not economically
viable, a further analysis of process design which considers a higher detail level as:
energy cogeneration, heat integration, and the use of new technologies, among others,
could help to improve the process efficiency which would be reflected directly in the
production cost of biodiesel.
Described technology is a perfect representation of the biorefinery concept applied to the

biofuels production, in which oil palm is used as a sole feedstock for producing mainly
biodiesel, and ethanol as an important co-product which is widely used by the transport
sector. Therefore, it is expected that this integrated technology for biodiesel production
which uses efficiently the lignocellulosic residues and crude glycerol to produce the
159
required ethanol as feedstock, can be regarded as a valuable opportunity for biofuels

production. Moreover, the two proposed ways for ethanol production should eliminate the
need of using other feedstock which is important as foods, such as: sugar cane, sugar
beet, corn, wheat, sorghum, etc.
8.7.3 PHB production

The total production costs of PHB at 99.9 wt % from raw glycerol are shown in Table 8.32.
This table was obtained using glycerol at 88 or 98 wt % in the fermentation stage. Also,
the three downstream processes were compared. In all cases the cost for raw material
represents the glycerol purification cost. In this way, the glycerol purification process
represents only between 4.8 to 5.6 % of the total PHB production cost when glycerol at 88
wt % is used and this values increased between 6.3 to 7.7 % when glycerol at 98 wt % is
used.
Table 8.32. Total PHB production costs from crude glycerol through raw glycerol (88 wt
%) and pure glycerol (98 wt %)
Item
Cost (US$/kg) *DSP I
*DSP II
*DSP III
and Share (%) 88 wt % 98 wt % 88 wt % 98 wt % 88 wt % 98 wt %

Cost
0.118
0.149
0.118
0.149
0.118
0.149
Share
5.6
7.71
4.84
6.27
5.42
7.07
Cost
0.7787
0.658
0.9809
0.953
0.8678
0.841
Share
36.94
33.96
40.23
39.97
39.87
39.77
Cost
0.0752
0.066
0.0752
0.068
0.0752
0.064
Share
3.57
3.41
3.08
2.85
3.45
3.03
Maintenance and
Cost
0.2296
0.21
0.2402
0.236
0.2451
0.223
operating charges
Share
10.89
10.84
9.85
9.9
11.26
10.55
Plant overhead, general
Cost
0.2128
0.186
0.2287
0.218
0.2091
0.191
and administrative costs Share
10.09
9.6
9.38
9.14
9.61
9.03
Cost
0.6938
0.668
0.7951
0.76
0.6616
0.646
Share
32.91
34.48
32.61
31.87
30.39
30.55
Cost
2.108
1.937
2.438
2.384
2.1767
2.114
Share
100
100
100
100
100
100
Raw material
Utilities
Operating labor
Total
*DSP: Downstream process
160
In general terms the lower production costs are obtained when glycerol at 98 wt % is used
in the fermentation process, due to the higher PHB yield in the fermentation stage and the
lower energy requirements in the spray drying process. On the other hand, the lower
glycerol purification cost obtained for glycerol at 88 wt % generates a lower share in the
total PHB production costs compared with glycerol at 98 wt %. In both cases (88 and 98
wt %) the higher value in the total production cost was obtained for the Downstream
Process II, which uses a solvent extraction stage. This extraction requires heating the
expensive solvent DES up to 110 C which increase the utility costs.
The total PHB production costs obtained are between 2.11 and 2.44 US$/Kg when
glycerol at 88 wt % is used in the fermentation process, and between 1.94 and 2.38
US$/Kg when glycerol at 98 wt % is used. These production costs are near to the lower
sale prices reported in the literature (involving profits) and were obtained from other
substrates (see Table 8.33). In economical terms the best technological scheme for PHB
production from crude glycerol includes three important features as follows: i) purification
of crude glycerol up to 98 wt %, ii) a two continuous fermentation stages and finally iii) the
PHB recovering performed with the Downstream Process I, which is similar to the
BIOPOL flow sheet [45-46].
Table 8.33. Main producers of PHA in the world

PHB:
Product Company
name
Biomer: P(3HB)
Biotechnoly Co.
Germany
Biocycle: P(3HB)
PHB industrial
S/A company,
Brazil
Biogreen: P(3HB) Mitsubishi GAS
Chemical,
Japan
P(3HB)
Metabolix, USA
(BASF, ADM)
Substrate
Price (US$/kg)
Small
scale
production [148]
Sugar
cane[149]
25 (2003)
3.75-6.25 (2010)
12.5-15 (2003)
3.12-3.75 (2010)
Methanol [148]
2.75 (2010)
Corn,
[149]
sugar -
Production
(t/y)
50 (2003)
1400(2003)
30-60000(2010)
-
Pure glycerol has a higher cost than raw glycerol, but the yield of PHB is higher when
pure glycerol is used as feedstock. Moreover, each downstream process should be
adjusted to the conditions of the fermentation broth. Thus, using the blocks process
161
analysis (glycerol purification, fermentation, and PHB isolation and purification) the best
technological scheme for the production of PHB was found.
In all the cases assessed in this study the total production costs (see Table 8.32) were
lower than those reported in the literature (see Table 8.33) using feedstocks different to
glycerol. Generally, it has been suggested that the higher share to the total PHB
production cost is the substrate cost which is up to 45%. Meanwhile, if glycerol is used as
feedstock this share is below 8 %. These results indicate that using crude glycerol as
feedstock to produce PHB could be a profitable alternative to develop biorefineries in the
biodiesel industry.
8.7.4 D-Lactic acid production

The five scenarios were economically compared as shown in Table 8.34. The total
production cost was discriminated, in general terms, by raw materials, services,
operatives, depreciation, and products and co-product sales.
If raw glycerol is used for the D-lactic acid production by mean of engineered E. coli
strains this value is lower than 8 % of the total production cost. The values here obtained
do not consider the transportation costs, since the D-lactic acid production process was
assumed to be a biorefinery adjacent the biodiesel production process. Both utilities and
capital costs represent the highest production cost which combined vary between 69.04
% for the Scenario 3 to 73.12 % for the Scenario 1. Also, it was noticed that utilities costs
increased when glycerol concentration decreased, which makes sense due to the higher
requirement of both water and heat for the fermentation stage. The opposite behavior
occurred for the capital costs.
In order to obtain more detailed information about the D-lactic acid production from raw
glycerol, the total production cost was divided in two processing sections: (i) Glycerol
purification plus glycerol fermentation and (ii) D-lactic acid recovery and purification, as
shown in Table 8.32. The first section (i.e., glycerol purification and fermentation)
represents in all cases between 21 to 26 %, while the second section (i.e., D-lactic acid
purification) is between 74 to 79 %. The D-lactic acid purification cost was estimated to be
between 0.789 to 0.915 USD$/kg of L.A. Although these values are lower than that ones
162
reported by Joglekar et al. [55] for a similar downstream process (see: Table 8.15, Route
1), the esterification and hydrolysis by reactive distillation processes were not here
considered. Also, a rigorous economic analysis was carried out meanwhile the purification
costs obtained by Joglekar et al. [55] were based only on reported literature.
Table 8.34. Economic results for raw glycerol conversion to D-lactic acid: Cost (USD$/kg)
and Share (%)
Scenario
1
Raw materials
Utilities
Operating labor
Cost
0.081
0.0812
0.0789
0.0791
0.0252
Share
6.93
6.78
7.16
7.79
2.43
Cost
0.4556
0.3932
0.2956
0.2844
0.2736
Share
38.95
32.85
26.83
28.02
26.35
Cost
0.0542
0.0583
0.0565
0.0565
0.0581
4.63
4.87
5.13
5.57
5.60
0.0488
0.0628
0.0659
0.0579
0.069
4.17
5.25
5.98
5.70
6.65
0.0136
0.0146
0.0141
0.0141
0.0146
1.16
1.22
1.28
1.39
1.41
0.0515
0.0606
0.0612
0.0572
0.0635
4.40
5.06
5.56
5.64
6.12
0.0707
0.0788
0.0699
0.0681
0.0703
6.04
6.58
6.34
6.71
6.77
Cost
0.3997
0.453
0.465
0.4468
0.4696
Share
34.17
37.85
42.21
44.02
45.23
-0.0055
-0.0057
-0.0054
-0.0492
-0.0056
Share
-0.47
-0.48
-0.49
-4.85
-0.54
Cost
0.304
0.282
0.274
0.226
0.219
Share
25.98
23.56
24.86
22.27
21.10
Cost
0.866
0.915
0.828
0.789
0.819
Share
74.02
76.44
75.14
77.73
78.90
Cost
1.1696
1.1968
1.1017
1.0149
1.0383
1.327
1.297
1.409
1.530
1.495
Share
Maintenance
Cost
Share
Operating charges
Cost
Share
Plant Overhead
Cost
Share
G and A cost
Cost
Share
Co-products sales
L.A. purification
Total
Scenario 2 Scenario 3 Scenario 4 Scenario 5
Cost
163
High differences can be observed for the total production cost of D-lactic acid from raw
glycerol, which range from 1.0149 to 1.1968 USD$/Kg of L.A. The increasing order for the
total production cost is: Scenario 4 < Scenario 5 < Scenario 3 < Scenario 1 < Scenario 2,
which is completely related to the order found to the D-Lactic acid production. Also, the
commercial sale price for D-lactic acid was compared to the production cost and it was
found that this ratio (i.e., sale price/production cost) is higher than the unity for all
scenarios, and for the Scenario 4 this ratio was 1.53. These results indicate that D-lactic
acid production from raw glycerol by mean of engineered E. coli could be a profitable
alternative for glycerol usage.
The lowest total production cost of D-lactic acid from raw glycerol was obtained for the
Scenario 4 in which three fermentative advantages are joined simultaneously: (i) use of
crude glycerol (85 wt %), (ii) high glycerol concentration in the fermentation media (40 g/l),
and (iii) total consumption of glycerol. Thus, the economical successful of a
biotechnological scheme depends highly on the fermentation stage and the above
described three characteristics could lead to high process performance. In this order of
ideas, one of the main purposes of metabolic engineering should be to develop specific
strains able to consume completely raw substrates at high concentrations.
8.7.5 Succinic acid production

The five scenarios were economically assessed and the results are shown in Table 8.34.
These production costs were discriminated by raw materials, services, operatives,
depreciation, and products and co-product sales. The values here obtained do not
consider the transportation costs, since the succinic acid production process was
assumed to be a biorefinery adjacent the biodiesel production process.
In all cases the raw material costs represent only between 2.8 to 3.91 %, while the utilities
are the highest share of the total production costs with a weight between 65.9 to 70.12 %
followed by the capital costs with a share among 11.23 to 14.55 %.
164
Table 8.35. Economic results for raw glycerol conversion to succinic acid: Cost (USD$/kg)
and Share (%)
Scenario
1
Raw materials
Utilities
Operating labor
Maintenance
Operating charges
Plant Overhead
G and A cost
Co-products sales
S.A. purification
Total
Cost
0.0906
0.0908
0.0885
0.0885
0.0908
Share
3.91
3.00
3.41
3.33
2.79
Cost
1.5673
2.0850
1.7877
1.8644
2.1486
Share
67.60
68.95
68.79
70.12
65.93
Cost
0.0583
0.0587
0.0556
0.0503
0.0584
Share
2.51
1.94
2.14
1.89
1.79
Cost
0.0366
0.0549
0.0446
0.0458
0.0669
Share
1.58
1.82
1.72
1.72
2.05
Cost
0.0146
0.0147
0.0139
0.0126
0.0146
Share
0.63
0.49
0.53
0.47
0.45
Cost
0.0474
0.0568
0.0501
0.0481
0.0626
Share
2.05
1.88
1.93
1.81
1.92
Cost
0.1720
0.2882
0.2547
0.2561
0.4072
Share
7.42
9.53
9.80
9.63
12.50
Cost
0.3373
0.3804
0.3097
0.2986
0.4152
Share
14.55
12.58
11.92
11.23
12.74
Cost
-0.0055
-0.0055
-0.0060
-0.0055
-0.0055
Share
-0.24
-0.18
-0.23
-0.21
-0.17
Cost
0.3104
0.3205
0.2837
0.2516
0.3104
Share
13.39
10.60
10.92
9.46
9.52
Cost
2.008
2.703
2.315
2.407
2.949
Share
86.61
89.40
89.08
90.54
90.48
Cost
2.319
3.024
2.599
2.659
3.259
1.0747
0.8241
0.9589
0.9373
0.7647
Also, it was noticed that utilities costs increased with both the global fermentation yield
(see Table 8.20) and the low concentration of glycerol in the fermentation media. This
behavior is due to both the inefficient usage of glycerol during the fermentation process
and the higher requirement of serves.
165
The total production costs were divided in two processing sections: (i) Glycerol purification
plus glycerol fermentation and (ii) succinic acid recovery and purification, as shown in
Table 8.32. Thus, more detailed information about the succincic acid production from raw
glycerol can be obtained. In this way, glycerol purification and fermentation represent, in
all cases, lower than 13.4 % indicating that most of the total production cost of succinic
acid from raw glycerol are in the recovery and purification processes (> 86.5 %).
Regarding to the succinic acid purification costs, high differences were found among the
five scenarios since this value varies from 2.008 to 2.949 USD$/Kg of S.A, being its
relative difference almost 50%. These differences found in the purification costs are
mainly related to the complex behavior of the fermentation process, due to variables such
as glycerol concentration, glycerol purity, glycerol consumption, and yield to succinic acid
affects the performance of the global process. Thus, the increasing order for succinic acid
recovery and purification costs and for total production costs of succinic acid from raw
glycerol are the same, it is Scenario 1 < Scenario 3 < Scenario 4 < Scenario 2 < Scenario
5.
On the other hand, the commercial sale price for succinc acid was compared to its
production cost and it was found that this ratio (i.e., sale price/production cost) was only
higher than the unity for the Scenario 1, but this value is still too close to the unity. Thus, it
can be stated that the succinic acid production from glycerol still requires performing high
effort in order to make this process profitable since the its production costs is high yet.
8.7.6 Propionic acid production

The five analyzed scenarios were economically assessed as shown in Table 8.36 and the
production costs results were discriminated by raw materials, services, operatives,
depreciation, and products and co-product sales. The values here obtained do not
consider the transportation costs, since the propionic acid production process was
assumed to be a biorefinery adjacent the biodiesel production process.
Low raw material costs were obtained in all cases when raw glycerol is used, representing
only around 5 % of the total production costs. But both utilities (between 45.8 to 58.9 %)
and capital (between 20.8 to 29.9 %) costs represent the highest production cost which
166
combined vary between 75.76 % for the Scenario 3 to 79.69 % for the Scenario 1 and the
Scenario 5. Also, it was found that the utilities costs depend mainly on the glycerol
concentration in the fermentation media.
Table 8.36. Economic results for raw glycerol conversion to propionic acid: Cost
(USD$/kg) and Share (%).
Scenario
1
Raw materials
Utilities
Operating labor
Maintenance
Operating charges
Plant Overhead
G and A cost
Co-products sales
L.A. purification
Total
Cost
0.0851
0.0851
0.0851
0.0851
0.0851
Share
4.41
5.05
4.71
4.38
4.59
Cost
1.1355
0.8192
0.8275
0.9709
0.9497
Share
58.89
48.67
45.85
49.99
51.22
Cost
0.0561
0.0624
0.0706
0.0745
0.0691
Share
2.91
3.71
3.91
3.83
3.73
Cost
0.0342
0.0651
0.0783
0.0841
0.0762
Share
1.78
3.87
4.34
4.33
4.11
Cost
0.0140
0.0156
0.0176
0.0186
0.0173
Share
0.73
0.93
0.98
0.96
0.93
Cost
0.0452
0.0638
0.0744
0.0793
0.0727
Share
2.34
3.79
4.12
4.08
3.92
Cost
0.1625
0.1161
0.1171
0.0636
0.0618
Share
8.43
6.90
6.49
3.27
3.33
Cost
0.4011
0.4614
0.5398
0.5717
0.5279
Share
20.80
27.41
29.91
29.43
28.47
Cost
-0.0055
-0.0055
-0.0055
-0.0055
-0.0055
Share
-0.28
-0.33
-0.30
-0.28
-0.30
Cost
0.3100
0.3108
0.3104
0.2959
0.3104
Share
16.07
18.46
17.20
15.23
16.74
Cost
1.618
1.372
1.495
1.647
1.544
Share
83.93
81.54
82.80
84.77
83.26
Cost
1.928
1.683
1.805
1.942
1.854
0.985
1.129
1.053
0.978
1.025
167
In addition, the total production cost was divided in two processing sections: (i) glycerol
purification plus glycerol fermentation and (ii) propionic acid recovery and purification, as
shown in Table 8.36. The glycerol purification and glycerol fermentation stages only
represented between 15.23 to 18.46 %, which indicates that most efforts are required for
the downstream process since a high share of the total production costs (between 81.54
to 84.77 %) is consumed for the propionic acid purification.
Total production costs of propionic acid from raw glycerol range from 1.683 to 1.942
USD$/Kg of P.A., which represents a relative difference of 15.4 %. Being the increasing
order for the total production costs: Scenario 2 < Scenario 3 < Scenario 5 < Scenario 1 <
Scenario 4.
The ratio between the sale price to the production cost was calculated for the five
analyzed scenarios, and this ratio was higher than the unity only for the Scenario 2,
Scenario 3, and Scenario 5. Thus, it is stated that this process could be profitable only
when high concentration of glycerol is used in the fermentation media and high yield to
propionic acid is obtained.
8.8 Conclusions
In the past, important efforts have been made in order to introduce the biotechnological
production of 1,3-propanediol from glycerol to the industry. However, research tendencies
were focused on microorganism development and on the analysis of some specific
process conditions. On the other hand, the drastic increment in the use of biodiesel has
caused an oversupply of glycerol in the market. Thus, in order to suitably use the glycerol
obtained from the biodiesel industry, the mass production of 1,3-propanediol from glycerol
needs additional analysis. Here, a complete technological scheme for its production has
been not only proposed by also assessed. Even more, the fermentation stage was
optimized by three different ways and as result it was possible to assess economically
three scenarios where the third one had the lowest production cost. The relative
differences respect to the first and the second scenarios were 7.2% and 13.0 %. The
obtained results provide enough information to understand the different possibilities for
process intensification using this technology and also to compare it with other new
industrial alternatives for the utilization of glycerol as a raw material.
168
Due to the low cost of raw glycerol, methanol recovery from glycerol implies low
purification costs. Meanwhile, the three possibilities assessed for glycerol bioconversion
to ethanol showed that the global production costs of fuel ethanol from raw glycerol are
lower than the commercial price of fuel ethanol. These facts show the potential for raw
glycerol bioconversion to fuel ethanol using E. coli. Also, the comparison carried out with
a previous paper (which considers the fuel ethanol production from sugarcane and corn in
the Colombian case) shows that the global production costs of fuel ethanol from raw
glycerol can be as profitable as the production of fuel ethanol from conventional raw
materials as sugarcane. The latter is a completely developed industry in Colombia.
Production of ethanol from glycerol is presented as an alternative which uses a renewable

resource that does not generate direct or indirect competition with the food industry,
property that do have the raw materials used in commercial (maize, cassava Beets, sugar
cane, molasses, etc.). The techno-economic feasibility of this process was demonstrated
through simulation and evaluation of processes and found that the cost of production is
similar to the process established commercially in Colombia from sugar cane. The
environmental performance of this process was not the best of those obtained for the
different raw materials, but significant higher compared with the process benefits from
corn.
Biodiesel and ethanol can be jointly produced using oil palm as sole source by mean of
processes integration, such as the biodiesel production with the ethanol production from
two feedstocks: lignocellulosic residues (empty fruit bunches and palm press fiber
produced during) and crude glycerol. Thus, alcohol is completely self-supplied by the
integrated process and low quantities of wastes are produced without any global
production of glycerol. Economical evaluation showed a higher biodiesel production cost
for the integrated process than the traditional biodiesel production process which uses
ethanol and palm oil as feedstocks. But the first one is a promising technology available to
build an autonomous biodiesel production plant with low waste levels. This process must
be economically improved by further analysis from a process design view point, based on
process simulation which showed be a powerful tool for performing processes integration.
Three technological schemes to produce PHB from crude glycerol were analyzed under
two fermentation conditions (i.e., using glycerol at 88 wt % and 98 wt %). In this work it
was found that it is better to use pure glycerol as feedstock for the production of PHB than
169
raw glycerol. This phenomenon is explained by the fact that the higher PHB yield reduces
the utility costs in the downstream process. The results shown here are important for the
industrial production of PHB using glycerol as a raw material. Currently, in the biodiesel
production the total profitability of any new project could be determined by the right use of
glycerol as a massive by-product. The proposed strategy to use pure glycerol as
substrate can be understood as a very interesting alternative since the final composition
in the glycerol streams depends on the source of the feedstock used for biodiesel
production. Also, most of the biotechnological alternatives to produce added value
compounds from glycerol are sensitive to contaminants in the raw material. Thus, several
technical and economical advantages as well as a more stable production of PHB are
obtained when a standardized raw material as pure glycerol is used.
Usage of raw glycerol, engineered Escherichia coli strains, and processes integration for
the production of optically pure D-lactic acid is an important alternative to transform the
by-produced glycerol during the biodiesel synthesis. Although five different configurations
for the fermentation stage were considered, in all cases the total production costs were
lower than its sale price. Thus, the whole process scheme for D-lactic acid production
could be considered as potentially profitable design. Also, it was found that the combined
effect of both high glycerol concentration and use of low quality glycerol in the
fermentation media, lead to the best economic performance. The results shown here are
important for the industrial production of D-lactic acid using glycerol as a raw material. On
the other hand, in the biodiesel production the total profitability of any new project could
be determined by the right use of glycerol as a massive raw material.
Otherwise, for succinic acid and propionic acid production results showed that most
efforts are required in order to reduce the downstream process since the total production
costs were too close to the sale price of these carboxylic acids.
References
[1] Gonzalez-Pajuelo, M., Meynial-Salles, I., Mendes, F., Andrade, J.C., Vasconcelos, I.,
and Soucaille, P.: Metabolic engineering of Clostridium acetobutylicum for the industrial
production of 1,3-Propanediol from glycerol. Metab. Eng. 7, 329-336 (2005).
170
[2] Ito, T., Nakashimada, Y., Senba, K., Matsui, T., and Nishio N.: Hydrogen and ethanol
process. J. Biosci. Bioeng.. 100, 260265 (2005).
[3] Zhao, Y.N., Chen, G., and Yao, S.J.: Microbial production of 1,3-propanediol from
glycerol by encapsulated Klebsiella pneumoniae. Biochem. Eng. J. 32, 9399 (2006).
[4] Menzel, K., Zeng, A.-P., and Deckwer, W.-D.: High concentration and productivity of
1,3-propanediol from continuous fermentation of glycerol by Klebsiella pneumoniae.
Enzyme Microb. Technol. 20, 8286 (1997).
[5] Xiu, Z.L., Song, B.H., Sun L.H., and Zeng, P.: Theoretical analysis of effects of
metabolic overflow and time delay on the performance and dynamic behavior of a twostage fermentation process. Biochem. Eng. J. 11, 101109 (2002).
[6] Xiu, Z.L., Song, B.H., Wang, Z.T., Sun, L. H., Feng, E. M., and Zeng, A.P.:
Optimization of dissimilation of glycerol to 1,3-Propanediol by Klebsiella pneumoniae in
one- and two-stage anaerobic cultures. Biochem. Eng. J. 19, 189-197 (2004).
[7] Edgar, T.F., Himmelblau, D.M., and Lasdon L.S.: Optimization of chemical processes.
Second Edition, McGraw Hill, New York (2001).
[8] Raval, K.N., Hellwig, S., Prakash, G., Ramos-Plasencia, A., Srivastava, A., and Biichs.
J.: Necessity of a two-stage process for the production of azadirachtin-related limonoids in
suspension cultures of Azadirachta indica. J. Biosci. Bioeng.. 96, 16-22 (2003).
[9] Janusz J. Malinowski. Reactive Extraction for Downstream Separation of 1,3Propanediol. Biotechnol. Prog. 2000, 16, 76-79.
[10] Jian Hao, Hongjuan Liu, and Dehua Liu. Novel Route of Reactive Extraction To
Recover 1,3-Propanediol from a Dilute Aqueous Solution. Ind. Eng. Chem. Res. 2005, 44,
4380-4385.
[11] Jian Hao, Feng Xu, Hongjuan Liu and Dehua Liu. Downstream Processing of 1,3Propanediol Fermentation Broth. J. Chem. Technol. Biotechnol. 81:102108 (2006).
[12] Serafimov L. A., Zharov V. T., Timofeyev V. S., Rectification of multicomponent
mixtures. I. Topological analysis of liquid vapor phase equilibrium diagrams, Acta Chim.
Acad. Sci. Hung. 1971, Tomus 69, 4, 383.
171
[13] Pisarenko Y. A., Serafimov L. A., Cardona, C. A., Efremov, D.L., Shuwalov, A.S.,
Reactive distillation design: analysis of the process statics, Reviews in Chemical
Engineering 17 (4), 2001.
[14] Serafimov L. A., Pisarenko Yu. A., Kulov N. N., Coupling Chemical Reaction with
Distillation: Thermodynamic Analysis and Practical Applications, Chemical Engineering
Science 54 (1999) 1383-1388.
[15] Giessler S., Danilov R. Y., Pisarenko R. Y., Serafimov L. A., Hasebe S., and
Hashimoto I., Feasible Separation Modes for Various Reactive Distillation Systems. Ind.
Eng. Chem. Res. 1999, 38, 4060-4067
[16] Giessler S., Danilov R. Y., Pisarenko R. Y., Serafimov L. A., Hasebe S., Hashimoto I..
Systematic structure generation for reactive distillation processes. Computers and
Chemical Engineering 25 (2001) 4960
[17] Quintero JA, Montoya MI, Snchez OJ, Giraldo OH, Cardona CA. Fuel ethanol
production from sugarcane and corn: Comparative analysis for a Colombian case. Energy
2008;33:385-399.
[18] Cardona CA, Snchez OJ. Fuel ethanol production: Process design trends and
integration opportunities. Bioresour. Technol 2007;98:2415-2457.
[19] Raskovic P, Anastasovski A, Markovska Lj, Mesko V, Process integration in
bioprocess indystry: waste heat recovery in yeast and ethyl alcohol plant. Energy
2010;35:704717.
[20] Yu S, Tao J. Energy efficiency assessment by life cycle simulation of cassava-based
fuel ethanol for automotive use in Chinese Guangxi context. Energy 2009;34:22-31.
[21] Yu S, Tao J. Simulation-based life cycle assessment of energy efficiency of biomassbased ethanol fuel from different feedstocks in China. Energy 2009;34:476484.
[22] Cardona CA, Snchez OJ. Energy consumption analysis of integrated flowsheets or
production of fuel ethanol from lignocellulosic biomass. Energy 2006;31:2447-2459.
[23] Cardona CA, Quintero JA, Paz IC. Production of bioethanol from sugarcane bagasse:
Status and perspectives. Bioresour Technol 2009;101:4754-4766.
172
[24] Gutirrez LF, Snchez J, Cardona CA. Process integration possibilities for biodiesel
production from palm oil using ethanol obtained from lignocellulosic residues of oil palm
industry. Bioresour Technol 2009;100:1227-1237.
[25] Felix E, Tilley DR. Integrated energy, environmental and financial analysis of ethanol
production from cellulosic switchgrass. Energy 2009;34:410436.
[26] Dharmadi Y, Murarka A, Gonzalez R. Anaerobic fermentation of glycerol by
Escherichia coli: a new platform for metabolic engineering. Biotechnol Bioeng
2006;94:821829.
[27] Yazdani SS, Gonzalez R. Engineering Escherichia coli for the efficient conversion of
glycerol to ethanol and co-products. Metab. Eng. 2008;10:340351
[28] Nielsen J, Villadsen J, Liden G. Bioreaction engineering principles. Kluwer
Academic/Plenum Publishers, New York, 2003.
[29] J. M. Hernndez-Salas, M. S. Villa-Ramrez, J. S. Veloz-Rendn, K. N. RiveraHernndez, R. A. Gonzlez-Csar, M. A. Plascencia-Espinosa, S. R. Trejo-Estrada.
(2009) Bioresource Technology 100 1238-1245.
[30] C. A. Cardona Alzate, O. J. Snchez Toro. (2006) Energy 31 2447-2459.
[31] M. Rivera, C.A. Cardona. (2004) Ingeniera y Competitividad 6
[32] L. F. Gutirrez (2008) Ph.D. thesis National University of Colombia at Manizales
Manizales, Colombia
[33] A. Abdul Aziz, M. Husin, A. Mokhtar. (2002) Journal of Oil Palm Research 14 9-14.
[34] A. Abdul Aziz, K. Das, M. Husin, A. Mokhtar. (2002) Journal of Oil Palm Research 14
10-17.
[35] M. W. Zahari, A.R. Alimon. (2004) Palm Oil Developments 40 5-9.
[36] S. Prasertsan, P. Prasertsan. (1996) Biomass and Bioenergy 11 387-395.
[37] Posada JA, Cardona CA. 2010. Anlisis de la refinacin de glicerina obtenida como
by-Product of biodiesel production). Ingeniera y Universidad 14:2-27.
173
[38] Posada JA, Cardona CA, Rincn LE. Sustainable biodiesel production from palm
using in situ produced glycerol and biomass for raw bioethanol. In 32nd symposium on
biotechnology for fuels and chemicals. Clearwater Beach, Florida. April 19-22. 2010.
[39] Mothes G, Schnorpfeil C, Ackermann CJU. Production of PHB from crude glycerol.
Eng Life Sci 2007;7(5);475479.
[40] Cavalheiro JMBT, de Almeida MCMD, Grandfilis C, da Fonseca MMR. Poly(3hydroxybutyrate) production by Cupriavidus necator using waste glycerol. Process
Biochem 2009;44(5);509-515.
[41] Jacquel N, Lo CW, Wei YH, Wu HS, Wang SS. Isolation and purification of bacterial
poly(3-hydroxyalkanoates). Biochem Eng J 2008;39;1527.
[42] Tamer M, Moo-Young M., Chisti Y. Disruption of Alcaligenes latus for recovery of poly
(-hydroxybutyric acid): comparison of high-pressure homogenization, beadmilling and
chemically induced lysis, Ind Eng Chem Res 1998;37;18071814.
[43] Chen Y, Chen Y, Chen J, Yang H. Kinetics of PHB-containing biomass disruption in
surfactantchelate aqueous solution. Process Biochem 2003:38;1173-1182.
[44] Hejazi P, Vasheghani-Farahani E, Yamini Y. Supercritical uid disruption of Ralstonia
eutropha for poly ( -hydroxybutyrate) recovery. Biotechnol Progr 2003;19;15191523.
[45] Asrar J, Grys KJ. Biopolymers Vol. 4: Polyesters III - Applications and commercial
products. Wiley-VCH, Munster; 2002. p 398.
[46] Harding KG, Dennis JS, von Blottnitz H, Harrison STL. Environmental analysis of
plastic
production
processes:
Comparing
petroleum-based
polypropylene
and
polyethylene with biologically-based poly-B-hydroxybutyric acid using life cycle analysis.

J. Biotechnol. 2007;130;5766.
[47] Kapritchkoff FM, Viotti AP, Alli RCP, Zuccolo M, Pradella JGC, Maiorano AE, Miranda
EA, Bonomi A. Enzymatic recovery and purification of polyhydroxybutyrate produced by
Ralstonia eutropha, J Biotechnol 2006;122;453462.
174
[48] Dien, B. S., N. N. Nichols, and R. J. Bothast. 2001. Recombinant Escherichia coli
engineered for production of L-lactic acid from hexose and pentose sugars. J. Ind.
Microbiol. Biotechnol. 27:259264.
[49] Okano, K., T. Tanaka, C. Ogino, H. Fukuda, and A. Kondo. 2010. Biotechnological
production of enantiomeric pure lactic acid from renewable resources: recent
achievements, perspectives, and limits. Appl. Microbiol. Biotechnol. 85:413423.
[50] Zhou, S., K. T. Shanmugam, and L. O. Ingram. 2003. Functional replacement of the
Escherichia
coli
D()-lactate
dehydrogenase
gene
(ldhA)
with
the
L(+)-lactate
dehydrogenase gene (ldhL) from Pediococcus acidilactici. Appl. Environ. Microbiol.

69:22372244.
[51] Zhou, S., T. B. Causey, A. Hasona, K. T. Shanmugam, and L. O. Ingram. 2003.
Production of optically pure D-lactic acid in mineral salts medium by metabolically
engineered Escherichia coli W3110. Appl. Environ. Microbiol. 69:399407.
[52] Zhu, Y., M. A. Eiteman, K. DeWitt, and E. Altman. 2007. Homolactate fermentation by
metabolically engineered Escherichia coli strains. Appl. Environ. Microbiol. 73:456464.
[53] Mazumdar, S., Clomburg, J.M., and Gonzalez, R. 2010. Escherichia coli Strains
Engineered for Homofermentative Production of D-Lactic Acid from Glycerol. Applied and
Environmental Microbiology. 76: 43274336.
[54] Hong, A.A.; Cheng, K.K.; Peng, F.; Zhou, S.; Sun, Y.; Liu, C.M.; Liu, D.H. Strain
isolation and optimization of precess parameters for bioconversion of glycerol to lactic
acid. J. Chem. Technol. Biotechnol., 2009, 84, 1576-1581.
[55] Joglekar, H.G., Rahman, I., Babu, S., Kulkarni, B.D., Joshi, A. 2006.Comparative
assessment of downstream processing options for lactic acid. Separation and Purification
Technology. 52:117
[56] Tung L.A., and C.J. King, Ind. Eng. Chem. Res. 33 (1994) 32173223.
[57] Kaufman E.N., S.P. Cooper, S.L. Clement, M.H. Little, Appl. Biochem. Biotechnol.
5152 (1995) 605620.
[58] Evangelista L.R., Z.L. Nikolov, Appl. Biochem. Biotechnol. 5758 (1996) 471480.
175
[59] Kulprathipanja S., A.R. Oroshar, US Patent 5,068,418, 1991

[60] Zheng Y.J., X.H. Ding, P.L. Cen, C.W. Yang, G.T. Tsao, Appl. Biochem. Biotechnol.
5758 (1996) 627632.
[61] Antonio G.R., G. Vaccari, E. Dosi, A. Trilli, M. Rossi, D. Matteuzzi, Biotechnol.
Bioeng. 67 (2000) 147156.
[62] Srivastava A., P.K. Roychoudhury, V. Sahai, Biotechnol. Bioeng. 39 (1992) 607613
[63] Cao X., H.S. Yun, Y.M. Koo, Recovery of lactic acid by anion-exchange resin
Amberlite IRA-400. Biochem. Eng. J. 11 (23) (2002) 189196.
[64] Tong W.-Y., X.-Y. Fu, S.-M. Lee, J. Yu, J.-W. Liu, D.-Z.Wei, Y.-M. Koo, Biochem.
Eng. J. 18 (2) (2004) 8996.
[65] Raya-Tonetti G., P. Cordoba, J. Bruno-barcena, F. Sineriz, N. Perotti, Biotechnol.
Tech. 13 (3) (1999) 201205.
[66] Vaccari G., A. Gonzalez-Vara, A.L. Campi, E. Dosi, P. Brigidi, Appl. Microbiol.
Biotechnol. 40 (1993) 2327.
[67] Senthuran A., V. Senthuran, B. Mattiasson, R. Kaul, Biotechnol. Bioeng. 55 (1996)
841853.
[68] Kaufman E.N., S.P. Cooper, S.L. Clement, M.H. Little, Appl. Biochem. Biotechnol.
5152 (1995) 605620.
[69] Lee H.-J., Y. Xie, Y.-M. Koo, N.-H.L.Wang, Biotechnol. Prog. 20 (2004) 179192.
[70] Chen C.-C., L.-K. Ju, Sep. Sci. Technol. 33 (10) (1998) 14231437.
[71] Min-tian G., M. Hirata, M. Koide, H. Takanashi, T. Hano, Process Biochem. 39 (2004)
19031907.
[72] Bailly M., H. Roux-de Balmann, P. Aimar, F. Lutin, M. Cheryan, J. Membr. Sci. 191
(2001) 129142.
[73] Bailly M., Desalination 144 (13) (2002) 157162.
176
[74] Habova V., K. Melzoch, M. Rychtera, B. Sekavova, Desalination 163 (2004) 361
372.
[75] Li H., R. Mustacchi, C.J. Knowles,W. Skibar, G. Sunderland, I. Dalrymple, S.A.
Jackman, Tetrahedron 60 (2004) 655661.
[76] Hong, Y. K. and W. H. Hong (2000) Reactive extraction of succinic acid with
tripropylamine (TPA) in various diluents. Biopro. Eng. 22: 282-284
[77] Hong Y.K., Hong W.H., and Han D.H., Application of Reactive Extraction to Recovery
of Carboxylic Acids. Biotechnol. Bioprocess Eng. 2001, 6: 386-394.
[78] Tamada J.A., A.S. Kertes, and C.J. King. Extraction of Carboxylic Acids with Amine
Extractants. 1. Equilibria and Law of Mass Action Modeling. Ind. Eng. Chem. Res. 1990,
29, 1319-1326.
[79] Yabannavar V.M., D.I.C.Wang, Biotechnol. Bioeng. 37 (1991a) 716722.
[80] Yabannavar V.M., D.I.C. Wang, Biotechnol. Bioeng. 37 (1991b) 10951100.
[81] Seo Y., W.H. Hong, T.H. Hong, Korean J. Chem. Eng. 16 (5) (1999) 556561
[82] Kim J.Y., Y.J. Kim, W.H. Hong, G. Wozny, Biotechnol. Bioprocess Eng. 5 (2000)
196201.
[83] Kim Y.J., W.H. Hong, G. Wozny, Korean J. Chem. Eng. 19 (5) (2002) 808814.
[84] Choi I., W.H. Hong, J. Chem. Eng. Jpn. 32 (1999) 184189.
[85] Nielsen J, Villadsen J, Liden G. Bioreaction engineering principles. New York: Kluwer
Academic/Plenum Publishers; 2003.
[86] Posada JA, Cardona CA. 2010. Design and analysis of fuel ethanol production from
raw glycerol. Energy. 35(12):5286-5293.
[87] Posada JA, Cardona CA. 2010. Anlisis de la refinacin de glicerina obtenida como
by-Product of biodiesel production). Ingeniera y Universidad 14:2-27.
177
[88] Posada JA, Naranjo JM, Lpez JA, Higuita JC, Cardona CA. 2011a. Design and
Analysis of PHB Production Processes from Crude Glycerol. Process Biochemistry.
46:310-317.
[89] Datta R (1992) Process for the production of succinic acid by anaerobic fermentation.
US patent 5,143,833
[90] Datta R, Glassner DA, Jain MK, Vicky Roy JR (1992) Fermentation and purification
process for succinic acid. US patent 5,168,055
[91] Hermann BG, Patel M (2007) Todays and tomorrows bio-based bulk chemicals from
white biotechnologya techno-economic analysis. Appl Biochem Biotechnol 136: 361388
[92] Berglund KA, Yedur S, Dunuwila D (1999) Succinic acid production and purification.
US patent 5,958,744
[93] Yedur S, Berglung KS, Dunuwila DD (2001) Succinic acid production and purification.
US patent 6,265,190
[94] Glassner DA, Satinwood RD, Datta R (1992) Process for the production and
purification of succinic acid. US patent 5,143,834
[95] Zeikus JG, Jain MK, Elankovan P (1999) Biotechnology of succinic acid production
and markets for derived industrial products. Appl Microbiol Biotechnol 51:545552
[96] Berglund KA, Elankovan P, Glassner DA (1991) Carboxylic acid purification and
crystallization process. US patent 5,034,105
[97] McKinlay JB, Vieille C, Zeikus JG (2007) Prospects for a biobased succinate industry.
Appl Microbiol Biotechnol. doi: 10.1007/s00253-007-1057-y
[98] Jaquet A, Quan R, Marison W, vain Stockar U (1999) Factors influenceing in
potentioluse of aliquat 336 for the in situ extraction of carboxylic acids from cultures of
Pseudomonas putida. J Biotechnol 68:185
[99] King CJ, Dtarr J (1990) Recovery of carboxylic acids from water by precipitation from
organic solutions. US patent 5,104,492
178
[100] King CJ, Poole LJ (1993) Craboxylic acid sorption regeneration process. US patent
5,412,126
[101] Kurzrock T. Weuster-Botz D. Recovery of succinic acid from fermentation broth.
Biotechnol Lett (2010) 32:331339.
[102] Tamada JA, King CJ (1990a) Extraction of carboxylic acids with amine extractants:
2. Chemical interactions and interpretation of data. Ind Eng Chem Res 29:13271333
[103] Tamada JA, King CJ (1990b) Extraction of carboxylic acids with amine extractants:
3. Effects of temperature, water coextraction and process considerations. Ind Eng Chem
Res 29:13331338
[104] Hong YK, Hong WH, Han DH (2001) Application of reactive extraction to recovery of
carboxylic acids. Biotechnol Bioproc Eng 6:386394
[105] Tung LA, King CJ (1994a) Sorption and extraction of lactic and succinic acids at
pH[pKa1. 1. Factors governing equilibria. Ind Eng Chem Res 33:32173223
[106] Hong YK, Hong WH (2000a) Equilibrium studies on reactive extraction of succinic
acid from aqueous solutions with tertiary amines. Bioproc Eng 22:477481
[107] Hong YK, Hong WH (2000b) Reactive extraction of succinic acid with tripropylamine
(TPA) in various diluents. Bioproc Eng 22:281284
[108] Hong YK, Hong WH (2000c) Extraction of succinic acid with 1-octanol/n-heptane
solutions of mixed tertiary amine. Bioproc Eng 23:535538
[109] Hong YK, Han DH, Hong WH (2002) Water enhanced solubilities of succinic acid in
reactive extraction using tertiary amines/alcohol systems. Korean J Chem Eng 19:8386
[110] Hong YK, Hong WH (2004) Influence of chain length of tertiary amines on
extractability and chemical interactions in reactive extraction of succinic acid. Korean J
Chem Eng 21:488493.
[111] Song H, Huh YS, Lee SY, Hong WH, Hong YK (2007) Recovery of succinic acid
produced
by
fermentation
of
metabolomically
succiniciproducens strain. J Biotechnnol 132:445452
engineered
Mannheimia
179
[112] Lee SY, Kim JM, Song H, Lee JW, Kim TY, Yang YS (2008) From genome
sequence to integrated bioprocess for succinic acid production by Mannheimia
succiniciproducens. Appl Microbiol Biotechnol 79:1122
[113] Juang RS, Huang RH (1997) Equilibrium studies on reactive extraction of lactic acid
with an amine extractant. Chem Eng J 65:4753
[114] Heyberger A, Prochazka J, Volaufova E (1997) Extraction of citric acid with tertiary
aminethird phase formation. Chem Eng Sci 97:0032100327
[115] Huh YS, Jun Y-S, Kong YK, Song H, Lee SY, Hong WH (2006) Effective purification
of succinic acid from fermentation broth produced by Mannheimia succiniciproducens.
Proc Biochem 41:14611465
[116] Li Q, Li WL, Wang D, Liu BB, Tang H, Yang MH, Liu Q, Xing JM, Su ZG (2008) pH
neutralization while succinic acid adsorption onto anion-exchange resins. Appl Biochem
Biotechnol. doi:10.1007/s12010-008-8355-4
[117] Kushiku T, Fujiwara K, Satou T, Sano (2006) Method for purifying succinic acid from
fermentation broth. US patent 2006/0276674A1.
[118] Jun YS, Huh YS, Park HS, Thomas A, Jeon SJ, Lee EZ, Won HJ, Hong WH, Lee
SY, Hong YK (2007) Adsorption of pyruvic and succinic acid by amine-functionalized
SBA-15 for the purification of succinic acid from fermentation broth. J Phys Chem C
200(111):1307613086.
[119] Pai RA, Doherty MF, Malone MF (2002) Design of reactive extraction systems for
bioproduct recovery. AIChE J 48:514526
[120] Blankschien M.D., Clomburg J.M., Gonzalez R. Metabolic engineering of
Escherichia
coli
for
the
production
of
succinate
from
glycerol.
Metabolic
Engineering12(2010)409419
[121] Scholten E. and
Dirk Dagele. Succinic acid production by a newly isolated
bacterium. Biotechnol Lett (2008) 30:21432146.
180
[122] Lee P.C., Lee W.G., Lee S.Y., Chang H.N. Succinic Acid Production with Reduced
By-Product Formation in the Fermentation of Anaerobiospirillum succiniciproducens Using
Glycerol as a Carbon Source. Biotechnology and Bioengineering, 72:2143-2146, 2001.
[123] Thompson, J.C., He, B.B., Characterization of crude glycerol from biodiesel
production for multiple feedstocks. Appl. Eng. Agric. 2006, 22(2):261-265.
[124] Huh YS, Hong YK, Hong WH, Chang HN (2004) Selective extraction of acetic acid
from the fermentation broth produced by Mannheimia succiniciproducens. Biotechnol Lett
26:15811584.
[125] Zhu Y., Li J., Tan M., Liu L., Jiang L., Sun J., Lee P., Du G., Chen J., Optimization
and scale-up of propionic acid production by propionic acid-tolerant Propionibacterium
acidipropionici with glycerol as the carbon source. Bioresource Technology 101 (2010)
89028906.
[126] Zhang A., Yang S.T., Propionic acid production from glycerol by metabolically
engineered Propionibacterium acidipropionici. Process Biochemistry 44 (2009) 1346
1351
[127] Hauer, E. and Marr, R. (1994). Liquid Extraction in Biotechnology, International
Chemical Engineering, 34(2), pp. 178-187.
[128] Timmer, J. K. M., Kromkamp, J. and Robbertsen, T. (1994). Lactic acid separation
from fermentation broth by reverse osmosis and nanofiltration, Journal of Membrane
Science, 92, pp. 185-197
[129] Nomura, Y., Iwahara, M. and Hongo, M. (1987). Lactic acid production by
electrodialysis fermentation using immobilized growing cells, Biotechnology and
Bioengineering, 30, pp. 788-793
[130] Sirman, T., Pyle, D. L. and Grandison, A. S. (1991) Extraction of organic acids using
a supported liquid membrane, Biochemical Society Transactions, 19 (3), pp. 274-279
[131] Dai, Y. and King, J. (1996). Selectivity between lactic acid and glucose during
recovery of lactic acid with basic extractants and polymeric sorbents, Industrial
Engineering and Chemistry Research, 35, pp. 1215-1224.
181
[132] Kertes, A. S. and King, C. (1986). Extraction Chemistry of Fermentation product

Carboxylic Acids, Biotechnology and Bioengineering, 28, 269-282.
[133] Keshav A., Wasewar K., Chand S., Extraction of Propionic Acid Using Different
Extractants (Tri-n-butylphosphate, tri-n-octylamine and Aliquat 336). Industrial &
Engineering Chemistry Research 2008, 47, 61926196.
[134] Keshav A., Chand S., Wasewar K.L., Equilibrium studies for extraction of propionic
acid using tri-n-butyl phosphate in different solvents. Journal of Chemical and Engineering
Data, 2008, 53, (7), 14241430
[135] Keshav A., Chand S., Wasewar K., Equilibrium and kinetics of extraction of
propionic acid using tri-n-octylphosphineoxide. Chemical Engineering and Technology
2008, 31 (9), 1290-1295.
[136] Keshav A., Wasewar K., Chand S., Reactive extraction of propionic acid with trinoctylamine in different diluents. Separation and Purification Technology 2008, 63, 179183.
[137] Keshav A., Wasewar K.and Chand S., Study of binary extractants and modifier
diluents systems for reactive extraction of propionic acid, Fluid Phase Equilibria 2008,
275, 2126.
[138] Keshav A., Chand S., Wasewar K., Recovery of propionic acid from aqueous stream
by reactive extraction: Effect of diluents. Desalination 2009, 244, 12-23.
[139] Keshav A., Chand S., Wasewar K., Recovery of propionic acid by reactive extraction
using tri-n-butyl phosphate in petroleum ether: Equilibrium study, Chemical and
Biochemical Engineering Quarterly 2008, 22(4), 433-437.
[140] Keshav A., Wasewar K.L., Chand S., Extraction of acrylic, propionic and butyric acid
using Aliquat 336 in oleyl alcohol: Equilibria and effect of temperature, Industrial &
Engineering Chemistry Research 2009, 48 (2), 888893.
[141] Keshav A., Wasewar K.L., Chand S., Reactive extraction of propionic acid using trinoctylamine, tri-n-butyl phosphate and Aliquat 336 in sunflower oil as diluent, Journal of
Chemical technology and Biotechnology. Published Online: 14 Oct (2008).
182
[142] Keshav A., Chand S., Wasewar K., Recovery of propionic acid by reactive extraction
using quaternary amine (Aliquat 336) in various diluents. Chemical Engineering Journal.
2009, 152, 95-102.
[143] Keshav A., Wasewar K.L., Chand S., Uslu H., Inci I., Thermodynamics of reactive
extraction of propionic acid, i-managers Journal on Future Engineering and Technology.
2009, 4(2), 41- 49.
[144] Keshav A., Wasewar K., Chand S., Extraction of propionic acid from model
fermentation broth: Effect of pH, salts, substrate, and temperature, AIChE Journal, 2009,
55(7), 1705-1711.
[145] ICISpricing report. See also: http://www.icispricing.com
[146] Johnson DT, Taconi KA. The glycerin glut: options for the value-added conversion
of crude glycerol resulting from biodiesel production. Environ Prog 2007;26(4):338-348.
[147] McAloon, A., et al., Technical Report NREL/TP-580-28893. 2000, National
Renewable Energy Laboratory: Golden, CO (USA). p. 35
[148] Mitsubishi Gas Chemical Company, Inc; Tokyo, Japan. Annual Report 1999.
[149] Shen L, Haufe J, Patel MK. Product overview and market projection of emerging
bio-based plastics PRO-BIP 2009: Final report. Utrecht University. Commissioned by
European Polysaccharide Network of Excellence (EPNOE) and European Bioplastics;
2009 p. 243.
9. Experimental Setup for Glycerol Fermentation to

PHB
Polyhydroxyalkanoates (PHAs) have been recognized as good substitutes for the nonbiodegradable petrochemically produced polymers. However, their high current
production cost limits their industrial applications. Carbon source can be represent
between 25% to 45% of the total PHB production costs. Glycerol, a by-product from the
biodiesel industry, can be used as primary carbon source for cell growth and PHB
synthesis and it is an interesting alternative for to increase the PHB feasibility economic
process. Currently, PHB is produced in an industrial scale using Gram negative bacteria.
Nevertheless, Gram-negative organisms contain lipopolysaccharides (LPS) which
copurify with the PHAs and induces a strong immunogenic reaction. Gram-positive
bacteria lack LPS and are hence potentially better sources of PHAs when used for
biomdical purposes. In this work, the conditions and capability of poly (hydroxybutyrate) (PHB) production by a Bacillus megaterium (Gram Positive bacteria
isolated from superficial sediments of Baha Blanca Estuary (Buenos Aires, Argentina))
using glycerol as only carbon source were studied. This microorganism was adapted
and tested at different initial glycerol concentrations and compared with other substrates
as glucose. Aspen Plus and Aspen Icarus were used for the processes simulation and for
the economic assessment, respectively.
9.1 Generalities
Polyhydroxyalcanoates
are
attractive
substitute
biopolymers
for
conventional
petrochemical plastics which have similar physical properties to thermoplastics and

elastomers. PHAs are homo or heteropolyesters and can be synthesized and stored
intracellularly by many bacterias in the form of granules and can account for up to 80% of
the total bacterial dry weight [1] [2]. They can be produced from renewable resources
through a fermentation process under restricted growth conditions for nitrogen,
184
phosphorus, sulfurs and/or oxygen in the presence of an excess carbon source [3]; PHAs
are also completely biodegraded and biocompatible [1], [2], [3], [4].
Polyhydorxybutyrate (PHB) was the first type of PHAs discovered and the most widely
studied.
PHB
has
similar
mechanical
properties
to conventional
plastics
like
polypropylene or polyethylene, but its production cost are higher than the petrochemical
plastic. The PHB production cost depends on the microorganism (yield and productivity),
carbon and nitrogen sources (substrates), fermentation conditions (temperature,
aeration), recovery and purification of the PHB. Carbon source could represent between
25 to 45 % of the total production costs [4][5].
Many researches have been developed in order to find cheaper carbon sources. Agroindustrial wastes are attractive candidates because they have desired characteristics:
such as: low prices and high availability. Moreover, when these wastes are used an
environmental problem is avoided.
There are different kinds of microorganisms able to produce PHB from diverse
agroindustrial wastes. Substrates such as whey, lignocellulosic materials and glycerol
from biodiesel have been studied (Table 9.1).
Glycerol is by-generated during the biodiesel production. With every 100 lbs of biodiesel
produced by the transesterification of vegetable oils or animal fats, 10 lbs of crude
glycerol are generated (10 wt %) [16]. Although pure glycerol is an important industrial
feedstock used in foods, drugs, cosmetics, pharmaceuticals, pulp and paper, leather,
textile and tobacco industries, the growth on biodiesel industry has carried out a glycerol
surplus and its consequent price has decreased. Thus, the economy of biodiesel industry
has been directly affected.
Chemical and biological transformations have been analyzed in order to convert glycerol
to added-valuable products. Biological conversion offers the opportunity to synthesize a
large array of products and functionalities. Glycerol can be used such carbon source in
microbiological process substituting sugars owing to the highly reduced nature of carbon
atoms. Different works have shown that some strains of microorganisms can produce
PHB using crude glycerol as carbon and energy source. This microorganisms can be wild
9. Experimental Setup
185
strains such as Cupriavidus necator [12], Methylobacterium rhodesianum [13] or

recombinant microorganism such as E. coli recombinant [15-16]. In the group of PHB
producer bacteria there are both Gram positive and Gram negative strains. Currently,
PHB is produced in an industrial scale using Gram negative bacteria. Nevertheless,
Gram-negative organisms contain lipopolysaccharides (LPS) which copurify with the
PHAs [17] and induces a strong immunogenic reaction. Therefore it is undesirable for
biomedical applications. Those purification processes increase the PHB production costs.
Gram-positive bacteria lack LPS and are hence potentially better sources of PHAs when
used for biomedical purposes [18].
Table 9.1. Some microorganisms PHB producer from different agroindustrial wastes.
Agroindustrial
Waste
Lignocellusic
Whey
Cane
bagasse
Xilosa +
Glucose
Glycerol
Microorganism
Methylobacterium sp.
ZP24
Pseudomonas
hydrogenovora
Thermus thermophilus
HB8
Recombinant
Escherichia coli
Burkholderia sacchari
IPT 101
Burkholderia cepacia IPT
048
Ralstonia eutropha
Productivity
(g PHB l-1h-1)
1.18
0.18
Reference
[5]
[6]
[7]
0.90
[8]
0.11
[9]
0.09
[9]
[10]
B. cepacia ATCC 17759
0.47
[11]
Cupriavidus necator
DSM 545
Methylobacterium
rhodesienum MB 126
Osmophilic organism
Escherichia coli CT1061
1.51
[12]
0.26
[13]
0.05
0.18
[14]
[15]
Bacillus megaterium is Gram positive, strict aerobic, non motile, rod shaped, spore
forming, citrate positive, bacteria hydrolyzing gelatin and casein [19]. We continue the
study of B. megaterium isolated from superficial sediments of Bahia Blanca Estuary
186
(Buenos Aires, Argentina) after to check in other work [20] that this microorganism is a
PHB producer from glucose. In this paper, we report the total adaptation of B. megaterium
to glycerol as carbon source.
9.2 Materials and Methods

9.2.1 Bacterial strain and its maintenance
The Bacillus megaterium was isolated from superficial sediments of Bahia Blanca Estuary
(Buenos Aires, Argentina) and characterized as a PHB producer in the presence of
excess carbon source with the restriction of nitrogen. This strain was used for the current
study. Stock cultures were grown at 33 C in nutrient broth and maintained at 4C after
growth on nutrient agar during the activation level. After adaptation to glycerol as sole
carbon source, the stock culture was maintained at 4C after growth on formulated agar
with glycerol. B. megaterium cells were stored at -80 C in 2 ml cryovials containing 300
l of glycerol and 700 l of a previously prepared growth liquid culture.
9.2.2 Culture medium

The seeding medium was prepared with the following concentrations: (NH 4 ) 2 SO 4 , 1g/l;
KH 2 PO 4 , 1.5 g/l; Na 2 HPO 4 , 9 g/l; MgSO 4 7H 2 O, 0.2g/l; and 1 ml of trace element
solutions composed by: FeSO 4 7H 2 O, 10 g/l; ZnSO 4 7H 2 O, 2.25 g/l; CuSO 4 5 H 2 O,
1g/l; MnSO 4 4H 2 O, 0.5; CaCl 2 2H 2 O, 2 g/l; H 3 BO 4 , 0.23; (NH 4 ) 2 Mo 7 O 24 , 0.2 g/l; and
HCl, 10ml. The carbon sources analyzed are both glycerol and glucose. The carbon
source and MgSO 4 .7H 2 O were autoclaved separately and added aseptically to the
medium after cooling.
9.2.3 Microorganisms adaptation and culture conditions

B. megaterium cells were used to inoculate nutrient agar plates supplemented with 20 g/l
of glycerol previously incubated at 30C until growth and then stored at 4C. Single
colonies from the grown nutrient agar plates were inoculated in 100ml erlenmeyer flasks
containing 10 ml of sterile nutrient broth medium supplemented with 20 g/l of glycerol and
incubated at 30C in an orbital shaker at 200 rpm during a period of 2-3 days. 1 ml of
these cultures was again transferred to 10 ml of seeding medium with glycerol.
Successive subculturing was performed 2-3 times, to assure a good adaptation of cell
187
growth on glycerol as carbon source. The next step was the seed of the culture in the
formulated medium. 1 ml of culture growth in nutrient broth supplemented with 20 g/l of
glycerol was transferred to 10 ml of formulated medium with 20 g/l of glycerol. Successive
subculturing was performed 2-3 times, to assure a good adaptation of cell growth
formulated medium and glycerol. Those tests were incubated at 30C in an orbital shaker
at 200 rpm during a period of 2-3 days.
9.2.4 Batch cultivations

The PHB fermentation was carried out for 36 h in a 3.7 liters Lab Fermenter
(Bioengineering, Switzerland) by using a grown formulated medium. Two different carbon
sources are studied: glucose and glycerol. For both, the formulated medium and
fermenter are the same. The fermentations with glycerol were carried out at 30C and
33C and 200rpm. Two different initial glycerol concentrations are used 20 and 50 g/l. The
fermentation with glucose was carried out at 33C and 200 rpm. The initial glucose
concentration was of 20g/l. The culture volume was of 1.5 l for all batch fermentations.
9.3 Analytical Methods

9.3.1 Biomass
The biomass of the culture was determined using the optical density measurement at
600nm (Spectronicspectrophotometer, Thermo SCIENTIFIC
GENESYS 20) and by
gravimetric.
9.3.2 PHB extraction

After fermentation, the cells were harvested by centrifugation at 18C and 6.000 rpm for
20 min and then the intracellular PHB was extracted by using the Chloroform
hypochlorite dispersion extraction. The dispersion media contains 50ml of chloroform and
50ml of a diluted (30 wt %) sodium hypochlorite solution in water, in an orbital shaker at
100 rpm. The cell powder was treated at 38C for 1 h. The mixture obtained was then
centrifuged at 4000 rpm for 10 min, which resulted in three separate phases. PHB was
recovered from the bottom phase that contains PHB dissolved in chloroform. PHB is
precipitated using 10 volumes of ice-cold methanol [21].
188
9.3.3 PHB quantification

Dried biomass is used for methanolysis of monomers according to the method described
by Braunegg et al. [22] and modified by Lageveen al. [23]. Approximately 10 mg of cells
mass was reacted in a small screw-cap test tube with a solution containing 1 ml of
chloroform, 0.85 mL of methanol, and 0.15 mL of sulfuric acid for 140 min at 100 C. After
reaction, 0.5 mL of distilled water was added and the test tube was shaken vigorously for
1 min. After phase separation, the organic phase (bottom layer) was removed and
transferred to a small screw-cap glass vial. 50 l from this organic phase were taken and
added to a test tube and injected in the GC-MS. And an Agilent Technologies 6850 series
II gas chromatograph was used. The gas chromatograph was equipped with a HP-5MS
capillary column (25 m length, 0.32 mm internal diameter). Helium (5cm/min) was used as
the carrier gas. Injector and detector were operated at 230 C and 275 C, respectively. A
temperature program was used for efficient separation of the esters (120 C for 5min,
temperature ramp of 8 C per min, 180 C during 12 min). An Agilent Technologies 5975B
mass spectrometer was used.
9.3.4 Glycerol quantification

Glycerol concentration was determined off-line by HPLC (Hitachi LaChrom Elite)
equipped with an auto sampler (Hitachi LaChrom Elite L-2200), a Bio-Rad Aminex
Fermentation Monitoring Column (150 mm x 7.8 mm), a column oven (Hitachi LaChrom
Elite L-2300), a HPLC pump (Hitachi LaChrom Elite L-2130) and a Hitachi LaChrom Elite
L-2490 refraction index detector. Injection volume was 20 ml and elution was achieved
using a 50 mM solution of H 2 SO 4 . The column was kept at 65C and the pump was
operated at a flow rate of 0.8 ml min-1.
9.3.5 PHB characterization

Fourier transform infrared spectroscopy (FTIR): Freeze-dried, precipitated PHB from B.
cereus SPV was used to prepare KBr discs (sample:KBr, 1:100). An FTIR spectrum
1720X spectrometer (Perkin Elmer, USA) was used under the following conditions:
spectral range, 4000400 cm1; window material, CsI; 16 scans; resolution 4 cm1; the
detector was a temperature-stabilized, coated FRDTGS detector [24].
189
9.4 Results and discussion

Glycerol was used as only carbon source and we studied the productivity and yield of the
microorganism. The fermentation profile of glycerol to PHB and biomass, in formulated
medium by B. megaterium are shown in Figures 9.1 and Figure 9.2. Each one of
fermentation was carried out with different initial glycerol concentrations (20g/l and 50 g/l,
respectively). Both fermentations were carried out at 30C and 200 rpm, in the 3.7 liters
Lab Fermenter (Bioengineering, Switzerland). The culture volume was of 1.5 l. Final
concentration of PHB was 1.116 g/l with a productivity of 0.0248 g/l*h when glycerol at 20
g/l was used, while the final concentration of PHB was 2.356 g/l with a productivity of
0.7365 g/l*h when glycerol at 50 g/l was used.
Therefore, the best initial glycerol
concentration is the 50 g/l.
5
4.5
4
3.5
g/l
3
2.5
2
1.5
1
0.5
0
0
10
15
20
25
30
35
40
45
50
Time (hours)
Figure 9.1. Accumulation profile I of Bacillus megaterium cultivated in the 3.7 liters Lab
Fermenter (Bioengineering, Switzerland) with a culture volume of 1.5 l, initial glycerol
concentration of 20 g/l at 30C and 200 rpm.
Biomass,
PHB.
Then, the influence of temperature on the fermentation process was analyzed. The
temperature range for the growth of Bacillus was found to be from 25 to 45C [17]. The
temperatures analyzed in this work were 30C and 33C. Both fermentations were carried
out at 200 rpm, in the 3.7 liters Lab Fermenter (Bioengineering, Switzerland). The culture
volume was of 1.5 l, at the same initial glycerol concentration of 20g/l (Figures 9.1 and
Figures 9.3). Final PHB concentration was 1.116 g/l with a productivity of 0.0248 g/l*h at
30 C, while the final concentration of PHB was 3.4 g/l with a productivity of 0.0771 g/l*h
at 33 C. Therefore, the best temperature operation is the 33C.
190
10
9
8
7
g/l
6
5
4
3
2
1
0
0
10
15
20
25
30
35
40
Time (hours)
Figure 9.2. Accumulation profile II of Bacillus megaterium cultivated in the 3.7 liters Lab
Biomass,
PHB.
6
5
g/l
4
3
2
1
0
0
10
15
20
25
30
35
40
45
50
Time (hours))
Figure 9.3. Accumulation profile III of Bacillus megaterium cultivated in the 3.7 liters Lab
Biomass,
PHB.
In the same form, glucose was used as only carbon source in order to compare the use of
glycerol as raw material. The initial glucose concentration used was of 20g/l at 33C
(Figure 9.4). Final PHB concentration was 2.83 g/l with a productivity of 0.0783 g/l*h. A
comparison between obtained results for PHB production by mean of B. megaterium
using glucose and glycerol as substrates was made as shown in Table 9.2.
191
25
20
g/l
15
10
5
0
0
10
15
20
25
30
35
40
Time (hours)
Figure 9.4. Accumulation profile IV of Bacillus megaterium cultivated in the 3.7 liters Lab
Fermenter (Bioengineering, Switzerland) with a culture volume of 1.5 l, initial glucose
Biomass,
Glucose,
PHB
Table 9.2. Comparison between experimental results for glucose and glycerol
fermentation to PHB
GLUCOSE
Variable
GLYCEROL
20g/l-
20g/l-
50g/l-
20g/l-
T=33C
T=33C
T=30C
T=30C
DCW(g/l)
5.08g/l
5.7g/l
7.8 g/l
4.7 g/l
PHB(g/l)
2.83 g/l
3.4g/l
2.356 g/l
1.116g/l
% Accumulation
55.7%
62.3%
30.2%.
23.74%.
Time of Max. PHB
36
42
32
45
0.0786
0.0771
0.07365
0.0248
10.5
11.80
15.50
0.30
0.29
0.07
production (hours)
Productivity
gPHB/l*h
Total substrate
consumption (g /l)
Yield P/S
The produced PHB was characterized. The peak got form the FT-IR spectrum (1728 cm1
) corroborates with the peak reported, indicated the presence of PHB using glycerol as
sole carbon resource.
192
References
[1] Khanna S, Srivastava AK. Recent advances in microbial polyhydroxyalkanoates.
Process Biochemistry 2005;40:607619.
[2] Mahishi LH, Tripathi G, Rawal SK. Poly(3-hydroxybutyrate) (PHB) synthesis by
recombinant Escherichia coli harbouring Streptomyces aureofaciens PHB biosynthesis
genes: Effect of various carbon and nitrogen sources. Microbiol Res 2003;158:1927.
[3] Steinbchel A. Perspectives for Biotechnological Production and Utilization of
Biopolymers: Metabolic Engineering of Polyhydroxyalkanoate Biosynthesis Pathways as a
Successful Example. Macromolecular Bioscience 2001;1:1-24.
[4] Lee SY. Plastic bacteria? Progress and prospects for polyhydroxyalkanoate
production in bacteria. Tibtech 1996; (VOL 14).
[5]. A. Nath, M. Dixit, A. Bandiya, S. Chavda, A.J. Desai. Enhanced PHB production and
scale up studies using cheese whey in fed batch culture of Methylobacterium sp. ZP24.
Bioresource Technology 99 (2008); 57495755
[6]. Martin Koller, Rodolfo Bona, Emo Chiellini, Elizabeth Grillo Fernandes, Predrag
Horvat, Christoph Kutschera, Paula Hesse, Gerhart Braunegg. Polyhydroxyalkanoate
production from whey by Pseudomonas hydrogenovora. Bioresource Technology 99
(2008); 48544863
[7]. Pantazaki AA, et al. Production of polyhydroxyalkanoates from whey by Thermus
thermophilus HB8.Process Biochem (2009), doi:10.1016/j.procbio.2009.04.002
[8]. Beom Soo Kim. Production of poly(3-hydroxybutyrate) from inexpensive substrates.
Enzyme and Microbial Technology 27 (2000) 774777.
[9]. L. F. Silva, M. K. Taciro, M. E. Michelin Ramos, J. M. Carter, J. G. C. Pradella, J. G.
C. Gomez. Poly-3-hydroxybutyrate (P3HB) production by bacteria from xylose, glucose
and sugarcane bagasse hydrolysate. J Ind Microbiol Biotechnol (2004) 31: 245254
[10]. Jian Yu, Heiko Stahl. Microbial utilization and biopolyester synthesis of bagasse
hydrolysates. Bioresource Technology 99 (2008) 80428048.
193
[11]. L. F. Silva, M. K. Taciro, M. E. Michelin Ramos, J. M. Carter, J. G. C. Pradella, J. G.

C. Gomez. Poly-3-hydroxybutyrate (P3HB) production by bacteria from xylose, glucose
and sugarcane bagasse hydrolysate. J Ind Microbiol Biotechnol (2004) 31: 245254
[12]. Cavalheiro JMBT, et al. Poly(3-hydroxybutyrate) production by Cupriavidus necator
using waste glycerol. Process Biochem (2009),
[13]. Bormann EJ, Roth M. Production of polyhydroxybutyrate by Methylobacterium
rhodesianum and Ralstonia eutropha in media containing glycerol and casein
hydrolysates. Biotechnol Lett 1999;21:105963.
[14]. Koller M, Bona R, Braunegg G, Hermann C, Horvat P, Kroutil M, et al. Production of
polyhydroxyalkanoates from agricultural waste and surplus materials. Biomacromolecules
2005;6:5615
[15]. Pablo I. Nikel, M. Julia Pettinari, Miguel A. Galvagno, Beatriz S. Mndez. Poly(3hydroxybutyrate) synthesis from glycerol by a recombinant Escherichia coli arcA mutant in
fed-batch microaerobic cultures. Appl Microbiol Biotechnol (2008) 77:13371343.
[16] Syed Shams Yazdani and Ramon Gonzalez. Anaerobic fermentation of glycerol: a
path to economic viability for the biofuels industry. Current Opinion in Biotechnology 2007,
18:213219
[17]. S.P. Valappil, S.K. Misra, A.R. Boccaccini, T. Keshavarz, C. Bucke, I. Roya. Largescale production and efficient recovery of PHB with desirable material properties, from the
newly characterized Bacillus cereus SPV. Journal of Biotechnology 132 (2007), 251258
[18] Chen, G.Q., Wu, Q. The application of polyhydroxyalkanoates as tissue engineering
materials. Biomaterials 26 (2005), 65656578.
[19]. Sureshbabu K. P., Venkatraman D., Kalimuthu., Neelamegam R., Muniyandi J.,
Sangiliyandi G. Optimization and fed-batch production of PHB utilizing dairy waste and
sea water as nutrient sources by Bacillus megaterium SRKP-3. Bioresource Technology
101 (2010), 705711.
[20]. Naranjo, J. M., Vasquez, J. A., Higuita, J.C., Cardona, C.A. PHB production and
characterization from a Bacillus megaterium strain isolated from Marine Sediments.
194
[21]. Hahn, S.K., Chang, Y.K., Lee, S.Y. Recovery and characterization of poly(3hydroxybutyric acid) synthesized in Alcaligens eutrophus and recombinant Escherichia
coli. Appl. Environ. Microbiol. 61 (1995), 3439.
[22] Braunegg, G. Sonnleitner, B., Lafferty, R.M. A rapid gas chromatographic method
for the determination of poly-B-hydroxybutyric acid in microbial biomass. Eur. J.
Appl. Microbiol. Biotechnol. 6 (1978), 29-37.
[23]. Lageveen, R. G., Huisman, G. W., Preusting, H., Ketelaar, P., Eggink, G. & Witholt,
B. Formation of polyesters by Pseudomonas oleovorans : eect of substrates on
formation
and
composition
of
poly-(R)-3-hydroxyalkanoates
and
poly-(R)-3-
hydroxyalkenoates. Appl Environ Microbiol 54 (1988), 29242932.

[24]. S.P. Valappil, S.K. Misra, A.R. Boccaccini, T. Keshavarz, C. Bucke, I. Roy. Largescale production and efficient recovery of PHB with desirable material properties, from the
newly characterized Bacillus cereus SPV. Journal of Biotechnology 132 (2007) 251258
10. Conclusions
Commercially three qualities of glycerol were identified as the most important ones. Crude
glycerol with a purity ranging from 80-88 wt %, technical glycerol mainly found at 97 wt %,
and refined glycerol (USP or FCC grades) at 99.7 wt %. These three types of glycerol
differ significantly in the content of water, fatty acid residues, esters, and other organic
wastes. Also, some differences were found for the use of diverse feedstocks for biodiesel
production on the composition of the glycerol layer. Although, most of the first use oils
lead to not big differences in the glycerol layer, a completely different behavior was
observed for the glycerol obtained from WVO represented by low concentration of
glycerol and methanol with a high content of fats. On the other hand, based on the
traditional purification of glycerol, a flowsheet able to purify raw glycerol up to the three
commercial qualities above described was designed, simulated and economically
assessed. Results showed that not only quality requirements were successfully obtained
but also for the analyzed purification scale all the processes were profitable. Thus, a
homogenized raw material and a purification process were obtained in order to continue
the analysis of different possibilities of glycerol transformation to added-value products.
Acrolein, hydrogen, and 1,2-propanediol, are three of the most commercially important
products obtained from glycerol, due to their applications, established market, and sale
prices. Here the technological schemes to produce these compounds were designed,
simulated, and economically assessed. Thus, simulation results showed that all the
processes are technologically feasible reaching high purity of product. Also, acrolein
production was found to be viable at a purity of 92 wt %, but do not at a purity of 98.5 wt
%. Finally, both hydrogen and 1,2-propanediol production processes are also
economically viable, where the last one generates the highest profit margin.
In the past, important efforts have been made to introduce the biotechnological production
of 1,3-propanediol from glycerol to the industry. However, research tendencies were
196
focused on microorganism development and some process conditions analysis. The

drastic increment in the use of biodiesel caused an oversupply of glycerol in the market.
For this reason and in order to optimize its productivity, mass production of 1,3propanediol from glycerol needs additional analysis. Here, fermentation of glycerol with K.
pneumoniae was optimized using three different models and considering two
simultaneous goals: i) high volumetric productivity and ii) high 1,3-propanediol
concentration. In conclusion, the obtained results provide enough information to
understand the different possibilities for process intensification using this technology and
also to compare it with other new industrial alternatives for the utilization of glycerol as a
raw material.
Due to the low cost of raw glycerol, methanol recovery from glycerol implies low PCs.
Meanwhile, the three possibilities assessed for glycerol bioconversion showed that the
GPCs of fuel ethanol from raw glycerol are lower than the commercial price of fuel
ethanol. These facts show the potential for raw glycerol bioconversion to fuel ethanol
using E. coli. Also, the comparison carried out with a previous paper (which considers the
fuel ethanol production from sugarcane and corn in the Colombian case, [13]) shows that
the GPCs of fuel ethanol from raw glycerol can be as profitable as the production of fuel
ethanol from conventional raw materials as sugarcane. The latter is a completely
developed industry in Colombia.
Biodiesel and ethanol can be jointly produced using oil palm as sole source by mean of
processes integration, such as the biodiesel production with the ethanol production from
two feedstocks: lignocellulosic residues (empty fruit bunches and palm press fiber
produced during) and crude glycerol. Thus, alcohol is completely self-supplied by the
integrated process and low quantities of wastes are produced without any global
production of glycerol. Economical evaluation showed a higher biodiesel production cost
for the integrated process than the traditional biodiesel production process which uses
ethanol and palm oil as feedstocks. But the first one is a promising technology available to
build an autonomous biodiesel production plant with low waste levels. This process must
be economically improved by further analysis from a process design view point, based on
process simulation which showed be a powerful tool for performing processes integration.
10. Conclusions
197
Three technological schemes to produce PHB from crude glycerol were analyzed under
two fermentation conditions (i.e., using glycerol at 88 wt % and 98 wt %). In this work it
was found that it is better to use pure glycerol as feedstock for the production of PHB than
raw glycerol. This phenomenon is explained by the fact that the higher PHB yield reduces
the utility costs in the downstream process. The results shown here are important for the
industrial production of PHB using glycerol as a raw material. Currently, in the biodiesel
production the total profitability of any new project could be determined by the right use of
glycerol as a massive by-product. The proposed strategy to use pure glycerol as
substrate can be understood as a very interesting alternative since the final composition
in the glycerol streams depends on the source of the feedstock used for biodiesel
production. Also, most of the biotechnological alternatives to produce added value
compounds from glycerol are sensitive to contaminants in the raw material. Thus, several
technical and economical advantages as well as a more stable production of PHB are
obtained when a standardized raw material as pure glycerol is used.
Usage of raw glycerol, engineered Escherichia coli strains, and processes integration for
the production of optically pure D-lactic acid is an important alternative to transform the
by-produced glycerol during the biodiesel synthesis. Although five different configurations
for the fermentation stage were considered, in all cases the total production costs were
lower than its sale price. Thus, the whole process scheme for D-lactic acid production
could be considered as potentially profitable design. Also, it was found that the combined
effect of both high glycerol concentration and use of low quality glycerol in the
fermentation media, lead to the best economic performance. The results shown here are
important for the industrial production of D-lactic acid using glycerol as a raw material. On
the other hand, in the biodiesel production the total profitability of any new project could
be determined by the right use of glycerol as a massive raw material.
11. List of Publications and Submitted Papers

This chapter shows the published results throughout scientific meeting, papers, book
chapters, invited book chapters, and books. Also, a list containing the submitted papers
was made.
11.1 Published
Scientific Meetings
1. Posada JA, Higuita JC, Cardona CA. 2011. Optimal crude glycerol biorefinery
from
biodiesel
production
to
produce
poly-3-hydroxybutyrate.
World
Renewable Energy Congress 2011 Sweden. Linkping University.
2. Posada JA, Quintero JA, Cardona CA. 2010. Energy and environmental
comparison among the production of fuel ethanol from crude glycerol, sugar
cane, and crop. IV International congress of science and technology of the
biofuels. Bucaramanga, Colombia. November 30th - December 3rd.
3. Posada JA, Cardona CA, Rincn LE. 2010. Sustainable biodiesel production
from palm using in situ produced glycerol and biomass for raw bioethanol. In:
Society for Industrial Microbiology. 32nd symposium on biotechnology for fuels
and chemicals. Clearwater Beach, Florida. Abril 19th-22nd.
200
Papers
4. Posada JA, Naranjo JM, Lpez JA, Higuita JC, Cardona CA. 2011. Design
and Analysis of PHB Production Processes from Crude Glycerol. Process
Biochemistry. 46:310-317.
5. Posada JA, Cardona CA. 2010. Design and analysis of fuel ethanol
production from raw glycerol. Energy. 35(12):5286-5293.
6. Posada JA, Cardona CA. 2010. Anlisis de la refinacin de glicerina obtenida

como co-producto en la produccin de biodiesel (Validation of glycerin refining
obtained as a by-Product of biodiesel production). Ingeniera y Universidad
14:2-27.
7. Posada
JA,
Orrego
CE;
Cardona
CA.
2009.
Biodiesel
production:
Biotechnological approach. International Review of Chemical Engineering

(I.Re.Che.), 1(6):571-580.
Book Chapters
8. Posada JA, Cardona CA, Cetina DM, Orrego CE. 2009. Bioglicerol como
materia prima para la obtencin de productos de valor agregado (Bioglycerol
as raw material to obtain added value products). En: CARDONA CA (ed).
Avances investigativos en la produccin de Biocombustibles (Reasearching
advances for biofuels production). Manizales: Artes Graficas Tizan. p. 103127. ISBN: 978-958-44-5261-0
11. List of Publicatiions
201
Books
9. Cardona CA, Posada JA, Quintero JA. 2010. Aprovechamiento de

subproductos y residuos agroindustriales: Glicerina y Lignocelulsicos (Use of
agroindustrial wastes and by-products: Glycerin and Lignocellulosics).
Manizales: Artes Graficas Tizan. p. 218. ISBN: 978-958-44-7611-1
Invited Book Chapters
10. Posada JA, Rincn LE, Cardona CA. Integral Use of Palm Oil: Production of
Biodiesel and Added Value Compounds from Glycerin. In: Oil Palm:
Cultivation, Production and Dietary Components. Editor: Susan A. Penna.
Book Chapter Ed. Nova Publisher. Series: Agriculture Issues and Policies.
ISBN: 978-1-61122-201-2.
11.2 Submitted
Papers
1. Posada JA, Jaramillo JJ, Cardona CA. Glycerol fermentation to 1,3propanediol: Comparison among four culture configurations. Submitted to
Biochemical Engineering Journal.
2. Posada JA, Higuita JC, Pisarenko YA, Cardona CA. Design and economical
analysis of the technological scheme for 1,3-propanediol production from raw
glycerol. Submitted to Theoretical Foundations of Chemical Engineering.
202

3. Posada JA, Cardona CA. Comparison among three chemical processes for
glycerol conversion: Acroleine, Hydrogen and 1,2-Propanediol. Submitted to
Bioresource Technology.
4. Posada JA, Rincn LE, Cardona CA. Sustainable biodiesel production from oil
palm: crude glycerol and biomass conversion to raw-bioethanol. Submitted to
Applied Biochemistry and Biotechnology.
5. Posada JA, Quintero JA, Cardona CA. Comparacin econmica y ambiental

entre la produccin de etanol a partir caa de azcar, maz y glicerol crudo.
Submitted to Revista de Ingeniera Qumica-UIS.
6. Posada JA, Quintero JA, Cardona CA. Comparison among three technologies
for biodiesel production from Jatropha seeds. Under review by the advisor.
7. Posada JA, Cardona CA. Possibilities of glycerol conversion as a sole raw

material: a review. Under review by the advisor.
8. Posada JA, Cardona CA, Gonzalez R. Design, simulation, and economic

assessment of D-lactic acid production process from raw glycerol using
engineered Escherichia coli strains. Under review by the advisor.
9. Posada JA, Naranjo JM, Lpez JA, Higuita JC, Cardona CA. Poly(3hydroxybutyrate) production by Bacillus megaterium using glycerol as
substrate: experimentation and process simulation. Under review by the
advisor.
10. Posada JA, Cardona CA. Design, simulation, and economic assessment of
succinic acid production process from raw glycerol. Under review by the
advisor.
11. List of Publicatiions
203
11. Posada JA, Cardona CA. Design, simulation, and economic assessment of
propionic acid production process from raw glycerol. Under review by the
advisor.

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Design and Analysis of

Technological Schemes for Glycerol

John Alexander Posada Duque

Universidad Nacional de Colombia

Diseo y Evaluacin de los

John Alexander Posada Duque

Universidad Nacional de Colombia Sede Manizales

Design and Analysis of

John Alexander Posada Duque

Universidad Nacional de Colombia

Palabras clave: Conversin de glicerol, diseo de procesos, simulacin de procesos,

Glycerol Conversion to Added Value Products

assessment; glycerol-based biorefineries.

2. Chapter 2: The glycerols world

3. Chapter 3: Methodology for processes design and analysis

4. Chapter 4: Separation and purification of glycerol

Glycerol Conversion to Added Value Products

5. Chapter 5: Chemical conversion of glycerol

6. Chapter 6: Biochemical conversion of glycerol

7. Chapter 7: Study cases for chemical conversion of glycerol

8. Chapter8: Study cases for biochemical conversion of glycerol

9. Chapter 9: Experimental setup for glycerol fermentation to PHB

10. Chapter 10: Conclusions

11. Chapter 11: List of Publications

Figure 1.2. Global biodiesel production by feedstock

Figure 3.1. Hierarchical decomposition according to the "onion diagram"

Figure 4.1. Flowsheet of conventional schemes for glycerol purification

Figure 4.2. Flowsheet of the Ambersep BD50 process

Figure 4.3. Simplified flowsheet for raw glycerol purification

Figure 5.1. Possible products for glycerol oxidation

Figure 6.1. Schematic representation of glycerol degradation process on the part

Figure 6.2. Main metabolic pathways for fermentative degradation of glycerol

Figure 6.3. Pathways involved in the microaerobic utilization of glycerol in E. coli

Figure 6.4. Products that can be synthesized from succinic acid

Figure 6.5. Pathways involved in the micro aerobic utilization of glycerol

Figure 7.1. Simplified flowsheet for acrolein production by glycerol dehydration

Figure 7.2. Simplified flowsheet for hydrogen production by gasification

Figure 7.3. Simplified flowsheet for 1,2-propanediol production by hydrogenolysis

Figure 8.1. Hysteresis loops and multiple steady states

Figure 8.3. 1,3-Propanediol productivity and concentration in the second

Figure 8.6. Acetylation reaction of 1,3-propanediol with iso-butyl aldehyde to

Figure 8.8. Residue map curves for the reactive system

Figure 8.9. Direct separation with fed 0.377645/0.6223552iP13DO/Water

Figure 8.10. P/W ratio, Direct Separation (XF: 0.377645/0.622355-2iP13DO/water)

Figure 8.11. iso-Volatility curve (Wateriso-Butyraldehyde2-iso-Propil-1,3-Dioxane) 101

Figure 8.16. Flowsheets for PHB production from glycerol (88 or 98 wt %)

Figure 8.18. Complex formed during the reactive extraction process of

Glycerol Conversion to Added Value Products

Figure 9.1. Accumulation profile I of Bacillus megaterium

Figure 9.2. Accumulation profile II of Bacillus megaterium

Figure 9.3. Accumulation profile III of Bacillus megaterium

Figure 9.4. Accumulation profile IV of Bacillus megaterium

Table 4.1. Quality specifications for the main qualities of glycerol

Table 4.2. Fatty acid profile of vegetable and used oils

Table 4.3. Composition of the glycerol layer obtained by decantation during

Table 4.4. Simulation results for raw glycerol purification process

Table 4.5. Purification costs (PC) of raw glycerol (US$/L)

Table 7.1. Simulation results for dehydration process from glycerol

Table 7.2. Simulation results for gasification process from glycerol

Table 7.3. Simulation results for hydrogenolysis process from glycerol

Table 7.4. Production costs for glycerol conversion to added-value

Table 7.5. Percentage of Production costs for glycerol conversion to added-value

Table 8.1. Results summary for each optimization model