The XXI century has brought industrial development and also an environmental

problem of great importance, leaving behind depletion of renewable and nonrenewable resources, pollution of water bodies and soil, climate change, among
others. However, although there are different rules governing industrial
processes, environmental awareness as a society just beginning to be born.
Therefore, it is necessary to develop new methods from friendly methods with
the environment in search of the planet's sustainability.

Internationally an alternative which we can speak from the industrial field, is

the implementation and development of bioprocesses, which are mainly based
on the reuse of waste or industrial by-products and the use of biomass as
feedstock for generation product of industrial importance.

Succinic acid is an organic acid and is synthesized as a metabolic product of

the tricarboxylic acid cycle (TCA) in plants, animals and microorganisms [10].
However, for industrial use in foods, pharmaceuticals, solvents, surfactants,
and others, is mainly generated from the chemical synthesis of petroleum, this
being a non-renewable resource [11] [2] [12]. Therefore the development of
sustainable solutions based on renewable resources for the production of
succinic acid has attracted attention in recent years, using microorganism as
Actinobacillus succinogenes, Anaerobiospirillum succiniciproducens,
Mannheimia succiniciproducens, and Escherichia coli exploiting sources of
carbon sugars such as lactose, xylose, arabinose, cellobiose, among others;
that cost has led to evaluate other sources such as corn fiber, rice husks, cane
molasses, stalk crops, crop waste, and others. [13].

One last recently evaluated carbon source, abundant and inexpensive is

glycerol (according to Oleoflores S.A.S, located in Codazzi, Cesar ton of crude
glycerol has an approximate cost per tonne of $ 83 USD). It is noteworthy that
glycerol is a byproduct of the biodiesel industry which is obtained from
vegetable oils or animal fats by a simple chemical process called
transesterification, having a ratio 10 to 1, that is, for every 10 tons 1 ton
biodiesel glycerol byproduct is generated [7]. According to current trends,
biodiesel production will continue to increase in the coming years and this will
result in a great deal of glycerol, the glycerol current markets can not absorb.
Therefore, the use of innovative technologies glycerol, given commercial value
of this product and create new markets for crude glycerine excess [14].

4.1 Bio-Based production of Organics Acids.

For some years it is well known that bacteria, yeasts and filamentous fungi are
candidates for the production of organic acid through metabolic engineering.
Bacteria such as E. coli hosts industrial production are well established [5]
taking into account growing fast, their genetic background are clear, and many
viable genetic tools have been developed. It is also known that organic acids
obtainable from the enzymatic hydrolysis of glycerol, particularly succinic acid,
fumaric and itaconic; and to achieve this, the microorganisms use various
metabolic pathways dependent enzymatic machinery we have, growth
conditions and certain factors such as temperature and pH.

Yin et al [5], conducted a study comparing different routes of synthesis of

organic acids. They also summarized metabolic engineering strategies as a tool
for bacteria, yeast and filamentous fungi improve performance, suggesting the
application of these methodologies associated computational biology. Some of
these applications are:

Deletion or gene to produce citric acid lipolytica Yarrowia: Deletion of the gene
encoding the trehalose-6-phosphate synthase A decreased the level of
trehalose-6-phosphate, an inhibitor of hexokinase. As a result, accumulation CA
started before.

or for the production of succinic acid a strain of Corynebacterium glutamicum

recombinant was constructed to express PYC was used. The LDHA gene
encoding lactate dehydrogenase, was also eliminated in the strain. Production
by this strain SA reached 146 g / L in less than 46 h with a yield of 1.4 mol
glucose / mol.

The TCA cycle or was introduced into S. cerevisiae by overexpression genes

encoding MDH and R. oryzae FUM and endogenous of PYC. Compared with
control, the recombinant strain expressing MDH and PYC produced a greater
degree of FA, but production was low: 3.18 g / L, due to the imbalance of power

Moreover Binns et al [6], conducted a study of analysis and control of metabolic

networks for the production of SA where they found that the step that has the
greatest positive effect on the production of SA is the uptake of glycerol, and
the enzymes malate to SA, and from pyruvate to malate are important steps
with positive control.

In a study by Huang et al [4] which was used to obtain a strain FA wild1.22 R.

oryzae derived oryzae ATCC 20344 R. and grown on potato dextrose agar

plates at 35 C for 7 days. It showed that the adaptability or evolutionary

engineering is a viable option for use glycerol as a carbon source in the
synthesis of organic acids tool.

Vlysidis, Binns, Webb, & Theodoropoulos [15] conducted a study using glycerol
as a carbon source for the microorganism Actinobacillus succinogenes, where
higher performance product obtained final succinate concentration and
productivity were equal: 1.23 succinate g / g glycerol, 29.3 g succinate / L and
0.27 g succinate / L / h, respectively (table 1). Also they carried out a
comparison of different studies to obtain succinic acid (Table 2).

Table 1. Experimental results for different initial conditions. From [16]

Table 2. Comparison of different studies to Obtain succinic acid from glycerol.

From [16]

4.2 Commercial Potential of Succinic Acid.

The biobased succinic acid is a versatile product with a huge commercial

potential in the market of intermediate chemicals. It can be used in various
applications, such as biopolymers (for example, polybutylene succinate or
PBS), polyurethanes, coatings and products used in biotechnology.

The global market for succinic acid in terms of revenue was estimated at a
value of $ 182.8 million in 2010 and is expected to reach $ 496.0 million in
2016, growing at a CAGR of 18.7% from 2011 to 2016. Europe and North
America dominates the world market for succinic acid, accounting for 66.0% of
global demand for succinic acid in 2010. it is expected that Asia and the Pacific
are the fastest growing market in the near future, due to the rapid
advancement and increasing demand from key countries such as China and
India [17]. Moreover, according Bioplastics [18] "It is expected that demand for
succinic acid to increase in the coming years, primarily marked by the
production of bioplastics ... Growth will be marked by the biobased PET, which
today represents 40% of bioplastics capacity, and 2016 represent 4.6 million
tons, ie, about 80% of production capacity "on the other hand the production of
plastic items in the country reaches a value of 4,000 million dollars (7.18 billion
pesos), according to the Colombian Association of Plastic Industries

(Acoplsticos). Being the PBS (Polybutylene succinate) or PET biobased a

perfect choice for insertion bioplastics market taking into account that is
because it is friendly to the environment to be biologically based.

Debabov [19] mentions:

"Succinic acid (butanedioic acid or 1,2 ethane dicarboxylic acid) is a dibasic
carboxylic acid. Was obtained (until 2009) on an industrial scale raw
petrochemicals, especially from maleic anhydride (Fig. 1) ... Microbiological
industrial production of succinic acid began in 2009, and has continued to grow
rapidly since then, while the chemical production remains at the same level
(Table 3) "

Table 3. World production of Succinic Acid. Kt / year. From [19]

Industrial Production Years
2008 2010 2013 2014
Total production 25 30 60 81
25 25.5 25 Chemical Synthesis 25
microbial synthesis 0 4.5 35 56
58.3% of production 15 69

Figure 1. Succinic acid production by simultaneous acidification and purification

by electro dialysis and crystallization. CSL: corn steep liquor, ED:
electrodialized, CIP: cleaning-in-place. From [11]

Purification of Succinic Acid 4.3.

Production of organic acids from microorganisms requires culture media

containing not only carbon source will be converted into the final product, but
also some other elements such as salts and nitrogen in the purification process
adversely affect the process separation of succinic acid. This process
represents 60-70% of the production costs in the process. [10] [20]

Some authors have economically evaluated the economic recovery of succinic

acid, and have reported several possible, such as precipitation, distillation,
electrodialysis and extraction (Table 3) [14] alternatives. Generally the recovery
method is based on succinic acid precipitation and crystallization technology,
but this process is very expensive.

Table 4. Comparison of different studies to purification of succinic acid

.
Author Description Ref
(Huh et al., 2006) Developed a new process That consisted of a reactive
extraction by pretreatment and a stage of vacuum distillation, later to
crystallization, This process allowed highly purified succinic acid with a 99.8%
purity and 73.1% return rate. [10]

(Pratiwi, Yokouchi, Matsumoto, & Kondo, 2015) Discussed a system of two

aqueous phases (ATPS) using Ionic Liquid and water-miscible alcohols / sales /
sales With the aim of extracting succinic acid is Reached Where results of up to
85.5% Extracted succinic acid and later recovered by crystallization. [twenty]

(Song & Lee, 2006) We EVALUATED the process of purification of succinic acid
by electro conventional dialysis followed by division of water of electro dialysis
membrane stacks, That Eliminates MOST of the cation salt and produces highpurity acid stream esta process Increases the concentration of succinic acid
from 51.5% to 79.6% and removes proteins and salts, acetic acid concentration
from 13.2% Increased to 19.9%. [14]

(Wang et al., 2014) In This study was integrated fermentation and separation
by membrane ultrafiltration. The overall procedure consisted of ferment, ultrafilter, absorbing, distill and crystallize.

Succinic acid of high purity (99.4%) Through This process of separation with a
recovery rate of 90% was Obtained. [twenty-one]

(Sosa, rock, & Velizarov, 2016) Three membrane assisted technologies Were
EVALUATED: electro dialysis, Donna Nano filtration and dialysis. All Processes
are above 90% rejections for succinate. I was to concentrate rich in ionic
species, treats including succinate, but leaving behind non-ionic and high
molecular mass species. Then was Treated by Nano filtration, THUS Achieving a

complete disposal almost Formate and acetate. Finally, Donnan dialysis was
used to replace the metal cations in salts of succinate in the Nano filtration
retained by H +. The results Showed approximately 76% of the removal of
cations, Which was maligned in the same percentage of succinic acid Obtained
free. [22]

(Lubsungneon, Srisuno, Rodtong, & Boontawan, 2014) We EVALUATED an

integrated membrane filtration procedure Consisting of Nano (NF) and the
permeation of steam (VP). The NF Showed ITS utility for protein and color
rather than the Elimination of Separation Between organic acids. Performance
of diethyl succinate (DES) was the initial relationship of reactive function while
operating temperature played an Important role in productivity. The
experimental results Showed That Became most of it succinic acid in DES at
the end of the esterification reaction With help of VP. After fractionation and
hydrolysis, high purity of succinic acid was Obtained. [2. 3]

(Efe, Pieterse, van der Wielen, & Straathof, 2011)

The separation of Its salts of succinic acid was EVALUATED through hydrophobic
zeolite sorbent adsorption columns. [24]

1. Methodology

5.1 Selection of the Microorganism.

E. coli KJ122 has been reported in the literature as a microorganism capable of

producing succinic acid from glucose [25] This microorganism was genetically
modified with the aim of directing the metabolic pathway for the production of
succinic acid as can be evidenced in the figure 2 and 3.

Figure 2. General Route anaerobic metabolism of E. coli AS1600a. The crosses

symbolize the deletions. The crosses in boxes symbolize key Deletions. From
[25]

Figure 3. Expanded portion of metabolism of E. coli AS1600a. The crosses

symbolize additional genes deleted. From [25]

5.2 Initial growth of E. coli AS1600a from glycerol.

E. coli was grown initially in LB medium AS1600a in a pre inoculum for an

overnight. Subsequently a growth curve was performed in order to compare
them with the growth in LB medium with glycerol as shown in Table 5. The
cultures containing glycerol were grown for three generations (each generation
represents a culture passage for 48 hours , 250rpm, 37 C) to make suits and
reduce the time lag phase.

Table 5. Concentrations of components of the culture medium.

ID Tryptone (g / L) NaCl (g / L) Yeast Extract (g / L) Glycerol USP
(G / L)
Initial 10 10 5 0
G1 10 10 5 October
G2 10 10 5 20
G3 10 10 5 30
G4 10 10 5 40
G4 10 10 5 50
G6 10 10 5 60

* All cultures contained also 50g / ml ampicillin and 34 g / mL

chloramphenicol.

Because glycerol is a viscous substance soluble in water equivalency in grams

we took considering the following formula:

M=D*V

Example:

G1 = 1.25 * 0,8mL = 1g

Where the density of glycerin is added 1.25 and volume for each 100 ml was
0.8, 1.6, 2.4, 3.2, 4.0 and 4.8 ml respectively.