Plácido and Capareda Bioresour. Bioprocess.

(2016) 3:23
DOI 10.1186/s40643-016-0100-1

REVIEW Open Access

Conversion of residues and by‑products

from the biodiesel industry into value‑added
products
Jersson Plácido* and Sergio Capareda

Abstract
Biodiesel, one of the most important sources of renewable energy, is produced in large quantities around the world;
however, its production generates different kinds of residues and by-products which raise economic and environ-
mental concerns. This review presents a compilation of the data on current state of transformation of residues and
by-products of biodiesel industry into products that are suitable for bio-refining. The review has analyzed glycerol,
biodiesel washing wastewaters, and solid residues. The technologies were described and the most significant experi-
mental results and variables were summarized to allow researchers an easy access to this information.
Keywords: Biodiesel, Waste glycerol, Biodiesel washing wastewaters, Fermentation, Thermal conversion

Background Renewable Fuels Alliance 2012) and its production has

The extensive use of fossil fuels has generated environ- been increasing constantly over the last few years (Rossi
mental issues, such as global warming and atmospheric et al. 2011).
pollution (Siles et al. 2010). The replacement of fossil The production of biodiesel in large quantities also
fuels for renewable biofuels is a necessity. The employ- results in the generation of abundant quantities of resi-
ment of biofuels from biological feedstocks is a signifi- dues or by-products which cannot be utilized in bio-
cant option because they can be generated sustainably diesel production process (Eliche-Quesada et al. 2012).
from sunlight, carbon dioxide, and water (Juang et al. Therefore, new applications to treat and use these com-
2011). Biodiesel is one of the most prominent biofuels in pounds have become an important topic. In biodiesel,
the world. Biodiesel is the name given to the fatty alkyl the most significant residues and by-products are glyc-
esters produced after transesterification of fatty acids erol, biodiesel washing wastewaters, methanol, and solid
using methanol or ethanol in the presence of a catalyst residues (Varanda et al. 2011). Glycerol is the by-product
such as sodium hydroxide. To produce biodiesel, oils that generates the largest interest, because it can involve
from different sources can be used. They are obtained the largest revenue for the biodiesel industry. Glycerol
from plants oil (palm, soy bean, sunflower, etc.) or ani- is the main by-product from biodiesel transesterifica-
mal fats (chicken, beef, and pork). Because biodiesel can tion (Nitayavardhana and Khanal 2011; Ethier et al.
be produced using different types of plant or animal oil, 2011); by 2016, the worldwide waste glycerol production
its production can be developed in most places around is expected to reach 4 billion gallons (Yang et al. 2012).
the world. The traditional technology is fully developed However, the glycerol produced from biodiesel has low
and technologically accessible. For example, in 2010, 19 quality and low price ($0.05 per pound) and its purifi-
billion L of biodiesel were produced worldwide (Global cation for further applications is economically unviable
(Nitayavardhana and Khanal 2011). The largest residue
produced by the biodiesel industry is the biodiesel wash-
*Correspondence: [email protected] ing wastewaters from the biodiesel washing process. The
Bio‑Energy Testing and Analysis Laboratory (BETA Lab), Biological projected production of biodiesel for 2016 is 37 billion
and Agricultural Engineering Department, Texas A&M University,
201 Scoates Hall, 2117 TAMU, College Station, TX 77843, USA
gallons, this amount of biodiesel will produce 43 billion

© 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made.
Plácido and Capareda Bioresour. Bioprocess. (2016) 3:23 Page 2 of 12

gallons of wastewaters (Yang et al. 2012; Siles et al. 2011). glycerol as a participant in the production of other bio-
This large amount of residues is related with disposal and products. Hydrothermal electrolysis transforms glycerol
environmental issues, because these wastewaters have a into lactic acid using aqueous alkaline conditions at high
high organic load that does not allow its direct disposal temperatures and pressures (Yuksel et al. 2011). In this
into the sewage system (Rattanapan et al. 2011). Solid case, a designed autoclave and a flow type reactor were
by-products are the residues that include pressed seed evaluated. The autoclave achieved a glycerol conversion
cakes, spent earth, and agricultural wastes. The biological of 83 %, whereas the flow type reactor accomplished a
solid residues are generally used in compost or for ani- 75 % (Yuksel et al. 2011). Other waste glycerol uses are
mal feeding; however, these compounds can increase the the production of castor oil glycerides and polyols. Glyc-
profitability of the biodiesel industry if they can be uti- erolysis of castor oil methyl esters and waste glycerol
lized as a substrate for the production of chemical com- generated castor oil monoglycerides (50.4 %) and diglyc-
pounds or energy. erides (35 %) which can be employed in the plastic indus-
The aim of this review was to summarize the current tries (Echeverri et al. 2013). On the other hand, polyols
state of art in the transformation of the most significant were produced from a sequential two-step liquefaction of
biodiesel by-products and residues. lignocellulosic material using crude glycerol as liquefac-
tion solvent. In this process, acid and base liquefactions
Biodiesel by‑products and residues promoted the esterification reaction between free fatty
Glycerol acids and glycerol and the condensation of polyols. These
1,2,3 propanetriol or commonly named as glycerol, glyc- biopolyols had similar properties as those of the petro-
erin, or glycerine is an organic compound with three leum-based polyols (Hu and Li 2014).
carbons and three hydroxyl groups. Glycerol is mainly Microbial transformation has been carried out for the
produced by saponification process and is widely utilized production of oxalic and docosahexaenoic acid (Ethier
in pharmaceutical and food industry (Ethier et al. 2011). et al. 2011), polymer-related molecules [polyhydroxybu-
Besides the saponification process, glycerol is an abun- tyrate (PHB) and 1,3-propanediol] (Dobroth et al. 2011),
dant by-product of biodiesel production process. Crude surfactants (Sousa et al. 2012), and animal feed (Nitaya-
glycerol is the name given to the glycerol produced dur- vardhana and Khanal 2011). Table 1 shows the microbial
ing biodiesel production (Dobroth et al. 2011; Ethier et al. transformation methods. To increase the concentration
2011) and is a by-product of transesterification reaction of bioproducts from crude glycerol, different strategies
that takes place between the fatty acids with methanol or have been used. The most significant ones are genetic
ethanol to produce methyl esters. Crude glycerol repre- engineering, media and environmental condition’s opti-
sents about 10 % of the product output in biodiesel pro- mization. Genetic engineering has been employed to
duction (Sousa et al. 2012); in fact, 1 kg of crude glycerol improve synthesis of different types of products. These
is produced per 12.6 L of biodiesel (Dobroth et al. 2011). improvements include genes insertion that allows con-
Crude glycerol is considered as a residue because residual sumption of crude glycerol and/or the production of
ethanol or methanol, fatty acid ethyl (or methyl) esters, different types of molecules. Escherichia coli was geneti-
and residual fatty acids are mixed with it (Dobroth et al. cally modified by inserting the aldehyde reductase and
2011). These impurities can be removed from crude glyc- aldehyde dehydrogenase genes, these genes improved the
erol to produce a raw material for pharmaceutical and generation of poly 3-hydroxybutyrate (Shah et al. 2014).
food industries. The crude glycerol purification and refin- Corynebacterium glutamicum was genetically modified
ing is very expensive to be sustainable in the biodiesel to increase the concentration of different types of amino
industry (Ethier et al. 2011; Sabourin-Provost and Hal- acids (Meiswinkel et al. 2013). Likewise, free fatty acid
lenbeck 2009). Therefore, the utilization of crude glycerol concentration was improved by using an E. coli with a
is important for the biodiesel industry future. The appli- modified OPLlTE gen. This modification increased free
cation of crude glycerol can be grouped into bioproduct’s fatty acids by 15.8 fold compared to the control (Lee
production (Rossi et al. 2012; André et al. 2010; Abad and et al. 2014). Recombinant engineering is an interesting
Turon 2012), renewable energy production (Yoon et al. option to improve the production of biobased products
2010; Ngo et al. 2011), and wastewater applications (Siles from waste glycerol. The cost of manipulating and main-
López et al. 2009; Bodík et al. 2009). taining these microorganisms needs to be considered.
This technology improves the up-stream process; how-
Bioproducts ever, the principal cost in biotechnological processes
The transformation of glycerol into bioproducts is per- is related with the recovery and purification costs. The
formed by physicochemical or microbial processes. Phys- developments in genetic engineering should be focused
icochemical process transforms glycerol, or it utilizes in improving the recovery and purification of these
Plácido and Capareda Bioresour. Bioprocess. (2016) 3:23 Page 3 of 12

Table 1 Bioproducts produced by microbiological transformation of crude glycerol

Product Microorganism Initial glycerol Yield References

Biosurfactants Bacillus sp. 2 % (v/v) – (Sousa et al. 2012)

Polyhydroxybutyrate Mixed microbial consortia 10 % (v/v) – (Dobroth et al. 2011)
1,3-Propanediol Klebsiella pneumoniae 10 % (v/v) 0.41 mol/mol (Rossi et al. 2012)
Docosahexaenoic acid Schizochytrium limacinum 90 g/L 1.74 g/L (Ethier et al. 2011)
Ethanol Enterobacter aerogenes 80 mM 0.96 mol/mol (Ito et al. 2005)
Oxalic acid Aspergillus niger NRRL 60 g/L 0.55 g/g (André et al. 2010)
Aspergillus niger LFMB 60 g/L 0.61 g/g
Animal feed Rhizopus microsporus 75 % 0.83 g/g (Nitayavardhana and Khanal 2011)
Docosahexaenoic acid Schizochytrium limacinum 75–100 g/L 4.91 g/L (Chi et al. 2007)
1,3-Propanediol P. agglomerans, C. freundii 60 g/L 0.56 g/g (Casali et al. 2012)
20 g/L 0.68 g/g
Poly 3-hydroxybutyrate bioethanol Escherichia coli 30 g/L 0.8 g (Shah et al. 2014)
30.2 %
1,3-Propanodiol (1,3-PD) C. butyricum 80 g/L 2.75 g/L/h (Szymanowska-Powałowska and Leja 2014)
Lipids Chlorella protothecoides 30.55 wt% 2.07 g/L/h (Feng et al. 2014)
Polyhydroxyalkanoates Mixed microbial cultures 30 mg/L 0.27 g/L/d (Moita et al. 2014)
p-Hydroxybenzoate Pseudomonas putida S12 18.4 g/L 6.0 g/L (Verhoef et al. 2014)
l-Glutamate Corynebacterium glutamicum 20 g/L 0.026 g/g (Meiswinkel et al. 2013)
l-Lysine 0.08 g/g
l-Ornithine 0.12 g/g
l-Arginine 0.03 g/g
Putrescine 0.03 g/g
Dihydroxyacetone Gluconobacter frateurii 15 g/L 125.8 g/L (Liu et al. 2013b)
Free fatty acids Escherichia coli 10 g/L 231 mg/g (Lee et al. 2014)
1,3-Propanediol Lactobacillus diolivorans 10 g/L 0.36 g/L/h (Pflügl et al. 2014)
Biomass Yarrowia lipolytica 25 g/L 12.3 g/L (Juszczyk et al. 2013)
1,3-Propanediol Mixed microbial consortia 7 g/L 0.65 mol/mol g (Liu et al. 2013a)
1,3-Propanediol Citrobacter freundii 176 g/L 66.3 g/L (Metsoviti et al. 2013)
Fumaric acid Rhizopus arrhizus 80 g/L 4.37 g/L (Zhou et al. 2014)
n-Butanol Clostridium pasteurianum 10 g/L 0.35 g/g (Khanna et al. 2013)
Organic acids Propionibacterium freudenreichii 20 g/L 0.42 g/g (Ruhal and Choudhury 2012)
Trehalose 0.90 mg/g
Lipids Rhodotorula glutinis 30 g/L 5.40 g/L (Yen et al. 2012)

bioproducts. The most important modification to the modification achieved a larger yield and rate than the
culture media was nitrogen concentration. The change in batch reactor without microfiltration (Szymanowska-
nitrogen concentration increased the quantity of 1,3-pro- Powałowska and Leja 2014).
panediols and docosahexaenoic acid (Sabourin-Prov- In the last few years, the microbiological transforma-
ost and Hallenbeck 2009; Chi et al. 2007). Bioproduct’s tion of crude glycerol has become an interesting option
microbial processes have been evaluated using differ- to utilize crude glycerol and to increase the efficiency of
ent types of reactors configurations. Packed bed reac- biodiesel production process. Today, an industrial pro-
tor was employed in the production of 1,3-propanediol cess using crude glycerol does not exist; however, sev-
(Casali et al. 2012), whereas stirred tank reactors have eral products have been generated from glycerol using
been operated for docosahexaenoic acid (Ethier et al. biological transformations. As an example, an estimate
2011) 1,3 propanediol (Rossi et al. 2012) and polyhy- of 19 tonnes per year of bioplastics (PHB) can be pro-
droxyalkanoates (PHA) (Dobroth et al. 2011). Fed-batch duced from a biodiesel plant of 38 million L per year
configuration produced dihydroxyacetone in quantities (Dobroth et al. 2011). The potential for bioproduct from
(2–5 fold) greater than batch culture (Liu et al. 2013b). waste glycerol is great; however, the industrial recovery
Microfiltration was coupled to a batch reactor to improve of these bioproducts is a major issue. Downstream pro-
the production of 1,3-propanodiol by C. butyricum. This cessing is expensive, especially for products that are in
Plácido and Capareda Bioresour. Bioprocess. (2016) 3:23 Page 4 of 12

low concentrations. In those cases, the bioproducts need glycerol has been gasified along with olive kernel, hard-
to have a high value, therefore, the capital and produc- wood chips, physic nut waste, and palm shell waste
tion costs can be recuperated by the process. The future (Sricharoenchaikul and Atong 2012; Wei et al. 2011;
industrialization of bioproducts from glycerol is depend- Rattanapan et al. 2011; Skoulou and Zabaniotou 2013).
ent of the economic aspects related with the advances in The co-gasification of crude glycerol and biomass incre-
product recovery. Until these developments are not avail- mented Syngas yield, heating value, and H2 concentration
able, the industrial production of biobased product from (Sricharoenchaikul and Atong 2012; Wei et al. 2011; Rat-
waste glycerol is distant. tanapan et al. 2011; Skoulou and Zabaniotou 2013). The
Syngas produced from the crude glycerol co-gasification
Renewable energy production can be further employed to generate electricity or to
Renewable energy can be produced from crude glycerol produce chemicals or biofuels (Skoulou and Zabaniotou
using thermochemical or biological processes. The prin- 2013). On the other hand, microwave plasma gasifica-
cipal thermochemical processes include pyrolysis and tion is a technology that utilizes plasma flames produced
gasification, whereas the most important biological pro- by external electrical sources to gasify substrates. Crude
cesses are biological fuel cells, hydrogen generation, and glycerol microwave plasma gasification demonstrated an
anaerobic digestion (biomethane production). increment in gasification efficiency and Syngas heating
value (Yoon et al. 2013). Another type of gasification is
Thermal conversion Thermal conversions (gasification supercritical water gasification. This technology utilizes
and pyrolysis) and biomass fuel cells are the two physico- supercritical water to improve the gasification efficiency.
chemical processes used to generate energy from crude Supercritical water has the ability to act as an acid/base
glycerol. Gasification and pyrolysis are performed at high catalyst, dissolve non-polar organic compounds, and
temperatures (>300 °C) in an atmosphere which lacks react with other compounds (Tapah et al. 2014). The
oxygen or has a small quantity of it. These processes gen- application of supercritical water gasification in crude
erate a gas phase (synthesis gas or Syngas), liquid phase glycerol has been evaluated in the presence of KOH and
(bio-oil or bio-liquor), and a solid phase (biochar). Each Fe2O3–Cr2O3 as catalysts (Yang et al. 2013; Tapah et al.
phase percentage is dependent of the reaction conditions. 2014). Catalytic supercritical water gasification improved
In these processes, the most important conditions are the Syngas quality by reducing biochar impurities and
temperature, pressure, residence time, air to O2 ratio, and increasing the amount of combustible gases, especially
catalyst. Table 2 shows the thermal processes used in the H2. However, KOH did not act as a catalyst; in fact, KOH
transformation of crude glycerol into biofuels. acted as a reactant in the process and generated K2CO3
Pyrolysis studies have been performed using crude as the product (Yang et al. 2013).
glycerol as an auxiliary compound to pyrolyze differ- Thermal conversion is an option for the transformation
ent types of feedstocks. The mixture of crude glycerol of glycerol into energy and other chemicals. In this type
and swine manure improved the quality of bio-oil and of process, crude glycerol is an interesting option as a co-
its distillated fractions compared with the pyrolysis of substrate for gasification or pyrolysis processes. There-
swine manure alone (Cheng et al. 2014a). The addition of fore, the combination of a thermal conversion plant and
glycerol has been found as a factor to improve hydrogen biodiesel production can be an option for the biodiesel
(H2) concentration in the Syngas. The addition of 20 % of industry. The thermal conversion plant can use the agri-
glycerol to lignite generated H2 sixfold higher than lig- cultural residues (stems, leafs, stalks, husk, etc.) and the
nite pyrolysis (Manara and Zabaniotou 2013). Similar to pressed seeds as main substrate, whereas waste glycerol
lignite, olive kernel pyrolysis exhibited an increment of can be employed as a co-substrate to increase the qual-
11.6 % in the concentration of H2 when 25 % of the crude ity of the Syngas or the bio-oil produced. In this case, the
glycerol was added (Skoulou et al. 2012). Besides the viability will depend on the economic and logistic analy-
increment in H2 concentration, glycerol also increased ses for the thermal conversion plant.
the concentration of light hydrocarbons in Syngas. A
mixture 1:1 or 3:1 of crude glycerol and corn straw pro- Microbial fuel cells Microbial fuel cell is other approxi-
duced a twofold increment in C4 and C5 hydrocarbons mation to physicochemical energy generation. This pro-
compared with corn straw pyrolysis (Delgado et al. 2013). cess employs microbial strains to catalyze the oxidation
Crude glycerol gasification used traditional processes; of organic or inorganic matter for electricity generation
however, microwave plasma gasification and Super- (Feng et al. 2011). Feng et al. (2011) found that it is possible
critical Water Gasification have also been employed to to produce electricity using crude glycerol as substrate for
gasify crude glycerol. Traditional crude glycerol gasifi- microbial fuel cells. In their research, a maximum cath-
cation is performed in a co-gasification process. Crude ode power density of 2110 mW/m2 was obtained from the
Plácido and Capareda Bioresour. Bioprocess. (2016) 3:23 Page 5 of 12

Table 2 Crude glycerol thermal conversion

Process Co-substrate Glycerol concentra- Conditions Products References
tion

Batch pyrolysis Swine manure 1:3 swine 340 °C for 15 min Bio-oil (Cheng et al. 2014b)
manure:crude
glycerol
Co-gasification Olive kernel 49 wt% 750–850 °C Syngas (10.5–52.2) (Skoulou and Zabani-
Air ratio: 0.2–0.4 Bio-oil (2.4–19.5) otou 2013)
Biochar (37.9–78.3)
Co-gasification Hardwood chips 20 (wt%) 850 °C CO (19.73 % v) (Wei et al. 2011)
Air ratio: 0.293 CH4 (3.82 % v)
H2 (19.38 % v)
CO2 (11.67 % v)
Pyrolysis Greek lignite 15–20 wt% 850 °C H2 (65.44 v/v %) (Manara and Zabani-
otou 2013)
Slow co-pyrolysis Corn straw 1:1 glycerol:corn straw 30 °C/min, 550 °C Syngas (25 %) (Delgado et al. 2013)
Bio-oil (55 %)
Biochar (15 %)
Co-gasification Physic nut waste (pnw) 30 % 700–900 °C pnw Syngas (95.41 (Sricharoenchaikul and
Palm shell waste (psw) Air ratio: 0–0.6 wt%) Atong 2012)
psw Syngas (94.70
wt%)
Pyrolysis Olive kernel 25 wt% 720 °C H2 (45 %) (Skoulou et al. 2012)
Microwave plasma – 100 % Air ratio: 0–0.4 H2 (57 %) (Yoon et al. 2013)
gasification 2 kW microwave CO (35 %)
generator
Catalytic supercritical – 30 wt% glycerol 600 °C at 300 bar H2, (Tapah et al. 2014)
water gasification CO,
CO2,
CH4
Supercritical water – 7 wt% 500 °C, 45 MPa H2 (27.9 %) (Yang et al. 2013)
gasification
Gasification – 60 % 950–1500 °C H2 (38–42 %) (Yoon et al. 2010)
CO (39–41 %)
CO2 (9–15 %)
CH4 (1–3 %)

heat-treated anode of the microbial fuel cell with biodiesel H2 generation and glycerol consumption. In dark fermen-
waste medium (Feng et al. 2011). tation, culture medium has achieved improvements in the
hydrogen production. Rossi et al. (2011) found that bio-
Hydrogen generation Hydrogen is the most studied bio- mass pretreatment before glycerol conversion improved
fuel from crude glycerol. Hydrogen is produced by dark the hydrogen yield by fivefold. This process reduced the
fermentation and photofermentation processes (Rossi methanogenic bacteria in the sludge which was added
et al. 2011; Ghosh et al. 2012b). Table 3 describes the dif- as the inoculum (Rossi et al. 2011). Ngo et al. (2011)
ferent processes used in hydrogen production from crude improved the hydrogen yield (1.55–2 fold) compared
glycerol. with the un-supplemented culture by using a buffer solu-
Dark fermentation employs anaerobic or facultative tion (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic
microorganisms in a process similar to anaerobic diges- acid) and N2 sparging conditions (Ngo et al. 2011). Simi-
tion. This fermentation has been improved by modifying larly, Sarma et al. (2013a) found that slaughterhouse liq-
the microorganisms, the culture media, or the reactor uid waste, brewery waste biomass, and urea increased
conditions. The bacteria applied normally came from the H2 generation by Enterobacter aerogenes NRRL B
methanogenic fermentation including several types of 407 from 18 to 38 % (Sarma et al. 2013a). The addition of
hydrogenic bacteria (Rossi et al. 2011); however, the KH2PO4 and NH4Cl maximized the hydrogen (0.27 mol/
strains have been improved using molecular techniques mol) and ethanol (0.63 mol/mol) yields from crude glyc-
(transgenic strains) (Gonzalez et al. 2008) or selection erol using Klebsiella sp. TR17 (Chookaew et al. 2014b).
techniques (eco-biotechnological) (Varrone et al. 2013b), The modification of the growth media can be introduced
and these methodologies have allowed the increment of by reducing the inhibitor concentrations. Methanol and
Plácido and Capareda Bioresour. Bioprocess. (2016) 3:23 Page 6 of 12

Table 3 Hydrogen production from crude glycerol

Product Microorganism Initial glycerol Yield References

Photofermentation Rhodopseudomonas palustris 20 mM 6.1 mol/mol (Ghosh et al. 2012b)

Dark fermentation Klebsiella pneumoniae 10 % (v/v) 31.16 mol/g (Rossi et al. 2011)
Photofermentation Rhodopseudomonas palustris 10 mM 4.0 g/g (Sabourin-Provost and Hallenbeck 2009)
Dark fermentation Clostridium pasteurianum 10 g/L 0.775 mol/mol (Lo et al. 2013)
Dark fermentation Klebsiella sp. TR17 10 g/L 44.27 mmol/g (Chookaew et al. 2014a)
Photofermentation Rhodopseudomonas palustris 30 mM 7 mol/mol (Ghosh et al. 2012a)
Dark fermentation Enterobacter aerogenes NRRL B-407 10 g/L 2.51 mmol/L (Sarma et al. 2013b)
Dark fermentation Microbial mixed culture 90 % 0.66 mol/mol (Varrone et al. 2013b)
Dark fermentation Enterobacter aerogenes 20 g/L 25 mmol/L (Sarma et al. 2014)
Photofermentation Rhodopseudomonas palustris 10 mM 34 ml/g/h (Pott et al. 2013)
Dark fermentation Enterobacter aerogenes NRRL B 407 10 g/L 116.41 mmol/L (Sarma et al. 2013a)
Dark fermentation Klebsiella sp. TR17 11.14 g/L 0.27 mol/mol (Chookaew et al. 2014b)
Photofermentation Rhodopseudomonas palustris 50 mM 23–27 ml/g/h (Pott et al. 2014)
Dark fermentation Microbial mixed culture 20 g/L 9 L/L (Varrone et al. 2013a)
Dark fermentation Microbial mixed culture 50 % 0.55 mol/mol (Chookaew et al. 2014a)

saponified-free fatty acids (SFFA) were found as inhibi- light intensity (175 W/m2) (Ghosh et al. 2012a). Similar
tors of hydrogen production by Enterobacter aerogenes to dark fermentation, photofermentation is highly inhib-
NRRL B 407 (Sarma et al. 2013b). The SFFA was also ited by SFFA. Pott et al. (2013) demonstrated that SFFA
found as the most important inhibitor. The removal of is the most important inhibitor in crude glycerol, gener-
this inhibitor was evaluated using salting out and MgCl2 ating a reduction in the growth rate of R. palustris (Pott
reactions. Salting out removed 42 % of SFFA; however, it et al. 2013). To reduce the SFFA concentration in the
also decreased the carbon/nitrogen ratio, producing an crude glycerol, Pott et al. (2014) evaluated different tech-
inhibition of the H2 production. On contrary, addition of niques (ethanol and activated carbon, pH adjustment,
MgCl2 transformed SFFA into its inactive form (scum), solvent extraction, and precipitation of the fatty acids
increased H2 cumulative production (34.70 %), and glyc- with calcium) to reduce SFFA concentration. The best
erol utilization (2.5-fold) (Sarma et al. 2014). The reactor two treatments included pH adjustment and SFFA pre-
type and configuration have been evaluated to increase cipitation with calcium salts. These two treatments (23–
the H2 production from glycerol. The employment of a 27 ml/g/h) generated similar hydrogen yields as highly
continuous reactor in the culture of Clostridium pasteu- purified glycerol (29 ml/g/h) (Pott et al. 2014). Besides
rianum augmented the production of H2 compared to the large hydrogen yields, photofermentation also needs
the batch reactor (Lo et al. 2013). The immobilization of to improve light use by R. palustris and this raises the
Klebsiella sp. TR17 in an upflow anaerobic sludge blan- need to develop economic hydrogen permeable photo-
ket (UASB) reactors increased the H2 yield and hydraulic bioreactors (Ghosh et al. 2012a; Sabourin-Provost and
retention time (Chookaew et al. 2014a). Hallenbeck 2009).
Photofermentation is the bio-hydrogen production
from organic matter in the presence of light. Photofer- Anaerobic digestion In anaerobic digestion, crude glyc-
mentation is normally performed using organic acids; erol is employed as the principal carbon source (Hutňan
however, some purple non-sulfur photosynthetic bacte- et al. 2013) or as a complementary carbon source (Siles
ria can also transform glycerol into bio-hydrogen directly López et al. 2009) to improve denitrification processes
(Ghosh et al. 2012a; Sabourin-Provost and Hallenbeck (Bodík et al. 2009) or methane generation (Álvarez et al.
2009). Rhodopseudomonas palustris, a purple non-sulfur 2010). Table 4 shows the details of the crude glycerol
photosynthetic bacterium, incremented the hydrogen anaerobic digestion. Hutňan et al. (2013) evaluated the
yield 6 times compared with Enterobacter aerogenes or efficiency of using crude glycerol in an UASB reactor.
E. coli (Ghosh et al. 2012b). Rhodopseudomonas palus- They improve the methane yield by diluting previously
tris increased hydrogen production in cultures with high acidified crude glycerol, these modifications generated
nitrogen concentration (Sabourin-Provost and Hallen- high chemical oxygen demand (COD) removal, and meth-
beck 2009) and by optimizing crude glycerol concentra- ane production (Hutňan et al. 2013). In the work of Siles
tion (30 mM), glutamate concentration (4.5 mM), and López et al. 2009, the stoichiometry reaction for crude
Plácido and Capareda Bioresour. Bioprocess. (2016) 3:23 Page 7 of 12

Table 4 Crude glycerol anaerobic digestion

Co-substrate Glycerol pH/temperature/ °C Retention time (days) Methane yield References
concentration (%)

Sewage sludge 1 (v/v) 5/35 33 2353 mL/day (Fountoulakis et al. 2010)

Sewage sludge 3 7/37 12.3 0.8 L/g TVS (Athanasoulia et al. 2014)
Sewage sludge 0.63 (v/v) 7/35.0 20 1.3 m3/L crude glycerol (Nghiem et al. 2014)
Wastewaters 47 6.88/35.0 30 2.17 g/L/day (Watanabe et al. 2013)
– 80 7.6–8.9/37 1 0.9 L/mL (Hutňan et al. 2013)
Orange peel waste 7.19/35 8.5–30.0 330 mL/g VS (Martín et al. 2013)
Pig manure 3 55 15 0.47 L/g−1 VS (Astals et al. 2013)
Pig manure and fish 8 7.0–7.2/35 15.6 16.4 L/kg COD/day (Álvarez et al. 2010)
waste
VS volatile solids, TVS total volatile solids, COD chemical oxygen demand

glycerol was found: C3H8O3 + aNH3 → bCH4 + cCO2 price (Bodík et al. 2009). However, anaerobic digestion
+ dC5H7NO2 + eNH4HCO3, where the values of a, b, c, did not produce products with higher value.
d, and e were 0.663, 1.648, 0.526, 0.041, and 0.622 mol, Anaerobic digestion is an option to utilize waste glyc-
respectively (Siles López et al. 2009). The evaluation of erol and produce energy. Similar to thermal conversion,
anaerobic treatment of crude glycerol was made using glycerol is not used as a main substrate; however, it can
pretreated glycerol (distilled and acidified) and granular be sold or supplied to other anaerobic digestion plants
or non-granular sludge. In this, work the highest methane or the biodiesel plant that can include anaerobic diges-
yield was obtained by granular sludge-distilled glycerol; tion reactors to treat the organic wastes produced from
even so, granular sludge and acidified glycerol achieved the biodiesel production. Anaerobic digestion is a more
the highest glycerol degradation (100 %) and an appreci- developed and less expensive technique than thermal
able methane yield (Siles López et al. 2009). conversion.
Crude glycerol has been used principally as a co-sub-
strate for different types of compounds such as sewage Biodiesel washing wastewaters
sludge, manure, and food wastes. Large concentration of After the transesterification reaction, the methyl esters
crude glycerol has been inhibitory in the anaerobic diges- are separated from glycerol and the biodiesel is subjected
tion of sewage sludge; in fact, the recommended concen- to purification process to remove impurities (Suehara
tration is around 0.6–3 %. However, the crude glycerol et al. 2005). The principal impurities are the remaining
addition increased the methane yield around 2–4 times oil and methanol, residual catalyst, soap, and glycerin
compared with the control (Fountoulakis et al. 2010; (Phukingngam et al. 2011). Impurities removal con-
Athanasoulia et al. 2014; Nghiem et al. 2014). Similar sumes approximately 20 L of water to 100 L of biodiesel
to sewage sludge, pig manure anaerobic digestion was (Suehara et al. 2005); this process is repeated 2–5 times
improved by the addition of crude glycerol. Alvarez et al. depending on the amount of impurities present in the
(2010) found that methane generation can be maximized methyl esters (Phukingngam et al. 2011). At the end, the
by anaerobic co-digestion of crude glycerol with pig washing process generates between 20–120 L of waste-
manure and fish wastes. The highest methane produc- water from 100 L of biodiesel produced (Rattanapan
tion rate (16.4 L CH4/kg COD/day) was reached mixing et al. 2011). Biodiesel washing wastewaters (BWW) are
89 % of pig manure, 4 % of fish wastes, and 8 % of crude alkaline (pH ≈ 9), have high chemical oxygen demand
glycerin (Álvarez et al. 2010). Likewise, Astals et al. 2013 (COD) (60,000–545,000 mg/L), and grease and oil (G&O)
incremented the methane generation in 180 % by adding (7000–44,300 mg/L) (Rattanapan et al. 2011). However,
3 % crude glycerol (Astals et al. 2013). Crude glycerol has these wastewaters will have low nitrogen and phospho-
been identified as a sustainable carbon source for denitri- rus compounds, which make their biological treatment a
fication processes. In the work of Bodik et al. (2009), the challenging topic (Suehara et al. 2005; Rattanapan et al.
addition of crude glycerol into a denitrification process 2011; Phukingngam et al. 2011). Biodiesel washing waste-
increased almost 2.5 fold the denitrification efficiency waters need to be treated before being released into the
with a reduction of 5 ppm NO3/100 L of crude glycerol environment because, without any such treatment, the
(Bodík et al. 2009). Crude glycerol anaerobic digestion existing compounds can produce drainage plugging,
manages large volumes, produces energy, and has a low and decrease biological activity of the sewage treatment
Plácido and Capareda Bioresour. Bioprocess. (2016) 3:23 Page 8 of 12

plants (Suehara et al. 2005). The general treatment meth- et al. 2011). Chemical coagulation utilizes an external
odology utilized over this residue starts with an initial coagulant agent to bond different particles of G&O or
pretreatment for G&O reduction followed by a treat- COD in small groups, which can be easily removed by
ment for COD reduction. However, in recent years, the the addition of a flocculating agent (Ngamlerdpokin et al.
application of adsorption technologies has modified the 2011). On the contrary, electrochemical coagulation
BWW treatment. Table 5 summarizes the treatment and utilizes an electrical field to generate its own coagulant
pretreatment technologies used for washing wastewaters. agent. In both coagulations, the most common flocculat-
BWW pretreatment avoids COD degradation inhi- ing agents produced or introduced are aluminum related.
bition (Phukingngam et al. 2011). The pretreatment Aluminum compounds are recognized by its best proper-
technologies are chemical or physicochemical. The ties in coagulation and flocculation processes (Siles et al.
chemical pretreatment utilizes strong acids to remove 2011). The most important factors in electrocoagulation
G&O (Ngamlerdpokin et al. 2011). In this pretreatment, are voltage, pH, and reaction time. In chemical coagula-
the protons from the acid produce a coalescence effect tion, the important factors are concentration, pH, and
over the oil drops making them to move until they reach reaction time (Ngamlerdpokin et al. 2011; Rattanapan
upper part of the solution (Rattanapan et al. 2011). Addi- et al. 2011). Coagulations have showed efficient removal
tionally, protons can neutralize residual alkali catalyst of G&O when they are utilized as pretreatment or treat-
in biodiesel wastewater and substitute the Na atom in ment agents. However, coagulation only could achieve
soap molecules formed in the transesterification reaction substantial COD removal when it is performed after a
(Ngamlerdpokin et al. 2011). Strong acids such as H2S04, previous pretreatment (Chavalparit and Ongwandee
HCl, and HNO3 have been employed in these pretreat- 2009; Jaruwat et al. 2010).
ments; however, the best results were obtained by H2S04 Biodiesel washing wastewater anaerobic treatment is
with a pH of 2 or lower (Rattanapan et al. 2011; Ngam- another option besides coagulation. Similar to coagu-
lerdpokin et al. 2011). The most used physicochemical lation, anaerobic digestion needs to pretreat BWW to
pretreatment is electrocoagulation. In this pretreatment, reduce G&O and COD (Bezerra et al. 2011). Pretreat-
the wastewater is placed in a reactor which produces ment processes allowed efficient COD and G&O reduc-
an electric field that generates flocculating agents (alu- tion using anaerobic treatment; nevertheless, high
minum related) capable of removing the G&O or COD concentrations of some chemicals from the pretreatment
particles (Chavalparit and Ongwandee 2009). can be harmful for the anaerobic process (Siles et al.
After pretreatment, BWW have been treated using 2011). In addition to COD and G&O removal, anaerobic
anaerobic digestion or coagulation agents. Coagulation is process produces considerable amount of methane, with
a physicochemical treatment that reduces G&O or COD removals around 278–305 mL CH4/g COD removed,
concentrations in the BWW. The principal types are elec- which is about 88 % of the possible methane yield from
trochemical and chemical coagulation (Ngamlerdpokin COD (Siles et al. 2011; Bezerra et al. 2011).

Table 5 Treatments applied to wastewater from biodiesel washing process

Pretreatment Treatment Initial COD COD Initial G&O References
(g/L) removal G&O removal
(%) (g/L) (%)

Dilution Anaerobic process 14.8 100 15.1 98 (Suehara et al. 2005)

Acidification Anaerobic process 56.4 99 3.3 84 (Phukingngam et al. 2011)
Acidification and Dissolved air flotation 150 90 15 90 (Rattanapan et al. 2011)
electrocoagula- Electrocoagulation 30 55 6 97 (Chavalparit and Ongwandee 2009)
tion
Acid protonation Chemical coagulation 312 99.6 4.2 97 (Ngamlerdpokin et al. 2011)
Acid protonation Electrocoagulation 312 97.5 4.2 98.2 (Ngamlerdpokin et al. 2011)
Acidification, Anaerobic process 300 100 – – (Siles et al. 2010)
electrocoagulation
Acid protonation Electrochemical coagulation 588 100 22 100 (Jaruwat et al. 2010)
Electrospun polystyrene 445 75 – – (Shirazi et al. 2013)
membrane
Acidification Chitosan flakes adsorption 0.015, 0.71–0.73, 0.12 90 0.68–1.19 67 (Pitakpoolsil and Hunsom 2013)
Acidification Chitosan flakes adsorption 0.021, 0.24–31, 0.3 97.6 0.680–1.130 95.8 (Pitakpoolsil and Hunsom 2014)
Plácido and Capareda Bioresour. Bioprocess. (2016) 3:23 Page 9 of 12

In the last few years, BWW has been treated using used dark fermentation to produce hydrogen from pre-
adsorption and membrane technology to reduce the treated and untreated jatropha seed cake. The highest spe-
COD and G&O concentrations. Shirazi et al. 2013 evalu- cific bio-hydrogen yield (68.2 mL H2/g VS) was achieved
ated electrospun microporous membranes before and using 2.5 g VS of untreated jatropha seed cake; however,
after surface modification to reduce the COD from the largest cumulative concentration was achieved with 10
BWW. The modified membrane achieved the largest g VS (Lopes et al. 2015). Likewise, Panagiotopoulos et al.
reduction in COD (75 %). This technology did not use (2013) evaluated dark fermentation of cotton-seed cake.
any type of pretreatment to reduce COD or G&O con- Caldicellulosiruptor saccharolyticus produced significantly
centration, which is an advantage over the other BWW higher hydrogen yield (84–112 %) than the control. How-
treatments (Shirazi et al. 2013). Similar to membranes, ever, hydrogen was only generated when the cotton-seed
chitosan adsorption is another alternative for treating cake was pretreated with NaOH (10 %) (Panagiotopoulos
BWW. Chitosan flakes considerably reduce the con- et al. 2013). In a different approach, seed cakes have been
centrations of COD (90 %) and G&O (67 %), at 3.5 g/L used in solid-state fermentation to produce edible fungus
chitosan, pH 4, and 3 h of incubation (Pitakpoolsil and and enzymes (Veerabhadrappa et al. 2014; da Luz et al.
Hunsom 2013). To improve this treatment, four succes- 2013). da Luz et al. (2013) described the production of
sive treatments were tested. This modification enhanced the edible fungus Pleurotus ostreatus from jatropha seed
BOD, COD, and G&O removal up to 93.6, 97.6, and cake; this fungal strain degraded the antinutritional fac-
95.8 %, respectively (Pitakpoolsil and Hunsom 2014). tors in the jatropha seed cake (da Luz et al. 2013). Simi-
Anaerobic process and coagulation have similitudes in larly, Veerabhadrappa et al. (2014) reported proteases and
the degradation level; both processes achieved values up lipases from Aspergillus versicolor CJS-98 in the solid-state
to 85 %. The duration of the process is variable; anaero- fermentation of jatropha seed cake. The fermentation of
bic treatment is a slow process which takes 30–40 days jatropha seed cake with medium supplementation (malt-
(Bezerra et al. 2011); however, coagulation takes only ose and peptone 2 %), 40 % moisture, and 25 °C produced
20 min to 1 h (Ngamlerdpokin et al. 2011). Anaerobic maximum amount of proteases (3366 U/g) and lipases
process and membrane technologies have a reduced cost (1288 U/g) and removed jatropha seed cake anti-nutrients
compared with coagulation; the first ones do not have any (Veerabhadrappa et al. 2014). Pressed seed cakes and agri-
additional price and anaerobic digestion has the advan- cultural wastes from biodiesel crops have also been stud-
tage of producing energy from methane which could be ied for activated carbon production (Foo and Hameed
sold or utilized in the biodiesel plant. On the other hand, 2009; Nunes et al. 2009). Activated carbons from biofuel
coagulation processes need the presence of coagulants solid residues are produced via thermochemical conver-
and large quantities of energy to run the reactors; these sion, in a two-step process: first, the removal of residual
factors eventually increase the process costs. oil and second, the thermochemical activation using tem-
peratures between 600 and 800 °C under constant N2 flow
Treatment of other biodiesel residues (Nunes et al. 2009). From the biofuel agricultural residues,
Besides glycerin and BWW, biodiesel production pro- it is possible to produce low-cost activated carbons with
duces other residues. These are produced in lower quan- wide superficial area and good uptake capacity; however,
tities or have characteristics allowing the utilization of more research is needed to develop an activated carbon
conventional treatment techniques. Methanol is one of which can be applied in water purification or wastewater
these residues, and it can be easily recovered using dis- treatments (Foo and Hameed 2009).
tillation techniques. Methanol recovery systems such as Bentonites soaked in biodiesel also called spent earth
flash distillation (Wang et al. 2011) or vacuum distillation are a residue produced from biodiesel polishing process.
(Varanda et al. 2011) are available in the market. These In this step, saturated bentonites with a biodiesel concen-
techniques remove the methanol efficiently without tration of 20–50 % are obtained. The oil in the bentonites
affecting the transesterification process because of the can be treated by chemical extraction; similar to biodiesel
differences between the boiling points in their compo- production from vegetable oil. Besides that, this residue
nents (Varanda et al. 2011). has been evaluated to produce environmentally friendly,
Pressed seed cakes, spent earth, and agricultural wastes low-cost, and lightweight construction materials. In the
from biodiesel crops are also residues from the biodiesel work of Eliche-Quesada et al. (2012), glycerin and spent
production. Pressed seed cakes are the seed biomass left- earth were mixed with clay to produce lightweight bricks
over from oil extraction. This solid residue is normally (Eliche-Quesada et al. 2012). The addition of oil contain-
used as an organic amendment, animal feed, or is com- ing bentonites (at 15 %) into the clay generated ceramic
posted (Varanda et al. 2011). Recently, seed cakes have insulation bricks with reduced thermal conductivity and
been evaluated for hydrogen generation. Lopes et al. 2015 reduced energy costs (Eliche-Quesada et al. 2012).
Plácido and Capareda Bioresour. Bioprocess. (2016) 3:23 Page 10 of 12

Summary Bodík I, Blšťáková A, Sedláček S et al (2009) Biodiesel waste as source of

organic carbon for municipal WWTP denitrification. Bioresour Technol
Biodiesel is one of the most important sources of renew- 100(8):2452–2456. doi:10.1016/j.biortech.2008.11.050
able energy; however, biodiesel production is associated Casali S, Gungormusler M, Bertin L et al (2012) Development of a biofilm
with by-products and residues generation. Numerous technology for the production of 1,3-propanediol (1,3-PDO) from crude
glycerol. Biochem Eng J 64:84–90. doi:10.1016/j.bej.2011.11.012
techniques have been developed to utilize or to treat bio- Chavalparit O, Ongwandee M (2009) Optimizing electrocoagulation
diesel by-products or residues. These techniques have process for the treatment of biodiesel wastewater using response
shown that the above residues can be used to generate surface methodology. J Environ Sci 21(11):1491–1496. doi:10.1016/
S1001-0742(08)62445-6
bioproducts and renewable energy. These processes and Cheng D, Wang L, Shahbazi A et al (2014a) Catalytic cracking of crude bio-oil
techniques can also reduce environmental pollution and from glycerol-assisted liquefaction of swine manure. Energy Convers
increase the opportunities to generate additional income Manag 87:378–384. doi:10.1016/j.enconman.2014.06.084
Cheng D, Wang L, Shahbazi A et al (2014b) Characterization of the physi-
or reduce the costs of production of biodiesel. Inven- cal and chemical properties of the distillate fractions of crude bio-oil
tion of advanced technologies to deal with these residues produced by the glycerol-assisted liquefaction of swine manure. Fuel
could also make the biodiesel production process more 130:251–256. doi:10.1016/j.fuel.2014.04.022
Chi Z, Pyle D, Wen Z et al (2007) A laboratory study of producing docosahex-
sustainable and competitive against fossil fuels. aenoic acid from biodiesel-waste glycerol by microalgal fermentation.
Process Biochem 42(11):1537–1545. doi:10.1016/j.procbio.2007.08.008
Chookaew T, Prasertsan P, Ren ZJ (2014a) Two-stage conversion of crude
Abbreviations glycerol to energy using dark fermentation linked with microbial fuel cell
BWW: biodiesel washing wastewaters; COD: chemical oxygen demand; G&O: or microbial electrolysis cell. New Biotechnol 31(2):179–184
grease and oil; PHA: polyhydroxyalkanoates; PHB: polyhydroxybutyrate; SFFA: Chookaew T, O-Thong S, Prasertsan P (2014b) Statistical optimization of
saponified-free fatty acids; TVS: total volatile solids; UASB: upflow anaerobic medium components affecting simultaneous fermentative hydro-
sludge blanket; VS: volatile solids. gen and ethanol production from crude glycerol by thermotolerant
Klebsiella sp TR17. Int J Hydrogen Energy 39(2):751–760. doi:10.1016/j.
Authors’ contributions ijhydene.2013.10.141
JP was responsible of manuscript drafting and writing. SC was responsible da Luz JMR, Paes SA, Torres DP et al (2013) Production of edible mushroom
of final manuscript approval. Both authors read and approved the final and degradation of antinutritional factors in jatropha biodiesel residues.
manuscript. LWT Food Sci Technol 50(2):575–580. doi:10.1016/j.lwt.2012.08.006
Delgado R, Rosas JG, Gómez N et al (2013) Energy valorisation of crude
glycerol and corn straw by means of slow co-pyrolysis: production and
Acknowledgements characterisation of gas, char and bio-oil. Fuel 112:31–37. doi:10.1016/j.
The Colombian government and the Fulbright organization (FULBRIGHT- fuel.2013.05.005
COLCIENCIAS) are acknowledged for the financial support granted to Jersson Dobroth ZT, Hu S, Coats ER et al (2011) Polyhydroxybutyrate synthesis on
Plácido in the USA. biodiesel wastewater using mixed microbial consortia. Bioresour Technol
102(3):3352–3359. doi:10.1016/j.biortech.2010.11.053
Competing interests Echeverri DA, Perez WA, Rios LA (2013) Synthesis of maleated-castor oil glyc-
The authors declare that they have no competing interests. erides from biodiesel-derived crude glycerol. Ind Crops Prod 49:299–303.
doi:10.1016/j.indcrop.2013.05.008
Funding Eliche-Quesada D, Martínez-Martínez S, Pérez-Villarejo L et al (2012) Valoriza-
The Colombian Administrative Department of Science, Technology and Inno- tion of biodiesel production residues in making porous clay brick. Fuel
vation (COLCIENCIAS) and the Fulbright organization. Process Technol 103:166–173
Ethier S, Woisard K, Vaughan D et al (2011) Continuous culture of the micro-
Received: 27 February 2016 Accepted: 2 May 2016 algae Schizochytrium limacinum on biodiesel-derived crude glycerol
for producing docosahexaenoic acid. Bioresour Technol 102(1):88–93.
doi:10.1016/j.biortech.2010.05.021
Feng Y, Yang Q, Wang X et al (2011) Treatment of biodiesel production wastes
with simultaneous electricity generation using a single-chamber
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