Biodiesel biorefinery: opportunities and challenges for microbial production of fuels and chemicals from glycerol waste
Biotechnology for Biofuels
Biodiesel biorefinery: opportunities and challenges for microbial production of fuels and chemicals from glycerol waste
Joo R M Almeida 0
Lia C L Fvaro 0
Betania F Quirino 0
0 Embrapa-Agroenergy , Parque Estacao Biologica S/N, Av. W3 Norte (final), 70770-901, Brasilia, DF , Brazil
The considerable increase in biodiesel production worldwide in the last 5 years resulted in a stoichiometric increased coproduction of crude glycerol. As an excess of crude glycerol has been produced, its value on market was reduced and it is becoming a waste-stream instead of a valuable coproduct. The development of biorefineries, i.e. production of chemicals and power integrated with conversion processes of biomass into biofuels, has been singled out as a way to achieve economically viable production chains, valorize residues and coproducts, and reduce industrial waste disposal. In this sense, several alternatives aimed at the use of crude glycerol to produce fuels and chemicals by microbial fermentation have been evaluated. This review summarizes different strategies employed to produce biofuels and chemicals (1,3-propanediol, 2,3-butanediol, ethanol, n-butanol, organic acids, polyols and others) by microbial fermentation of glycerol. Initially, the industrial use of each chemical is briefly presented; then we systematically summarize and discuss the different strategies to produce each chemical, including selection and genetic engineering of producers, and optimization of process conditions to improve yield and productivity. Finally, the impact of the developments obtained until now are placed in perspective and opportunities and challenges for using crude glycerol to the development of biodiesel-based biorefineries are considered. In conclusion, the microbial fermentation of glycerol represents a remarkable alternative to add value to the biodiesel production chain helping the development of biorefineries, which will allow this biofuel to be more competitive.
Glycerol; Fermentation; Biofuels; Metabolic engineering; Biodiesel
Production of biofuels and chemicals from renewable
feedstocks is necessary to meet the energy demand in a
world where petrol fuels are becoming scarce and more
expensive. One of the main problems associated with
biofuels is still the production costs, which can be
reduced if residues of biofuels production processes are
converted into valuable coproducts [1,2]. Biodiesel is an
alternative fuel that reduces net greenhouse effects and
its use has become mandatory in many countries . It
is mainly obtained by the transesterification of fat and
vegetable oils in the presence of a catalyst by a primary
alcohol (usually methanol) leading to a fatty acid methyl
ester (FAME), which is used as a biofuel. Sunflower,
rape, soybean and palm oils are the main substrates to
make biodiesel worldwide, however, there are local
variations on which is the main source. In Brazil, for
example, 80% of the biodiesel produced in 2010 was from
soybean oil .
Biodiesel production increased considerably in the past
few years and so did the amount of residues generated
during its production (Figure 1A). Europe is still the
biggest biodiesel producer, whereas Brazil had the highest
increase in production rate in the last years when
compared with United States and Europe, i.e. from 736 in
2005 to 2,400,000 m3 in 2010 (Figure 1B). Production of
the two main types of residues, pies and crude glycerol,
is increasing concurrently with the biodiesel industry.
Pies, which are produced by pressing of palms, seeds
and others for oil extraction, are usually used as feed for
Figure 1 A: World biodiesel (bars) and crude glycerol (lines) production between 2005 and 2010. Biodiesel production was grouped by
continents; whereas crude glycerol represents the total production in the world over the years. B: Top ten biodiesel producing countries in 2010.
Their production corresponds to approximately 71.3% of the total 19.21 million cubic meters of biodiesel. Production percentage is shown for
each country. The production of crude glycerol was estimated assuming 0.106 L of crude glycerol per liter of biodiesel. The above figures were
derived from an interactive table generated on January 11, 2012 from U.S. Energy Information Administration, International Energy Statistics,
Biofuels Production (http://www.eia.gov/cfapps/ipdbproject/iedindex3.cfm?tid=79&pid=81&aid=1&cid=regions,
animals or as fertilizers, consequently adding value to
the biodiesel production chain. Crude glycerol, which is
derived from the transesterification reaction of fat and
vegetable oils (triglycerides) to produce biodiesel,
contains methanol, salts, soaps and water as the main
contaminants. Concentration and presence of each
contaminant will vary drastically from one industry to
another, due to a variety of parameters, including oil
source and reaction conditions. For instance, glycerol
and water content can vary, respectively, from 92% and
6%  to 65% and 26%  in crude glycerol samples.
The presence of these impurities in crude glycerol
samples is expected to influence negatively the
bioconversion process of this coproduct. However, it is important
to note that the excess of crude glycerol produced in the
biodiesel industry is leading to a decrease in glycerol
prices and glycerol is now considered a waste instead of
a coproduct . The production of crude glycerol
follows the increasing biodiesel production, since the
stoichiometry of the reaction dictates that for each 10 tons
of FAME, 1 ton of crude glycerol is formed (Figure 1).
Thus, the development of biorefineries based on crude
glycerol is expected to favor the biodiesel industry
economy, by reducing costs associated with the disposal of
residues and increasing production of value-added
In this review, we discuss the strategies to produce
fuels and chemicals of biotechnological interest by
microbial fermentation of glycerol. We highlight naturally
occurring and engineered bacteria, yeast and filamentous
fungi able to produce specific chemicals, as well as the
strategies to improve their performance during
fermentation. Market and industrial applications of these
microbial fermentation products are also discussed.
Microbial fermentation of glycerol
The excess of waste glycerol produced in the
biodiesel industry (Figure 1) may be used in
biotechnological processes to produce value-added chemicals to
avoid waste disposal and increase process economy.
Due to the reduced nature of the glycerol molecule,
microorganisms are able to convert it to a series of
metabolites (Figure 2), with yields similar to the ones
obtained when using sugars as substrates . Thus,
the valorization of the glycerol waste stream through
the production of microbial value-added metabolites
via fermentative processes has been substantially
evaluated. Yeasts and filamentous fungi have been tested
mainly aerobically for the production of organic acids
and polyols (Figure 2). On the other hand, production
of metabolites by bacteria, especially from the
Enterobacteriaceae and Clostridiaceae families, such as
Klebsiella, Enterobacter, Clostridium, has been tested
under anaerobic conditions. These bacteria have been
evaluated for the production of different chemicals,
including the alcohols 1,3-propanodiol, 2,3-butanediol,
butanol, and others (Figure 2). Engineered E. coli
strains have been used especially under microaerobic
conditions for the production of several chemicals.
Biotechnological applications of these metabolites and
how their production by microbial fermentation of
glycerol has been optimized by metabolic engineering
and fermentation strategies with different producing
strains are summarized and discussed below.
Figure 2 Examples of chemicals produced by microbial
fermentation of crude glycerol. Circles/positions indicate the
aerobiose conditions in which these chemicals can be produced by
microbial fermentation and the main microbial producing groups.
The most studied route of biotechnological valorization
of glycerol is related to its conversion to
1,3-propanediol (1,3-PDO). However, commercial production of
1,3-PDO from glycerol has not been reported. 1,3-PDO
can be utilized for the synthesis of the modern polymer
polytrimethylene terephthalate (PTT) that in turn can
be used to make carpets (CorterraW, Shell), special
textile fibers (SoronaW, DuPont), monofilaments, films,
and nonwoven fabrics. PTT is also used in the
engineering thermoplastics area . The demand of
polybutylene terephthalate (PBT), another potential 1,3-PDO
derivative, which is mainly consumed in the automotive
and electronic sectors, in 2005 was estimated at about
376 000 tons with an average annual growth rate of 5
PDO can be successfully produced fermentatively
from glycerol by bacteria of the Enterobacteriaceae
and Clostridiaceae families, mainly Klebsiella spp. and
Clostridium spp., respectively [2,10,11]. Two pathways
are necessary for the conversion of glycerol to
1,3PDO under anaerobic conditions by Enterobacteriaceae
(Figure 3). In the oxidative pathway, glycerol is
dehydrogenated by a NAD-dependent glycerol
dehydrogenase (GLY-Dhd) to dihydroxyacetone (DHA), which is
then phosphorylated by phosphoenolpyruvate (PEP)
and ATP-dependent DHA kinase (DHA-Kin). In the
parallel reductive pathway, glycerol is dehydrated by
the coenzyme B12-dependent glycerol dehydratase
(GLY-Dht) to 3-hydroxypropionaldehyde (3HPA), which
Figure 3 Different metabolic pathways for metabolism of glycerol. Production of 1,3-PDO by Enterobacteriaceae family members is shown
in red. DHA production by G. oxydans is shown in blue. Ethanol and formate production pathways in the fermentative utilization of glycerol by E.
coli are shown in gray. Proposed pathway for the conversion of glycerol to glyceric acid is shown in green. Dashed lines indicate multiple steps
or unknown enzyme(s) (GA pathway). Main products are highlighted in color- filled boxes. Abbreviations: GLY, glycerol; GLY-Dhd, GLY
dehydrogenase; DHA, dihydroxyacetone; DHA-Kin, DHA kinase; DHAP, DHA phosphate; PYR, pyruvate; GLY-Dht, GLY dehydratase; 3HPA,
3hydroxyapropionaldehyde; 1,3-PDO-Dhd, 1,3-PDO dehydrogenase; GLY-DhdE, membrane-bound GLY-Dhd; GLY-Kin, GLY kinase, G3P, glycerol
3phosphate, G3P-Dhd, G3P dehydrogenase; PPP, pentose phosphate pathway; PFL, pyruvate formate-lyase; mAdh: membrane-bound alcohol
in turn is reduced to the major product 1,3-PDO by
the NADH-dependent 1,3-PDO dehydrogenase
(1,3PDO-Dhd), thereby regenerating NAD+ (Figure 3)(as
reviewed in [2,10,11]).
Crude glycerol consumption and 1,3-PDO production
are influenced by the purity and concentration of the
glycerol, as well as by fermentation conditions [6,12,13].
For instance, higher glycerol concentrations and
microaerobic conditions increases the substrate consumption
and 1,3-PDO productivity, however, without affecting
fermentation yield [6,13]. It was also shown that addition
of fumarate at low concentrations (i.e., 5 mM, equivalent
to 2.9 g/L) during glycerol fermentation increased in
35% glycerol consumption and 1,3-PDO production
rates without affecting product yield . Several genetic
engineering strategies have been employed to produce
1,3-PDO in both native and non-native microbial
producers (reviewed in ). Regarding native producers,
mainly K. pneumoniae, increased 1,3-PDO production
has been attempted by overexpression of genes directly
involved in the 1,3-PDO biosynthesis pathway and
inactivation of genes involved in byproduct formation.
Regarding non-native producers, a 1,3-PDO biosynthesis
pathway has been constructed in bacteria and yeasts that
are not naturally able to produce 1,3-PDO by
heterologous overexpression of genes from natural producers
[10,15,16]. Among the engineered strains, 1,3-PDO
production from glycerol using K. pneumoniae and
E. coli strains looks the most promising, as shown by the
high 1,3-PDO titers obtained (Table 1).
2,3-Butanediol (BDO) can be employed in many
chemical syntheses. For instance, it can be used to produce
plastics, anti-freeze solutions and solvent preparations.
In addition, it can be converted to methyl ethyl ketone
(a liquid fuel additive), 1,3-butadiene (used to produce
synthetic rubber), diacetyl (a flavoring agent), or to
precursors of polyurethane (used in the pharmaceutical and
cosmetics industries) [36,37]. BDO is chemically
obtained from petroleum; however, the development of a
microbial production route based on renewable
feedstocks is of interest. Fermentation of glycerol by several
strains of Klebsiella spp. [11,12,18], resulted in BDO
production. However, the main product of such
fermentations is 1,3-PDO, whereas BDO is a minor product
along with acetate, lactate, succinate, and ethanol.
Recently, a study demonstrated that BDO can be
obtained as a major product of glycerol fermentation by
K. pneumoniae G31 . Strain specificity and culture
pH were the main factors influencing BDO production.
Indeed, alternate production of BDO and acetic acid, the
second most produced metabolite by K. pneumoniae
G31 were demonstrated to be associated with pH
K. pneumoniae DSM 2026
K. pneumoniae LDH 526
G. frateurii NBRC103465
A. tropicalis NBRC16470
Y. lipolytica Wratislavia K1
Z. denitrificans MW1 Fed-batch Aerobic 0.25 g/g 1.09 g/L/h 54.3 g/L 
a calculated from the data presented; b Percentage of theorethycal maximum; c 72% D-GA enantiomeric excess (ee); d 99% D-GA enantiomeric excess (ee); n.d. not
control; i. e. during fermentation the microorganism
alternates production of BDO and acetic acid to adjust
pH of the cultures. In addition, BDO production was
influenced by medium composition and aeration. When
medium composition, aeration regime, and the initial
pH of the fermentation were optimized, the production
of BDO reached 70 g/L, the highest concentration
obtained from glycerol, with a maximum yield of 0.39 g/g
glycerol (Table 1) .
Ethanol is mainly used as fuel for transportation, but it
can also be utilized in industry as a solvent and chemical
intermediate. World fuel ethanol production, mainly
based on yeast fermentation of sugarcane sucrose and
corn starch, has reached 114 millions of cubic meters in
2011 . In addition, the development of technologies
for ethanol production based on lignocellulosic
feedstocks has been evaluated intensively [39,40] and should
also contribute to the increasing demand for this fuel.
On the other hand, ethanol production from glycerol
has gained modest attention in the last years.
Bacteria of the Enterobacteriaceae family and of the
Clostridium genus are able to convert glycerol to
ethanol, however, yields are relatively low because ethanol is
only a secondary product of the fermentation [18,19,41].
In these cases, the main product of fermentation is
1,3PDO [18,41] or 2,3-butanediol . For instance, during
pilot scale production of 1,3-PDO, concentrations of
ethanol from glycerol varied from 8 to 17 g/L during
fed-batch fermentation of K. pneumoniae [18,41].
Recently it was demonstrated that E. coli can convert
glycerol to ethanol anaerobically , as well as
aerobically  (Figure 3). Initially, it was demonstrated that
E. coli is able to ferment glycerol anaerobically in a
pHdependent manner, which is linked to CO2 availability.
Glycerol fermentation proceeds under acidic conditions
(pH6), however it is impaired under alkaline conditions
(pH8) because CO2 availability is reduced (i.e., most
CO2 is converted to bicarbonate) . Afterwards,
several fold improvements in the production of ethanol and
also of the coproducts hydrogen and formate was
achieved by genetic engineering . Byproduct
formation was reduced in the strains producing
ethanolhydrogen and ethanol-formate by mutations that
inactivated fumarate reductase (DfrdA) and phosphate
acetyltransferase (Dpta). To prevent the conversion of formate
to CO2 and H2, the strain producing ethanolformate
also contained a mutation that inactivated formate
hydrogen lyase (DfdhF). Finally, high rates of glycerol
utilization and product synthesis were obtained by
simultaneous overexpression of glycerol dehydrogenase
(gldA) and dihydroxyacetone kinase (dhaKLM). The
production of ethanol from glycerol attained a yield of 85%
of the theoretical maximum (the maximum theoretical
yield is 1 mol each of H2 and ethanol per mol of glycerol
fermented) in both strains . Fermentation
performances of these strains were further improved by
elimination of lactate production by deletion of ldhA, which
encodes a lactate dehydrogenase, followed by
fermentation under microaerobic conditions . Conversion
rates of glycerol to ethanol as high as 85% of the
theoretical maximum in mineral medium were obtained
(Table 1). These results highlight the potential for using
E. coli as a host for the production of ethanol from
Production of ethanol from glycerol by the
methylotrophic yeast Hansenula polymorpha was recently
evaluated . A recombinant H. polymorpha yeast strain
expressing the pdc and adhB genes, which encode,
respectively, pyruvate decarboxylase and aldehyde
dehydrogenase II, from Zymomonas mobilis exhibited a
3.3-fold increase in ethanol yield from glycerol
fermentation under microaerobic conditions. However, the final
concentration of ethanol obtained was still very low, i.e.
2.74 g/L .
Butanol has been identified as an alternative fuel and as
a key chemical platform that can be industrially
converted to acrylates, ethers, and butyl acetate, which
in turn are utilized in paints, lacquers, and resin
formulations . Butanol has been produced by Clostridium
spp. fermentation of sugar for years. Clostridium
pasteurianum can produce 1,3-PDO and butanol
through anaerobic fermentation of glycerol .
However, only recently the production of butanol from crude
glycerol generated during biodiesel production was
evaluated . C. pasteurianum was shown to produce
butanol when grown in crude glycerol, although butanol
yields and productivity on this substrate was
considerably lower than on glycerol. Indeed, yields were
approximately 20% higher on glycerol than in crude glycerol
(from 0.36 g/g to 0.30 g/g (g butanol/glycerol
consumed), whereas fermentation time was 2.5 times longer
(i.e., 25 days instead of 10 days) on crude glycerol. The
reduced yield and productivity on crude glycerol may be
caused by contaminants in the crude glycerol, which
contained approximately 9095% glycerol, 510%
methanol and/or water, and 35% sodium sulfate .
These results indicate that C. pasteurianum may be used
to produce butanol from biodiesel-derived crude glycerol
to produce butanol, but its immediate industrial
applicability is limited because of its slow growth and low
Ketone and organic acids
Another important chemical produced from glycerol
fermentation is dihydroxyacetone (DHA), the main active
ingredient in all sunless tanning skincare products
[47,48]. DHA also serves as a versatile building block for
the organic synthesis of a variety of fine chemicals. It is
produced through oxidative fermentation by
Gluconobacter oxydans via a membrane-bound glycerol
dehydrogenase (GLY-DhdE) (Figure 3) [47,48]. This appears to
be the only reaction responsible for DHA synthesis and
employs oxygen as the final acceptor of reduced
equivalents, without NADH involvement. While DHA is
produced by a GLY-DhdE, growth of G. oxydans on glycerol
is ensured by a cytoplasmic pathway. In this pathway
glycerol is phosphorylated to glycerol-3-phosphate (G3P)
and then dehydrogenated to DHAP by glycerol kinase
(GLY-Kin) and G3P-Dehydrogenase (G3P-Dhd),
respectively. Finally, DHAP is catabolized in the pentose
phosphate pathway (PPP) (Figure 3).
G. oxydans fermentation of glycerol for the production
of dihydroxyacetone (DHA) is a process used in
industry, however, there are problems related to microbial
DHA production . For example, increased
concentrations of glycerol inhibit DHA production and
bacterial growth. In addition, production of glyceric acid (GA)
by membrane-bound alcohol dehydrogenases (adhA)
reduces production of DHA. To overcome these
problems, a strain with disrupted AdhA with improved
ability to grow in a higher concentration of glycerol
(220 g/L) and to produce DHA compared to a wild-type
strain (G. oxydans NBRC 12528) was generated.
While the wild-type strain produced 38 g/L of DHA and
47 g/L GA when cultivated in 150 g/L of glycerol, the
AdhA-disrupted strain produced only 2.2 g/L of GA and
increased production of DHA to 108 g/L . Recently,
an independent study demonstrated that optimization of
growth medium and fermentation conditions to increase
glycerol conversion to DHA by G. oxydans ZJB09112
lead to the production 161.9 g/L of DHA at a conversion
rate of 88.7% in a fed-batch process (Table 1) .
While glyceric acid (GA) coproduction in the DHA
production process is a problem, GA production by itself is
of commercial interest. GA may be used in the chemical
and pharmaceutical industries as a building block and
for the production of polymers and surfactants .
However, GA is not bulk produced, probably because a
sizable number of applications for this chemical have
not yet been developed. Because the GA molecule has
three functional groups, it has a huge potential as a
chemical that will add value to glycerol.
Like DHA, GA is mainly biotechnologically produced
by bacteria, more specifically by the family of acetic
acid bacteria (Acetobacteraceae), such as Gluconobacter
sp., Acetobacter sp., and Gluconacetobacter sp. A
membrane-bound Adh was shown to be involved in
GA production by acetic acid bacteria (Figure 3) .
Recently, the ability of 162 acetic acid bacterial strains
to produce glyceric acid was evaluated regarding
productivity and enantiomeric composition of the product
. Productivity of glyceric acid varied from less than
10 g/L up to 40 g/L, whereas enantiomeric purity
varied from less than 70% up to 99%. After optimization
of glycerol concentration and aeration, two selected
strains, Gluconobacter frateurii NBRC103465 and
Acetobacter tropicalis NBRC16470, were able to
produce more than 100 g/L of glyceric acid in fed-batch
fermentation. G. frateurii NBRC103465 produced
136.5 g/L of glyceric acid with a 72% D-GA
enantiomeric excess (ee), with yields of GA and DHA of
0.58 mol/mol and 0.12 mol/mol glycerol, respectively.
On the other hand, A. tropicalis NBRC16470 produced
101.8 g/L of glyceric acid with 99% of enantiomeric
excess (Table 1) . Identification of pathways
responsible for the conversion of glycerol in these
microorganisms is expected to allow the use of
metabolic engineering strategies to reduce byproduct
formation and lead to an industrial producer strain [24,51].
Lactic acid has been used in the food industry for several
years but has many other applications. Lactic acid can
be processed to make acrylic acid or 1,2 propanediol
used in polyester resins and polyurethane used as deicer
or antifreeze . Lactate esters are used as green
solvents for coating and in the cleaning industry, it can also
be polymerized into the biobased polymer PLA
(polylactic acid). As lactic acid became an important building
block in the chemical industry, its microbial production
has advanced significantly. Indeed, the availability of
cheap and abundant residues from the biofuels industry
is leading to a shift from chemical to microbial
production of lactic acid.
Lactic acid bacteria fermentation processes in the food
industry for production of lactic acid has a long history.
Current processes for D-lactate production based on
native lactic acid bacteria use sugars as carbon source and
may also be applicable to lignocellulosic feedstocks
[53,54]. However, the utilization of lactic acid bacteria in
industrial processes requires complex nutrients for cell
growth and may not result in high product selectivity
and enantiomeric purity .
Alternatively, lactic acid can also be produced from
glycerol by other naturally producing microorganisms,
including E. coli, Klebsiella, Clostridia, Bacillus and the
filamentous fungi Rhizopus oryzae [46,56-59], although
at very low concentrations and productivity. To
overcome these issues, new strains have been screened and
natural producers have been metabolically engineered to
increase yield and production rate. In a screening of soil
bacteria, the strain E. coli AC-521 was isolated based on
its ability to use glycerol as carbon source and to grow
quickly under aerobic conditions. This strain was able to
produce lactic acid with a productivity of 0.49 g/g/h and
a yield of 0.9 mol/mol glycerol (Table 1) .
Only recently E. coli has been engineered for
homofermentative production of D-lactate from glycerol .
Several enzymes leading to lactic acid production were
overexpressed, while pathways leading to production of
ethanol, succinate and acetate were blocked. The
engineered E. coli strain was able to produce optically pure
(99.9%) D-lactic acid from glycerol in minimal salts
medium with only few supplements. In addition, this
strain produced 32 g/L of D-lactate from 40 g/L of
glycerol at a yield of 0.83 g/g glycerol, and with specific
productivity for D-lactate production of 1.25 g/g cell
Succinic acid is largely used for manufacturing
healthrelated products, including pharmaceuticals, antibiotics,
amino acids, and vitamins. In addition, it is an important
building-block chemical that could be used to produce
important precursors for chemical synthesis such as
tetrahydrofuran, g-butyrolactone, 1,4-diaminobutane,
1,4-butanediol that are converted into a wide variety of
products, including green solvents, pharmaceutical
products, and biodegradable plastics . Natural
succinate-producing rumen bacteria, such as
Anaerobiospirillum succiniciproducens, can produce succinate
from glycerol . But these bacterial strains require
complex nutrients that increase production costs,
purification, waste treatment and, consequently, hinder their
utilization in an industrial process. Alternatively, the
fermentative production of succinic acid from glycerol has
been evaluated using either metabolically engineered
Escherichia coli [27,61,62] or yeast strains (Table 1)
Different strategies have been utilized to construct
metabolically engineered E. coli strains for production of
succinic acid from glycerol [27,61,62]. In a recent study,
a strain was constructed in two steps and the use of a
heterologous enzyme was needed . Initially,
byproducts formation was avoided by blocking pathways
leading to ethanol, acetic and lactic acid. Then, Lactococcus
lactis pyruvate carboxylase (pyc) was expressed in this
strain to drive the generation of succinate from the
pyruvate node. The resulting strain was able to
convert glycerol to succinic acid with a specific
productivity of ~ 4 g/g/h and a yield of 0.69 g succinate/g
glycerol, which is similar to yields obtained with
glucose (0.78 g/g). However, fermentation under
microaerobic conditions was necessary to overcome the
need for rich nutrients and keep a net production of
ATP. On the other hand, the use of microaerobic
conditions may be a problem because it will lead to
glycerol loss in form of CO2 .
In a concurrent study, E. coli was engineered to
produce succinate from glycerol using only native genes
. Production of succinate was increased in 3 steps: i)
coupling energy generation, i.e. ATP formation, to
succinate production by increasing the activity of
gluconeogenic phosphoenolpyruvate carboxykinase to perform
the carboxylation of phosphoenolpyruvate; ii) avoiding
formate and ethanol accumulation by inactivating
pyruvate formate-lyase (pflB) and; iii) deleting ptsI (part of
the intracellular phosphorelay system) which disrupts
the primary pathway for anaerobic glycerol metabolism.
With these modifications assembled, a strain able to
produce succinate from glycerol with yields as high as
0.8 mol/mol glycerol was obtained. However, succinic
acid productivity at anaerobic conditions was very low
(i.e., 102 mM or 40 g/L of succinate was produced in
Although the engineering strategies attempted so far
resulted in strains with slow metabolism and growth on
glycerol and/or need of oxygen supply during
fermentation, they are important because they pave the way for
the development of biotechnology applications using
Alternatively, succinic acid can be produced by yeast.
In a recent study a recombinant strain of the aerobic
yeast, Yarrowia lipolytica, was able to produce succinic
acid when cultivated on glycerol at low pH . The
strain was constructed by deletion of the succinate
dehydrogenase (SDH) subunit, which induces
accumulation of succinic acid in Y. lipolytica, and also in
fermentative yeasts as S. cerevisiae and Kluyveromyces
lactis [28,63,64]. Unexpectedly, mutations in the
succinate dehydrogenase subunit were shown to prevent
Y. lipolytica growth on glucose, however, growth on
glycerol and production of succinate was possible. Indeed,
a strain with the SDH2 gene deleted was able to produce
succinate from glycerol at the level of more than 45 g/L
in shaking flasks with buffering and more than 17 g/L
without buffering .
Citric acid is a weak organic acid that is commercially
produced by fermentation of molasses (sucrose and
glucose) by the fungus A. niger. As the citric acid global
production has reached 1.6 million tons and keeps
increasing annually at 3.5 - 4.0% in demand , its
production from glycerol is also of interest. Among
different potential producers of citric acid, the yeast
Y. lipolytica has gained much attention in the last years
as it is able to metabolize several important industrial
and agro-industrial byproducts (i.e. saturated free fatty
acids, raw glycerol) to produce large amounts of Single
Cell Oil (SCO), organic acids and biosurfactant
carbohydrate moieties (for instance, mannose and, rhamnose)
. As expected, Y. lipolytica is able to synthesize citric
acid using different carbon sources and secret it into the
medium in conditions of excess carbon and nitrogen
Several groups reported citric acid production from
glycerol by Y. lipolytica but at low production levels
and yields [6,67,68]. High production levels (i.e., above
100 g/L) were only obtained when better strains were
selected [69,70] and the cultivation mode was
improved [5,29,71]. The importance of strain selection
was highlighted when 27 strains of Y. lipolytica were
compared based on the ability to produce citric acid
from glycerol. Citric acid production in a
nitrogenlimited medium varied from 1.4 g/l up to 21.6 g/L
according to the strain utilized . Thus,
identification of strains that produce not only lower levels of
byproducts, such as isocitric acid and biomass, but
also exhibit high yields and productivity will benefit
the citric acid production process [69,70].
During batch fermentation of glycerol to citric acid,
the yeast is mainly affected by high concentrations of
crude glycerol, its impurities, and the increasing
concentrations of citric acid present at the end of
fermentation. Indeed, productivity of citric acid was shown to
decrease over time during crude glycerol fermentation
by Y. lipolytica [5,29]. To overcome these problems, at
least partially, a repeated-batch strategy for the
production of citric acid by Yarrowia using crude glycerol
has been evaluated [5,29]. The crude glycerol was
composed by glycerol 76% (w/w), sodium salts 4%
(w/w), methanol 0.1% w/w, metals Cu 0.3, Mg 100,
Fe 13.7, Zn 2.9, and Ca 46 (ppm), other organic
materials 0.8% (w/w) and water 19.5% (w/w). In this
setup, cultivation was conducted in batch mode, then
a portion of the culture liquid was withdrawn, and
the same volume of the production medium was
added. With that, cells were able to keep the
production of citric acid as high as 124.2 g/L with a yield of
0.77 g/g and a productivity of 0.85 g/L/h for
approximately 1000 h (Table 1) .
Oxalic acid is an organic acid known for its ability to
leach iron oxides. It can be applied in industries, such in
the manufacture of paper and detergents, to clean or
bleach iron complexes . Production of oxalic acid by
Aspergillus niger growing in crude glycerol waste from
biodiesel production plants was recently demonstrated
[30,72]. Oxalic acid production reached approximately
21 g/L with a conversion yield of glycerol to oxalic acid
(g/g) ranging from 0.55 to 0.62. These results look
promising even considering the long production time of
approximately 10 days. More recently, the ability of
A. niger XP strain to produce oxalic acid was studied in
submerged cultures containing 50 g/L of unpurified
biodiesel-derived waste, which was composed of 45%
(w/w) glycerol, 49% (w/w) free fatty acids, a low amount
of fatty acid ethyl esters, and soaps from ethyl ester
production . After 7 days of biosynthesis, the quantity
of oxalic acid produced by A. niger XP reached 48.9 g/L
with yields of oxalic acid as high as 0.88 (g. g 1 glycerol
Polyols (also called sugar alcohols, polyhydric alcohols,
or polyalcohols) are carbohydrates with a carbonyl group
(aldehyde or ketone) reduced to a corresponding
hydroxyl group. Examples of commercially available sugar
alcohols include xylitol, sorbitol, mannitol, erythritol,
lactitol, maltitol, and hydrogenated starch hydrolysate
(HSH) . These compounds are used in a variety of
applications, especially in the food, pharmaceutical, and
medical industries, but they also serve as intermediates
in chemical synthesis . Sugar alcohols share many
attributes with sugars and have unique nutritional
properties. They are used to improve the nutritional profile
of food products due to their low caloric content,
low insulin-mediated response, and non-cariogenicity
[75-79]. These compounds and their derivatives also
have other industrial applications, including the
production of polyurethanes, plastifying agents, resins,
surfactants, and intermediates for producing hydrocarbons
In fact, sugar alcohols especially sorbitol, xylitol, and
arabitol have been identified as belonging to a group
of the 12 best chemical building blocks derived from
biomass in studies by the U.S. Department of Energy
. Sorbitol can be converted into chemicals such as
propylene glycol, ethylene glycol, isosorbide, and
anhydro sugars, whereas xylitol and arabitol can be
converted into xylaric/xylonic acid, arabinoic/arabonic acid,
and glycols [81,84-86]. Due to their wide variety of
applications, including their use in biorefineries, the
production of polyols has increased worldwide. According to a
recent study , the global market for polyols is
expected to reach 1.81 million tons by the year 2015.
Traditionally, industrial production of sugar alcohols
has mainly been achieved using chemical means
(Table 2), more specifically by sugar hydrogenation (i.e.,
glucose, xylose, fructose) with chemical catalysts under
high temperature and pressure , a process that
requires pure substrates and costly chromatographic
purification steps. In recent years, the need to develop
new polyol production methods has arisen, and much
attention has been paid to biochemical processes. In fact,
polyol production by biochemical means offers the
potential to produce an environmentally friendly product
with high specificity and absence of impurities , an
efficient and cost-effective approach compared to their
production by chemical means. To produce sugar
alcohols, fermentation processes employing glucose,
fructose, and maltose as carbon sugars sources have been
optimized and metabolic engineering strategies have
been applied to different microorganisms [74-80,88-91].
Due to the high cost of using certain pure sugars (for
example, xylose, fructose, erythrose) for catalytic
hydrogenation in comparison to the final product (sugar
Table 2 Market and current production processes of selected chemicals
Chemical conversion of sugars
alcohols), many studies have instead described the use of
cellulosic and hemicellulosic biomass hydrolysates as
sources of carbon for fermentative polyol production
. Although most sugar alcohols are industrially
produced by chemical means, commercial production using
microorganisms in fermentative processes is becoming a
reality, as in the case for mannitol  and xylitol .
With the advent of an abundant and cheap carbon
source such as crude glycerol from biodiesel
production new efforts have sought to either microbially
produce sugar alcohols from this carbon source or employ
it as a complementary source to the sugars traditionally
used in the industry [31,99]. Next the results from using
glycerol or crude glycerol as sources of carbon for
production of the polyols mannitol, erythritol, and arabitol
with the aid of different microorganisms are discussed.
Some factors that influence the ability of two yeasts
(Torulopsis mannitofaciens CBS 5981 and T. versatilis
CBS 1752) to produce mannitol from glycerol have been
investigated . In optimal conditions, T.
mannitofaciens produced mannitol from glycerol consumed with a
yield of 31% of the theorethycal maximum. In addition, it
was shown that high concentrations of nitrogen sources
and KH2PO4 in the culture medium significantly reduced
mannitol yield despite consumption of glycerol. Previous
studies reported Candida magnoliae as excellent
mannitol producer using glucose and fructose mixtures as
carbon sources . A more recent study investigated
mannitol production from glycerol using resting cells of
this species . It was shown that C. magnoliae was able
to consume 100 g/L glycerol in 96 h, resulting in 51 g/L
mannitol, which corresponds to a yield of 0.51 g/g. In
addition, mannitol was the only metabolite detectable by
UVVIS or RI detector when samples from resting cells
of C. magnolia were analyzed using ion exclusion high
performance liquid chromatography. The absence of
other metabolites in solution may facilitate mannitol
recovery. Khan et al. (2009) also found that the mannitol
yield was negatively affected when glycerol was
supplemented with KH2PO4 and yeast extract.
Andr and co-workers  investigated the ability of
different strains of Yarrowia lipolytica to convert
residual crude glycerol (glycerol 70% w/w, impurities
composed of potassium and sodium salts (12% w/w),
nonglycerol organic material (1% w/w), methanol (2% w/w)
and water (14% w/w)) from biodiesel production into
chemical compounds with higher aggregate value. In this
study, the strains Y. lipolytica LFMB 19, Y. lipolytica
LFMB 20, and Y. lipolytica ACA-YC 5033 were cultured
in nitrogen-limited culture medium with a residual
crude glycerol concentration of 30 g/L. Under these
conditions, the main metabolic product synthesized by the
strains LFMB 19 and LFMB 20 was mannitol (6.0 g/L
maximum quantity and yield of 0.2 to 0.26 g per g of
crude glycerol consumed). This metabolite was produced
in negligible quantity by the ACA-YC 5033 strain .
In an independent study , other 15 strains of
different species of yeast and filamentous fungi of the class
Zygomycetes were compared on their ability to convert
crude glycerol into different chemical compounds.
Increasing concentrations of crude glycerol (30, 60, and
90 g/L) were used to screen the strains under
nitrogenlimited conditions. Y. lipolytica, Pichia membranifaciens,
and Thamnidium elegans were able to grow in culture
medium containing high initial glycerol concentrations
without inhibition by the substrate. In the case of
Y. lipolytica, the best producer of mannitol under the
conditions analyzed, there was a positive correlation
between the increase in crude glycerol concentration in
the initial culture medium and mannitol production,
with mannitol concentration and with yields reaching
19.4 g/L and 0.23 g/g, respectively .
These examples demonstrate the importance of
evaluating different microbial strains for mannitol production
from crude glycerol to select the most promising strains.
Although the production of mannitol on commercial
scale by bacteria using conventional carbon sources has
been accomplished , reports on the bioconversion
of crude glycerol into mannitol by bacteria are scarce.
Furthermore, the previous examples reveal the need for
further studies on the biochemical events that lead to
biosynthesis of mannitol and other polyols; for example,
Y. lipolytica strains grown in residual crude glycerol.
Commercial erythritol production occurs exclusively via
fermentation in substrates containing sugars, such as
glucose and fructose, from the hydrolysis of biomass.
Although fermentative erythritol production by different
microorganisms has been studied since 1960 [79,104],
few studies have investigated the production of this
polyol from different carbon sources . In the specific
case of using residual crude glycerol, containing 550 g/L
of glycerol and 50 g/L of KCl, as a carbon source, an
acetate-negative mutant of Y. lipolytica (Wratislavia K1)
was found to simultaneously produce significant
quantities of erythritol and citric acid . With an initial
crude glycerol concentration of 150 g/L in
nitrogenlimited culture medium and a pH of 5.5 favorable for
producing citric acid, a concentration of 81 g/L
erythritol was obtained after fed-batch fermentation for 97 h.
Subsequently, the effect of pH on erythritol production
by the Wratislavia K1 mutant of Y. lipolytica was
investigated . Erythritol concentration reached 170 g/L
(0.56 g/g yield) after fed-batch growth with a total
concentration of 300 g/L crude glycerol and pH of 3.0.
Under these conditions, citric acid was not produced,
demonstrating that this pH range is optimal for the
mutant to produce erythritol, as it prevents the channeling
of glycerol to citric acid production . This example
demonstrated that by using residual crude glycerol as
the carbon source, the yields of erythritol without the
generation of undesirable byproducts were comparable
to the reported yields with microorganisms used in
commercial erythritol production with glucose as substrate
. As previously reported for mannitol production, it
is noteworthy that approaches involving bioprospecting
microorganisms able to tolerate high osmolarity 
can lead to a higher yield relative to erythritol-producing
Arabitol polyol has many attributes of its enantiomer,
xylitol, making its use feasible in many known applications of
xylitol, such as in natural sweeteners, caries reducers, and
sugar substitutes for diabetic patients . Arabitol and
xylitol can be transformed into arabinoic/arabonic and
xylaric/xylonic acids, which in turn can be used to produce
unsaturated polyesters and polymers with new applications
[84,86]. This role indicates their importance in the
biorefinery context. In addition, arabitol can be biologically
converted into xylitol, for example, by Gluconobacter oxidans
[107,108], representing a possible efficient route for the
synthesis of xylitol of a higher purity and specificity.
In this scenario, Koganti  conducted an extensive
screening of over 214 yeast strains belonging to 25
different genera from the Agricultural Research Service
Culture Collection (NRRL) with regard to their ability to
produce arabitol/xylitol using crude glycerol from
biodiesel production. In this study, xylitol yield was low for
most strains; however, the Debaryomyces hansenii SBP-1
strain was selected for its ability to produce high arabitol
concentrations as the only polyol, thus facilitating
further separation of the product. Arabitol production by
D. hansenii SBP-1 was then investigated under different
conditions, and a yield of 0.5 g/g glycerol consumed was
attained with 150 g/L crude glycerol in the initial culture
medium under aerobic conditions at 30C.
Subsequently, culture conditions for the D. hansenii
SBP-1 strain were optimized , leading to an arabitol
yield of 0.6 g/g and volumetric productivity of 0.35 g/L/h.
A protocol was developed for separating arabitol from the
fermentation medium containing 2030 g/L glycerol and
4050 g/L arabitol, which led to a 60% arabitol yield with
biodegradable, and biocompatible thermoplastics .
They can be completely degraded by microorganisms
present in most environments, and can be produced
from different renewable carbon sources .
Poly3-hydroxybutyrate PHB belongs to the group of
polyhydroxyalkanoates (PHAs) and it is the best-studied example
of biodegradable polyesters. This polymer is naturally
synthesized by a wide variety of bacterial species as a
storage compound for carbon and energy . Nowadays,
commercial production of PHB is small and limited by
substrate cost . Thus, efficient conversion of cheap
crude glycerol from the biodiesel industry to PHB is an
interesting opportunity to reduce production costs and
make PHB an industrial biotechnological product [34,35].
Indeed, microbial PHB synthesis from glycerol has been
evaluated with several PHA producers and under different
fermentation conditions [34,35,113-116].
Conversion of glycerol to PHBs has reached high
production levels due to optimization of strains and
fermentations conditions. Implementation of fed-batch under
low O2 conditions appeared to be a suitable strategy for
the production of PHB by the genetic engineered E. coli
Arc2 strain . In microaerobic fed-batch cultures in
which glycerol was added to maintain its concentration
above 5 g/L, cell dry weight and PHB concentration
reached 21.17 and 10.81 g/L, respectively, after 60 h
(Table 1) . Similarly, fed-batch cultivation improved
PHB production by the newly isolated bacterium,
Zobellella denitrificans MW1, which was characterized as
producing large amounts of PHB from glycerol in
presence of NaCl [35,114]. Cultivation in the medium
containing 20 g/L NaCl, with optimized feeding of glycerol
and ammonia, resulted in a PHB content of 66.9% of cell
dry weight, and the polymer productivity and substrate
yield coefficient of 1.09 g/L/h and 0.25 g PHB/g glycerol,
Conversion of glycerol to D-xylulose has been achieved
using a two stage microbial fermentation. Initially,
glycerol was converted to D-arabitol by Candida
parapsilosis, then yeast cells were collected and finally the
D-arabitol from the culture supernatant was converted
to D-xylulose by G. oxydans . However, this
conversion process needs to be improved before increasing
production scale since from 170 g/L glycerol only
32.2 g/L of D-arabitol was produced.
Polyunsaturated fatty acids
Polyunsaturated fatty acids, such has docosahexaenoic
acid (DHA, 22:6), eicosapentanoic acid (EPA, 20:5) and
-linoleic acid (ALA, 18:3) have market presence as
nutraceuticals, because they are beneficial for human
health. The use of biodiesel-derived glycerol as carbon
source to produce such polyunsatured fatty acids,
especially DHA, was recently reviewed [118,119]. Thus we
do not further discuss this route for valorization of
crude glycerol in this work.
Biohydrogen and biomethane
Crude glycerol utilization to produce gaseous fuels,
biohydrogen and biomethane (biogas), has gained more
attention lately (as reviewed by [119,120]. Indeed, several
studies have reported biohydrogen and biomethane
production by microbial conversion processes from crude
glycerol using single microbial strains or bacterial
communities. Utilization of crude glycerol as the sole carbon
source or as a suitable co-substrate to improve the
efficiency of biodigesters has also been investigated. These
topics were currently reviewed and reported in recent
publications [119-122] and will not be further discussed
Biodiesel-based biorefinery: opportunities and challenges
Biorefineries are based on the integration of the biomass
conversion processes to produce power, fuels and
chemicals. In this context, the utilization of glycerol generated
in the biodiesel production process offers an excellent
opportunity to obtain chemicals by microbial
fermentation. Production yields of fuels and chemicals from
glycerol as high as 90% of theoretical maximum have been
obtained (Table 1). These high production yields of the
desired product should make the establishment of
bioprocesses easier. Even if the available producing strains
have not been evaluated at pilot scale yet (at least based
on the publicly available data), market demand for green
chemicals, new processes, and technologies for
lignocelulose biorefineries should facilitate the development of
industrial processes based on crude glycerol.
A vast range of fuels and chemicals can potentially be
produced by microbial fermentation of glycerol.
However, the expectation is that the primary products
obtained by fermentation in biorefineries are chemicals
with established demands and international markets. In
fact, from the fourteen compounds produced at lab scale
by microbial fermentation of glycerol showed in Table 1,
ten of them are nowadays produced by chemical or
microbial conversion of sugars or petrochemical processes.
More interesting, these chemicals are important
platform chemicals or products that have a consolidated
market demand of thousands of tons per year (Table 2).
Thus, once a production process based on glycerol
fermentation for any of these chemicals is developed, the
product could easily enter the market. On the other
hand, it is important that production costs of chemicals
from glycerol remain competitive with those obtained
from petroleum. It should be noted that demands for
sustainable development and volatile petroleum prices
should favor the use of greener chemicals in the long
term, even if their market price is not considerably
cheaper. Chemicals without or with a limited market,
like DHA and glyceric acid, may represent an
opportunity for the development of new products as there is no
direct benchmark production price they will need to
Manipulation of microbes to allow the cost
competitive commercial production of fuels and chemicals, such
as ethanol, butanol, isoprenoids and others, on sugar
and lignocellulose has advanced significantly in the last
years . Indeed, several processes based on
engineered microorganisms, especially yeast and bacteria
species, have been developed and implemented .
Two examples of success are given by the production of
1,3-PDO and succinic acid. The chemical 1,3-PDO is
traditionally made from fossil-derived ethylene oxide or
propylene, however, a bio-based process has been
developed and implemented by DuPont and Tate & Lyle.
Their process for production of 1,3-PDO relies on a
microbe expressing genes from several different
microorganisms to give the required productivity . The
process based on the use of a designer microbe to
produce 1,3-PDO from corn has been running in a
BioPDO plant in Tennessee (USA) with a capacity of 45
thousand tons a year since 2007. Similar approaches are
being developed for succinic acid. Bio-based succinic
acid production by a fermentative technology is the
focus of a joint venture between DSM and Roquette.
Indeed, the first testing volumes of this renewable and
versatile chemical produced from corn have already been
produced in a demonstration plant in Lestrem (France)
that was built in 2009. These developments are expected
to facilitate the establishment of glycerol-based
fermentation processes, since similar microorganisms and
pathways may be used.
Although the challenges of obtaining microbial strains
able to operate under industrial process conditions have
been overcome, another challenge for the production of
fuels and chemicals from biodiesel industry crude
glycerol is the supply chain. From the economic point of
view, it is important for the industry that the feedstocks
for biodiesel production, and consequently glycerol for
fuels and chemicals production, are abundant year
round. In this sense, some countries may not have
sufficient biomass, and consequently glycerol, to maintain an
industrial-scale production of biodiesel, fuels and
chemicals throughout the four seasons, especially due to
competition of biomass for other uses. One way to solve this
problem, especially in countries with vast territory and
mild climate year round, is the diversification of biomass
feedstocks in the industry and regionalization of
production plants. In this sense, the main feedstock for the
industry should be growing in the region of the
production plant, which may only be complemented by
alternative biomass from other regions. In countries like
Brazil, for example, where 80% of biodiesel is produced
from soybean oil, it might be advantageous to start using
oil from alternative biomass sources, like palm oil,
physic nut, castor bean and others. This would
guarantee the supply of biomass for the industry and at the
same time avoid transportation of soybean to production
plants around the country.
Increasing awareness of environmental issues and
consequent pressure from governments and public agencies to
reduce the emission of pollutants, together with the
increasing petroleum cost and demand for fuels and
chemicals worldwide have led to the development of
biomass conversion processes. Processes for production
of fuels and chemicals from crude glycerol waste from
the biodiesel industry have been evaluated and
developed at laboratory scale. Indeed, the strong potential of
crude glycerol use for the development of biorefineries
has been demonstrated by the production of several
chemicals using different routes and microorganisms.
Several yeasts and bacteria, especially E. coli and others
from the Enterobacteriaceae and Clostridiaceae families,
have been evaluated for fermentation of glycerol.
Efficient selection and construction of recombinant strains
based on biochemical and genomic data associated with
optimization of fermentation conditions resulted in
strains able to produce more than 100 g/L of the desired
products with yields above 50% of the theorethycal
maximum in laboratory scale.
Among the different routes to establish a fermentative
process based on microbial fermentation of glycerol, the
use of wild type natural producers and engineered
strains are the most considered. The later case is more
common for bacteria of the Enterobacteriaceae family
and the former one for engineered strains of yeast and
E. coli. When wild type strains are employed to produce
the desired chemical, efforts to improve production
generally concentrate on the optimization of fermentation
conditions, like aeration, pH, substrate concentration
and feeding. However, the use of these strains (species)
in industrial applications may be impaired by
pathogenicity, need for strict anaerobic conditions, or lack of
genetic tools. The use of engineered strains commonly
employed in industry, especially yeasts and E. coli, can
solve this problem. However, metabolic engineering
strategies have to be used to drive metabolite synthesis
through homologous and heterologous pathways.
Although in industry the most commonly used strains are
engineered producers of fuels and chemicals, a careful
screening of the biodiversity is generally advisable, since
microorganisms can naturally produce a vast range of
compounds. Once enzymes and metabolic pathways in
such microorganisms are identified and isolated they can
be used for the development of recombinant strains.
Considering the already remarkable advances for the
production of fuels and chemicals from glycerol using
microorganisms, it may be expected that strains for
industrial implementation of these bioprocesses should
become available in the next years. The technology for the
production of butanol, propanodiol and succinic acid
from simple renewable feedstocks such as starch and
sucrose is already available. Therefore, it is expected that
these chemicals will be the first to be produced from
more complex substrates such as biodiesel-derived crude
glycerol and other more complex biomasses. However,
before implementation of a pilot or industrial plant to
produce any fuel or chemical, two important aspects
should be considered: i) the cost of producing fuels and
chemicals by microbial fermentation should be
comparable with fossil-derived products; and ii) the feedstock
supply chain for the industry.
LCLF wrote the parts concerning polyols. BFQ participated in the design of
the review and revised the manuscript. JRMA participated in the design of
the review and wrote most of the manuscript. All authors read and
approved the final version.
This work was financially supported by the Brazilian National Council for
Scientific and Technological Development (CNPq), FINEP and Embrapa.
1Embrapa-Agroenergy, Parque Estao Biolgica S/N, Av. W3 Norte (final),
70770-901, Braslia, DF, Brazil. 2Universidade Catlica de Braslia, Genomic
Sciences and Biotechnology Program, 70790-160, Braslia, DF, Brazil.
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