Production of Aspergillus niger biomass on sugarcane distillery wastewater: physiological aspects and potential for biodiesel production
ChuppaT‑ostain et al. Fungal Biol Biotechnol
Production of Aspergillus niger biomass on sugarcane distillery wastewater: physiological aspects and potential for biodiesel production
Alain Shum Cheong Sing
Thomas Petit thomas.petit@univ‑reunion.fr
Background: Sugarcane distillery waste water (SDW) or vinasse is the residual liquid waste generated during sugarcane molasses fermentation and alcohol distillation. Worldwide, this effluent is responsible for serious environmental issues. In Reunion Island, between 100 and 200 thousand tons of SDW are produced each year by the three local distilleries. In this study, the potential of Aspergillus niger to reduce the pollution load of SDW and to produce interesting metabolites has been investigated. Results: The fungal biomass yield was 35 g L−1 corresponding to a yield of 0.47 g of biomass/g of vinasse without nutrient complementation. Analysis of sugar consumption indicated that mono‑ carbohydrates were initially released from residual polysaccharides and then gradually consumed until complete exhaustion. The high biomass yield likely arises from polysaccharides that are hydrolysed prior to be assimilated as monosaccharides and from organic acids and other complex compounds that provided additional C‑ sources for growth. Comparison of the size exclusion chromatography profiles of raw and pre‑ treated vinasse confirmed the conversion of humic‑ and/or phenolic‑ like molecules into protein‑ like metabolites. As a consequence, chemical oxygen demand of vinasse decreased by 53%. Interestingly, analysis of intracellular lipids of the biomass revealed high content in oleic acid and physical properties relevant for biodiesel application. Conclusions: The soft‑ rot fungus A. niger demonstrated a great ability to grow on vinasse and to degrade this complex and hostile medium. The high biomass production is accompanied by a utilization of carbon sources like residual carbohydrates, organic acids and more complex molecules such as melanoidins. We also showed that intracellular lipids from fungal biomass can efficiently be exploited into biodiesel.
Sugarcane distillery wastewater; Vinasse; Distillery spent wash; Aspergillus niger; Biomass production; Bioremediation; Biodiesel; Lipids
Sugarcane molasses fermentation and distillation into
rum lead to the production of wastewater called
stillage, vinasse, distillery wastewater or distillery spent
wash. Every produced litre of ethanol brings about from
10 to 18 litres of sugarcane distillery wastewater (SDW)
depending on distillation process and waste treatment
]. SDW is a dark brown effluent characterized by a
specific obnoxious odour, a high chemical oxygen demand
(COD) and a total organic carbon (TOC) that can reach
up to 120 and 17 g L−1 respectively [
]. According to
Wilkie et al. , COD is 4–5 times higher in sugarcane
molasse stillage as compared to sugarcane juice stillage.
Depending on the sugarcane origin and the industrial
process for ethanol production, intrinsic composition of
SDW can vary significantly. They generally have acidic
pH (from 3.8 to 5) due to the presence of organic acids
produced by the yeasts during the alcoholic
fermentation process [
]. A high mineral load was also reported
due to the presence of sulphur, potassium, phosphate,
calcium and sodium [
]. The high organic load of SDW
is mainly composed of melanoidins which are produced
through Maillard reactions between sugars and proteins
and caramels from overheated sugars that are responsible
for their colour and odour. Vinasse also contains other
refractory materials such as phenolic compounds,
anthocyanins, tannins and furfurans (for example hydroxyl
methyl furfural) which can reach up to 10 g L−1 [
The colloidal nature of caramels makes them resistant to
decomposition and toxic to microflora . SDW also
contains residual sugars and soluble proteins generated
by the fermenting yeasts [
All these characteristics combined with the high
volume of SDW produced worldwide cause significant
environmental issues. Over the last decades and due to
their high inorganic loads, SDW have been widely used
as agricultural fertilizer [
] but spreading is made now
statutory difficult due to their low pH, dark colour and
chemical content which may be responsible for
groundwater contamination and soil compaction [
high polluting loads lead to a modification of the soil
composition and can cause eutrophication of the
waterways because of the presence of proteins residues and
]. Moreover, melanoidins cause reduction
of sunlight penetration, of photosynthetic activity and of
dissolved oxygen concentration in natural aqueous
environment, whereas on land, they cause reduction of soil
alkalinity and inhibition of seed germination. In
consequence, phenolic compounds and melanoidins may
inhibit the activity of microorganisms contained in soils
and aquatic environments [
9, 10, 15
Several methods have been described in literature for
the use and disposal of SDW [
10, 15, 16
]. Among them,
aerobic treatment of SDW has been proposed for
decolourisation and COD reduction purposes. A number
of microorganisms, such as yeast and fungi were found
to be able to degrade melanoidins and to significantly
decrease the COD vinasse [
]. Preliminary experiments
performed in the lab (unpublished data) showed that
only a few molds are capable of growing on crude SDW,
such as Aspergillus strains and anamorphs. Aspergillus
niger is able to grow on a large variety of substrates, a
wide range of temperatures (6–47 °C) and pH (1.4–9.8),
explaining the ubiquitous occurrence of this species that
is encountered with a higher frequency in warm and
humid environments [
]. A. niger is also known to be a
good producer of extracellular enzymes with significant
industrial importance, including amylases, proteases,
pectinases, lipases as well as valuable molecules with
industrial interest such as citric, oxalic or gluconic acids
]. A. niger is also used for organic waste
enhancement  and its capacity to grow on diluted or
supplemented SDW was observed [
]. However, the
physiological growth characteristics of this
micro-organism cultured in crude sugarcane distillery spent wash
has not yet been reported. In addition, bioremediation
and potential valorisation of crude SDW were estimated
through the production of A. niger biomass as a valuable
source for biofuel.
Fungal strains, growth conditions and culture media
The strain used in this study was Aspergillus niger MUCL
28820 from BCCM (Brussels, Belgium) strain collection.
The strain was maintained routinely on potato dextrose
agar plates (PDA). A suspension of A. niger spores was
prepared as follow: spores, grown on PDA and incubated
at 28 °C for 72 h, were harvested using a glass loop and
suspended in sterile physiological water (NaCl 0.8%).
Cellular concentration was calculated using a
ThomaZeiss counting chamber. Growth experiments were
performed during 10 days, after inoculation with 100 µL of
spore suspension. Ten flasks containing 50 mL of
sterile SDW liquid medium at a starting concentration of
105 spores mL−1 were plugged with sterile cotton carded
and placed on a rotary shaker at 150 rpm at 28 °C. Assays
were performed in three independent biological
experiments. Every day, at the same hour, the biomass of three
flasks was harvested by filtration for further analysis (see
below) and this was repeated until day 10.
SDW medium was prepared as follows: raw SDW
(85 °C) was harvested in decontaminated barrel directly
from the output of the distillation column from
distillery “Rivière du Mât” (Saint-Benoit, Reunion Island) and
cooled to room temperature. SDW from the distillery
still contains the residual inactivated yeast biomass used
during alcohol fermentation. SDW samples were
harvested during the sugar production period (i.e. between
July and December) in 2012 and in 2014 and were frozen
and stored at − 20 °C until use. For each experiment, a
new batch of frozen DSW was thawed and then sterilized
by autoclaving for 20 min at 121 °C. Such autoclaved
DSW medium was microbiologically stable over time
(Additional file 1).
After filtration of 50 mL of SDW (through a cellulose
filter paper Whatman No. 1—porosity 11 µm), the
filtrates and filters containing the total suspended solids
(see example on Fig. 1) were both dried during 24 h in an
oven at 105 °C. The obtained dried masses were reported
to 50 mL allowing to determinate the corresponding
concentrations in total dissolved solids (TDS) and total
suspended solids (TSS), respectively. Mineral matters
present in the SDW filtrates were measured according
to Analytical Procedure of National Renewable Energy
Laboratory by incineration of 10 mL of SDW filtrates at
550 °C for 3 h [
]. The pH of the SDW filtrate was
measured using a pH-meter Denver Instrument. Soluble COD
and Total Nitrogen (TN) determinations were carried out
on the SDW filtrate using a DR 2800 spectrophotometer
(Hach Lange, Dusseldorf ) and the appropriate analytical
]. Samples were adequately diluted with sterile
deionized water and analysed according to
manufacturer’s instructions. SDW filtrates were diluted to 1/100 and
their optical density was measured at 475 nm using a
spectrophotometer Genesys 10 UV Deionised water was
used as blank.
Lipid accumulation medium (LAM) contained 30 g L−1
glucose, 1.5 g L−1 yeast extract, 0.5 g L−1 NH4Cl, 5.0 g L−1
Na2HPO4 (12H2O), 7.0 g L−1 KH2PO4, 1.5 g L−1 MgSO4
(7H2O), 0.1 g L−1 CaCl2 (2H2O), 0.01 g L−1 ZnSO4
(7H2O), 0.08 g L−1 FeCl3 (6H2O), 0.1 mg L−1 CuSO4
(5H2O), 0.1 mg L−1 Co(NO3)2 (6H2O), 0.1 mg L−1 MnSO4
(5H2O) and pH was adjusted to 5.5 according to [
Fungal biomass determination
The concentration of total suspended solids (TSS) in the
broth medium of each culture flask was determined by
filtration of 50 mL of SDW (treated or not by A. niger)
through a cellulose filter paper Whatman No. 1 (porosity
11 µm) previously dried for 24 h at 105 °C. The
insoluble suspended solids kept on the filter (see example on
Fig. 1) were dried during 24 h in an oven at 105 °C and
the obtained dry mass was weighed to provide TSS
concentration. Therefore, TSS contained the fungal biomass
produced during growth of A. niger as well as the initial
suspended yeast biomass contained in raw SDW. Fungal
biomass was thus estimated by subtracting the total
suspended matter of raw SDW to the total mass harvested
on the filter.
Determination of carbohydrates and organic acids
from filtrates of crude and pre‑treated SDW
The carbohydrate concentration of the filtrates
collected from crude SDW and SDW treated with A. niger,
were analysed by High-Pressure Liquid
Chromatography (HPLC) (Dionex Ultimate 3000) using an
Evaporative Light Scattering (ELS) detector (VARIAN) and
a Hi-plex Ca column (Varian, C18 bound—7.7 mm
of diameter × 300 mm of length). A mobile phase of
ultrapure water with a flow of 0.4 mL min−1 was used.
The oven temperature was programmed at 80 °C.
Alternatively, High-Pressure Ion Chromatography (HPIC)
(Dionex) using a pulsed amperometric detector and a
CarboPack PA1 column was used. A mobile phase of
NaOH (150 mM) at 1.5 mL min−1 was used at an oven
temperature of 30 °C. Analysis of organic acids was also
performed by HPLC (Dionex Ultimate 3000), using a
UV detector at 214 nm and an OA Acclaim column
(Varian, Silica, C18 bound, reverse phase, 4.6 mm of
diameter × 150 mm of length). The mobile phase was
composed of 100 mM Na2SO4 set at pH of 2.65 with
methanesulfonic acid (Sigma-Aldrich, CAS number
75-75-2) and the flow rate was 0.6 mL min−1. For all
analyses, 20 μL of samples diluted 100-fold for organic acids
and 50-fold for carbohydrates in water were injected
using an automatic autosampler. The identification and
the quantification of carbohydrates (mannitol, glucose,
fructose, sucrose) and organic acids (itaconic acid,
transaconitic acid, citric acid, isocitric acid, oxalic acid) were
made by determination of retention time of the
commercial standards and establishment of calibration curves
using external standard method. Treatment of the results
was done using Chromeleon 7.2 Chromatography Data
SEC profiles obtained from filtrates of crude and pre‑treated
A 5-days fermented SDW was chosen for this
experiment because at this stage of the growth
(mid-exponential phase), most of the sugars and organic acids remains
unchanged while biomass already reached more than
10 g L−1 DW, suggesting that others classes of molecules
were used preponderantly for growth of the cells. The
filtrates of crude SDW and 5-days treated SDW with A.
niger were ten times diluted with phosphate buffer (pH
7.0) and filtered (through a 0.45 µm filter) before
injection in a HPLC system Äkta-Purifier (GE Healthcare). As
previously described by [
], two SEC columns were
connected in series in order to obtain a wide resolving range:
the Superdex peptide 10/300 GL column with a
resolving range from 0.1 to 7 kDa was placed before the
second Superdex 200 10/300 column (GE Healthcare) with
a resolving range from 10 to 600 kDa. A similar volume
of 0.1 ml of the two samples previously diluted in PBS
was injected and elution of the molecules was performed
at room temperature using a 50 mM potassium
phosphate buffer (pH 7.0) as the mobile phase at a flow-rate of
0.4 mL min−1 and fractions of 2 mL each were collected.
The Unicorn 5.1 software (GE Healthcare) delivered on
the Akta purifier allows to either multiply or divide the
chromatograms with a constant factor: depending on
the total COD concentration of the sample, the obtained
chromatogram can be thus standardized per mg of COD.
Peak area integration of the standardized chromatograms
was performed by the Unicorn 5.1 software. The SEC
columns were calibrated for molecular weight
determination using a mixture of standard proteins of known
molecular weight between 12 and 669 kDa (HMW and
LMW calibration kits, GE Healthcare). Calibration
showed a linear relationship between the log of
molecular weight (MW) and the elution volume (Ve) of the
standards according to the following equation:
Log (MW) = −0.1536 Ve + 8.5794 (1)
R2 = 0.9976
with MW expressed in Da and Ve in mL.
EEM profiles obtained from filtrates of crude and pre‑treated
A three-dimensional excitation emission matrix (3-D
EEM) was determined on the SDW filtrates (raw or
treated with A. niger) and on the SEC fractions, using a
spectrofluorophotometer (Shimadzu RF-5301 PC) with
a 150-W Xenon lamp as the excitation source.
Excitation scans were performed from 220 to 450 nm at 10 nm
increments; emission scans were collected from 220 to
500 nm. The fluorescence data was processed using the
Panorama Fluorescence 3.1 software (LabCognition,
Japan). Prior to measurements, fractions of SEC samples
were diluted by 3–100 times using 50 mM phosphate
buffer (pH 7.0 ± 0.1) to avoid fluorescence signal
saturation. However, due to the impact of water noise, only
emissions obtained at excitation wavelengths
exceeding 275 nm were considered for a wavelength emission
exceeding 375 nm. Gallic acid (Sigma), used as
polyphenols standard [
] was also diluted in phosphate buffer
for analysis. Fluorescence was measured using a 1.0 cm
Lipid extraction from A. niger biomass and conversion into biodiesel
Intracellular lipids were extracted using a pressurized
liquid extraction method (PLE). 200 mg of lyophilized
biomass was mixed with Fontainebleau sand to fill a 10 mL
stainless steel vial suitable for PLE. The extraction was
carried out using chloroform/methanol (2/1) at 100 °C
during 10 min (three times), then 10 mL of water was
added to the extract and thoroughly mixed. Two phases
were obtained after overnight separation. The organic
phase was dried over anhydrous MgSO4, filtered and
concentrated using a rotative evaporator. Finally, the
concentrate was suspended in 3 mL CHCl3, transferred
to a pre-weighed bottle and evaporated overnight. The
bottle was weighted to determine the mass of extracted
lipids. Transesterification was performed according to a
procedure described by [
]. Briefly, 5 mL of 2% H2SO4/
CH3OH (v/v) was added to the extracted lipids and the
mixture was reflux heated at 70 °C during 1 h under
constant stirring. The flasks were then cooled at room
temperature. Next, 2 mL of hexane and 0.75 mL of
distilled water were added to the flasks and mixed. The two
phases were allowed to separate and the upper hexane
layer was recovered and dried over anhydrous
Analysis of the fatty acid composition was
carried out on a CP3800 Gas chromatograph
(Varian) equipped with a SG BPX-70 capillary column
(50 m × 0.22 mm × 0.25 µm) and a flame ionization
detector. The operating conditions were 240 °C injector
temperature, 260 °C detector temperature, 1.3 mL min−1
flow rate and oven temperature programmed from 120 to
230 °C at 3 °C min−1 then 230 °C for 17 min. 0.5 µL of
transesterification product was injected and subjected to
a split ratio of 5 at 0.5 min then 50 at 5 min. The
percentage of the peak area was assumed to be the percentage
content of the corresponding compounds.
Results and discussion
Physico‑chemical characteristics of SDW from Reunion
Physico‑chemical parameters of raw SDW
To characterize our raw materials, main
physico-chemical parameters of the collected SDW samples were
assayed. Results are presented in Table 1. pH value of
raw SDW (4.6 pH units) was comparable to average pH
values (3.8–4.6) reported by [
] for SDW from
others countries. Similarly, COD (107 g L−1) and TDS
(114 g L−1) of SDW from Reunion Island were in the
same range of order than the one reported for SDW
from different origins that ranged from 42 to 121 and
from 80 to 100 g L−1 respectively, with the outstanding
exception of TDS of Brazilian SDW that reached up to
152 g L−1 [
]. Chemical composition of SDW filtrate
SDW was incubated aerobically during 10 days with A. niger as explained in
TDS total dissolved solids, TSS total suspended solids, COD chemical oxygen
demand, TN total nitrogen, C/N carbon/nitrogen, OD475nm optical density
measured at 475 nm
a Except for TSS that were measured on insoluble suspended solids
showed a TN content of 2.32 g L−1 and a total mineral
content of 38.5 g L−1. The first parameter was globally in
good agreement with data of SDW from different
southern countries, i.e. 1.23–4.8 g TN L−1 whereas the latter
was higher than literature data namely 10.7–28.9 g L−1
]. Overall, these physico-chemical parameters
confirmed that SDW from Reunion Island are industrial
wastes with high polluting organic and mineral loads
that can be responsible for dangerous environmental
Physico‑chemical parameters of SDW after treatment with A.
To assess the bioremediation potential of A. niger, the
physico-chemical parameters of SDW were measured
10 days after the inoculation of the fungal spores in SDW.
As shown in Table 1, a pH increase (from 4.6 to 5.4) and
a decrease in OD475nm (linked to decolourisation) were
observed during aerobic fermentation of SDW. TDS were
significantly reduced from 114 to 89 g L−1 and this
essentially concerns organic matter reduction since the
mineral load was not significantly modified. A reduction of
COD and TN by 53 and 27% respectively were observed,
indicating a significant decrease of the organic
pollutant load of SDW. The pH increase could result from the
degradation of organic substances with peptidic moieties
or with amino group like humic substances,
melanoidins, peptides or amino acids initially contained in SDW
medium. The carbon/nitrogen (C/N) ratio remained
globally unchanged indicating that the fertilizing
potential of SDW remained the same after the fermentation
Bioremediation potential of A. niger on SDW was
partially described in literature. A maximal colour
elimination of 69% and a maximal COD removal of 75% were
obtained when MgSO4, KH2PO4, NH4NO3 and a
carbon source were added to SDW [
]. Also, immobilized
A. niger resulted in a 80% decolourisation of previously
anaerobically biodigested SDW [
]. Finally, the observed
COD and colour decrease suggested that refractory
molecules like melanoidins and other aromatic compounds
were hydrolysed into simple ones. Such hydrolysis of
some refractory compounds may contribute to the strong
decrease of the measured COD (− 53%) because of the
release of acidic moieties impacting on the oxidation
degree of the polymers. In this way, qualitative
characteristics of organic matter from raw and pre-treated SDW
Physiology of A. niger cultured on SDW
Global biomass production
Concomitantly to the modification of some
physicochemical parameters, important biomass production was
observed in the SDW medium after 10 days of A. niger
aerobic growth (Fig. 1 and Table 1). Fungal growth was
evaluated by measurement of the total suspended
solids in the broth medium that reached 43.4 g L−1 after
10 days. Given that the residual yeast biomass contained
in raw SDW amounted to 8.1 g L−1, a net production of
35.3 g L−1 of fungal biomass in SDW after 10
fermentation days could then be estimated. In addition to carbon
containing substrates, residual dead yeasts contained
in raw SDW (inactivated during the distillation
process and newly sterilised before use) are most likely to
play a role during growth such as bringing important
nitrogen source. Consequently, SDW was considered
as an interesting growth medium for A. niger biomass
production. In their study, Oshoma et al. [
demonstrated that the final concentration of A. niger biomass
could be increased from 1.63 to 2.75 g L−1 Dry weight
(DW) after nitrogen supplementation of cassava whey
by yeast extract (2 g L−1). By comparison, growth of A.
niger on SDW from Brazil distilleries in which the yeast
biomass was removed led to a biomass production of
8–13 g L−1 DW [
]. Here, the biomass production was
much higher since until 35 g L−1 of A. niger biomass can
be produced after 10 days on raw sugarcane vinasse
without any supplementation. Considering that total organic
matter of raw vinasse corresponds to TDS without ashes
(75.5 g L−1), a high biomass yield of 0.47 g g−1 on initial
organic compounds can be reached. This yield is similar
to that obtained by [
] that investigate the capability of
A. niger to utilize lignocellulose-derived compounds after
thermochemical pre-treatment of spruce wood chips.
However, because of the presence of fermentation
inhibitors, the pre hydrolysate medium had to be diluted 2 or 4
times to allow A. niger growth with a maximal
volumetric biomass yield of 7 g L−1 and a biomass yield on initial
carbon source of 0.46 g g−1.
To explain the physiological behaviour of A. niger on
raw SDW, carbohydrate content was monitored in the
medium during the 10 days of fermentation process
(Fig. 2). During the first 48 h, residual concentration of
glucose, fructose and mannitol was increased by a
factor of 2 and a maximal concentration of 7 g L−1 of
fructose, 1.6 g L−1 of glucose and 4 g L−1 of mannitol were
measured 2 days after inoculation of the fungal spores
in the SDW medium. In the meantime, total fungal
biomass increased slightly up to 4.37 g L−1. Accumulation of
these monosaccharides in the early stage of the
exponential growth phase strongly suggested that some complex
compounds were readily released by hydrolytic enzymes
secreted by A. niger. In a second period, from 48 h (day
2) to 192 h (day 8), fructose, glucose and mannitol were
gradually consumed, until complete exhaustion that
occurred at 168 h (day 7) for glucose and fructose, and at
192 h (day 8) for mannitol. Fungal biomass that increased
very weakly in the first period then suddenly increased
during the period of sugars assimilation (from day 2 to 8,
it increased from 0.8 to 25 g L−1) and yet increased even
after complete sugars exhaustion to reach 35.29 g L−1 at
day 10 (Fig. 2). Jin et al. [
] also observed that
monocarbohydrates were initially accumulated before being
taken up for conversion into mycelial biomass (from 7.5
to 9.2 g L−1) during aerobic fermentation of a raw starch
processing wastewater, with either Aspergillus oryzae or
Rhizopus oligosporus. This accumulation is most likely
occurring when the rate of complex polymers
hydrolysis is higher than the rate of carbohydrate uptake for cell
When looking more carefully at the biomass
production profile (Fig. 2), initial growth occurring during the
first 120 h (day 5) did not appear to occur exponentially,
but rather linearly. This observation would strengthen
the hypothesis that the growth is mainly limited by the
availability of fermentable sugars which are slowly and
linearly produced through the activity of specific
hydrolases from A. niger acting on complex polymers.
Organic acids utilization
It is known that SDW naturally contains large amount
of organic acids [
]. Some of them were assayed in raw
SDW filtrate and concentration of 5.7 ± 0.51 g L−1
for trans-aconitic acid, 2.8 ± 0.76 g L−1 for citric acid,
2.5 ± 0.47 g L−1 for isocitric acid, 0.7 ± 0.25 g L−1 for
itaconic acid and 0.6 ± 0.18 g L−1 for oxalic acid were
measured (Table 2). When sugars are being consumed by the
cells, the concentration of itaconic, isocitric and oxalic
acids tended to increase in the culture medium (+ 26,
+ 12 and + 136% respectively). An inverse relationship
between consumption of sugars and organic acid
production was already observed by [
] who reported that the
maximum acid production was found for 6 days old A.
niger cultures. Opposite tendency was noticed for citric
and trans-aconitic acids that were slightly consumed
during the first 7 days of growth. However, except for
itaconic acid for which concentration remained stable, all
organic acids were consumed partially or completely in
the remaining 3 days (Table 2). These data indicated that
organic acids were preferentially consumed during the
second period of fermentation after complete exhaustion
of monosaccharides. These carbon sources could be the
result of hydrolysis of melanoidins, polyphenols or
proteins present in crude SDW [
Taken together, these results showed that growth of A.
niger on SDW is a complex process. Free carbohydrates
initially present in the media (namely glucose,
fructose and mannitol) and other fermentable sugars
probably released from complex polymers through hydrolytic
activity of the fungal enzymes, are first consumed during
the early growth phase. When free sugars disappeared
from the medium (after 7–8 days of culture), growth
continued on the free organic acids accumulated in
the medium as well as other sugars released by A. niger
SDW biochemical fingerprints
Global EEM profiles of raw and pre‑treated SDW
Three-dimensional excitation emission matrixes (EEM)
were determined on the SDW filtrates in order to detect
potential modification of complex dissolved organic
matter like melanoidins or phenolic acids during A. niger
fermentation (Fig. 3). EEM is a widely used
non-degradative method for qualitative characterization of the
soluble substances of many effluents [
]. As proposed
], for typical wastewater spectra, the EEM can be
divided in at least two regions: (1) the region with
emission wavelength λEm < 380 nm which is associated with
fluorescent molecules types A and B containing a limited
number of aromatic rings like phenols, indole moiety and
free tryptophan; and (2) the region with λEm > 380 nm
which is associated with polycyclic aromatic
fluorophores (types C and D) such as Humic acid, flavonoid
and quinone. In addition, EEM of pure gallic acid was
performed to locate more precisely its associated zone:
EEM showed a fluorescent peak (250 < λEx < 275 nm and
325 < λEm < 370 nm) that was included in the phenolic
acid-like (PA-like) region (type B). The determination of
PA-like area zone in SDW is in accordance with [
worked on partially degraded food waste and undigested
Concerning the SDW media, both matrixes were
composed by four peaks with similar excitation/emission
wavelengths (λEx/λEm) position and intensity (Fig. 3a, b):
(1) for λEm < 380 nm, peak A and peak B were located
in the regions corresponding to protein-like (PN-like)
and phenolic acid-like (PA-like) compounds respectively
] and peak A was much more intense than peak B (2)
for λEm > 380 nm, peaks D and C were associated with
quinine-like components and could be related to humic
acid-like (HA-like) substances [
]. These results were in
good accordance with the results obtained by [
highlighted these groups of fluorophores (A, B and C-D
areas) in sugarcane vinasse. In this way, EEM determined
on the soluble SDW fractions (treated or not) did not
allow to clearly show EEM modifications pattern related
to A. niger metabolism (four independent replicates were
analysed; only one replicate was shown). This can be
explained by the complexity of the SDW medium that
contains highly fluorescent molecules possibly covering
the detection of other ones. Moreover, only specific
molecules with aromatic ring are detected by EEM.
EEM profiles after size fractionation of raw and fermented
In order to evaluate whether the SDW has been altered
by A. niger treatment, size fractionation of raw and 5-days
fermented SDW was chosen to provide a synthetic view
of their composition and size distribution. SEC
chromatograms were first monitored by absorbance detection at
210 nm and 280 nm but raw and fermented SDW filtrates
displayed similar profiles (data not shown). Regarding the
EEM spectra of the two SDW samples (Fig. 3a, b), high
fluorescence intensities could be noticed on the PN-like
region (peak A). One common couple of wavelengths
(λEx/λEm = 221/350 nm) that was previously described
] for detection of tryptophan containing PN-like
molecules was then selected for SEC monitoring.
Fluorescent compounds detected in this region (peak A) were
reported by [
] as soluble microbial products associated
to microbial activity or to cellular material.
Fractionation of the raw and fermented SDW filtrates
was performed by SEC and could be divided in seven
fractions from F1 to F7 corresponding to increasing
elution volume and to decreasing apparent molecular
size (Fig. 4a). Quantitative repartition of each fraction
among all the eluted molecules was also evaluated after
peak area integration (Table 3). It can be noticed that
fermented SDW showed some early eluted molecules in the
F1 fraction that were not present in raw SDW. According
to the calibration curve (see “EEM profiles obtained from
filtrates of crude and pre-treated SDW” section), these
PN-like molecules that eluted around 24 mL had high
apparent molecular weight around 100,000 Da. Also,
molecules with very small molecular weight, eluted in
the F6 and F7 fractions, were found in both profiles with
a similar repartition. The PN-like molecules included in
the F2 fraction (around 1000 Da) were not fully digested
during A. niger fermentation since they still represent
25.7% of the total molecules (Table 3). On the other hand,
a drastic diminution of F3 peak, and an increase of the F5
peak were observed in fermented compared to raw SDW
filtrate (Fig. 4a, Table 3). It is possible that the decrease
of F3 from 31.6% in the untreated SDW to 8.73% after A.
niger is recovered in the F5 peak that has increased from
7.7 to 18.63% of the total SEC area. These observations
suggest that molecules with intermediate size (F3) might
have been partially hydrolysed in small molecules (F5)
after 5 days of A. niger fermentation in SDW. This is in
agreement with the fact that after a first growth period
of A. niger on released monosaccharides, other complex
polymeric molecules need to be hydrolysed to provide
additional carbon sources. The high apparent molecular
weight molecules detected in the fermented SDW (F1)
might thus correspond to enzymes secreted by the
biomass for hydrolysis of SDW carbon-containing polymers.
To further investigate the effect of A. niger
fermentation on the biochemical characteristics of vinasse,
F1 to F7 fractions were collected and their EEM were
determined (Fig. 4b). For better specificity towards
PN-like detection, the ratios A/B and A/C of maxima
fluorescence intensity for these different zones were
calculated (Table 3). As shown in Fig. 4b, EEM fingerprints
were quite similar for raw and fermented SDW. Globally,
F2 fraction was more enriched in HA-like substances
(ratio peak C/A more important for F2 fraction than
for others) whereas F7 fraction was especially enriched
in PA-like molecules (ratio peak B/A more important
for F7 fraction and especially for SDW filtrate). EEM
fingerprints of F3, F5 and F6 fractions were slightly
impacted by fermentation. For F3 and F5, the A/C ratio
was increased by a factor of 2.8 and 1.7 after
fermentation respectively whereas A/B ratio was unchanged. That
might be linked to the increase in PN-like and/or the
hydrolysis of HA-like molecules during A. niger
fermentation. On the other hand, concerning F6 fractions, the
A/C ratio was reduced by a factor 1.9, decreasing from a
value of 3.5–1.8 after fermentation. So, fermentation has
decreased the level of HA-like substances in fractions
F3 and F5 whereas these substances were recovered in a
higher amount in F6 fractions.
According to these results, some physiological aspects
of A. niger fermentation of raw SDW can be proposed:
(1) production of high apparent molecular weight (F1
fraction) and hydrolysed (F3 and F5 fractions) PN-like
molecules (2) hydrolysis of HA-like substances (F3 and
F5 fractions) in smaller HA-like molecules, (F6
fraction) inducing vinasse decolourisation. This approach
also demonstrated that SEC coupled with fluorescence
monitoring at λEx/λEm = 221/350 nm is a good alternative
for determination of vinasse biochemical fingerprints.
This strategy was previously used to show the impact of
biological aggregate sludge and origin of aggregate on
exopolymeric substances fingerprint for which number
of peaks and their intensity were easily identified with the
specific PN-like fluorescence detection [
Lipid extraction from A. niger biomass and total Single Cell
In an attempt to explore the potential of fungal biomass
for biodiesel production, the lipid content of the biomass
produced on SDW was measured and compared with the
one produced on a lipid accumulation medium (Table 4).
With almost 3 times more biomass produced on SDW as
compared to LAM, the lipid content of the fungal
biomass grown on SDW (6.94%) was slightly higher as
compared to LAM (5.89%). Comparatively, Zheng et al. [
showed that A. niger grown on glucose or xylose as sole
carbon source led to a production of 5.8 and 4.6 g L−1 of
biomass with a lipid content of 9.6 and 8% respectively.
Similarly, A. niger grown on bagasse led to a fungal
biomass of about 1.9 g L−1 with a lipid content of 13.6% [
Also, André et al. [
] showed that two different A. niger
strains cultivated in crude glycerol could produce up to
8.2 g L−1 of biomass with a lipid content of about 50%
(meaning about 3 g L−1 of lipids). Although lipid content
of fungal biomass produced on SDW is rather low (circa
7%), the high A. niger biomass yield on this medium
suggested that SDW can therefore constitute a good
alternative and cheap medium for biodiesel production.
The composition of the lipids extracted from biomass
produced on LAM and SDW was respectively 18.2 and
24.9% for palmitic acid (16:0), 28.2 and 17.2% for oleic
acid (18:1, n-9) and 39.4 and 42.7% for linoleic acid (18:2,
n-6). Stearic (18:0) and α-linolenic (18:3, n-3) acids were
produced to a lesser extent by A. niger on both media
(Table 4). Singh [
] reported that A. niger biomass
grown on glucose medium contained mostly linoleic
acid (50%) and to a lesser extent, palmitic, stearic and
linolenic acids (8.3, 5.2 and 6% respectively). Whatever
the medium used, linolenic acid appeared as the major
intracellular lipid of A. niger biomass; however, it can
be noticed that A. niger grown on glucose medium and
on LAM were richer in oleic acid than biomass grown
on SDW (23.5 and 28.19 against 17.23%) [
comparison, biodiesel from Yarrowia lipolytica [
contained twice higher oleate esters but less than three times
linoleate esters than biodiesel from A. niger grown on
SDW. This suggests that lipids produced from A. niger
could be an interesting alternative to the ones produced
by microorganisms such as yeasts [
Finally, the main relevant physical properties to assess
fuel quality of biodiesel from A. niger are presented in
Table 5. Whatever the growth media used (SDW or
LAM), the biodiesel derived from A. niger showed
similar properties for all the tested physical parameters such
as cetane number (φ), viscosity (η), density (ρ), higher
16:0: palmitic acid; 18:0: stearic acid; 18:1 (n‑9): oleic acid; 18:2 (n‑6): linoleic acid; 18:3 (n‑3): linolenic acid
a Lipid content expressed in gram of lipids per 100 g of dry weight biomass
Each data is the mean of three independent biological experiments
CN cetane number, HHV higher heating value, CFPP cold filter plugging point (1in winter, 2in summer), n.a not available
a According to European and American specifications biodiesel fuel blendstocks (B100), standard specifications EN 14,214 and D 6751‑08 for biodiesel fuel
blendstocks (B100) established respectively by the European Committee for Standardization (CEN) and American Society for Testing and Materials (ASTM). Data from
], c [
], d [
heating value (HHV—δ) and cold filter plugging point
(CFPP). Cetane numbers of biodiesel produced from
SDW and LAM media (respectively 57.65 and 58.88)
were both at least 13% better than the minimal
requirement of biodiesel proposed by the European and
American standards. For comparison, the biodiesel derived
from A. niger had similar cetane numbers to biodiesels
produced from coconut, tallow or yellow grease
(respectively 59.3, 58.9 and 56.9) [
]. In comparison, cetane
number of biodiesel from Y. lipolityca was 64.37 [
Also viscosities of biodiesel produced from A. niger
(3.52 mm2 s−1 on LAM and 3.47 mm2 s−1 on SDW) were
globally in the range of values suggested by the
European Standards (between 3.5 and 5 mm2 s−1). Although
there are no European or American specification for this
parameter, HHV of biodiesel from A. niger grown on
SDW (40.01 MJ kg−1) is considered as acceptable given
that biodiesel from all kind of sources are generally 10%
less energetic than diesel from petroleum (49.65 MJ kg−1)
]. Finally, CFPP value of biodiesel obtained from A.
niger grown on SDW was lower than 0 °C, meaning that
this biodiesel could be used at low temperature.
This study demonstrated that raw SDW contains suitable
organic substrates for growth of A. niger including
monosaccharides, organic acids and complex polymers. The
growth reached up to 35.29 g L−1 DW fungal biomass
with a biomass yield of 0.47 g per g of SDW organic
compounds. Aerobic fermentation of raw SDW led to vinasse
decolourization with pH increase and COD decrease,
dropping thus significantly the pollutant load.
Biochemical fingerprints revealed that high molecular weight
PN-like components were secreted by A. niger during
growth while some PA and/or HA-like molecules were
consumed. Intracellular lipids from biomass showed
good physical characteristics for use as biofuel giving
new insights for concomitant bioremediation and carbon
reuse of SDW medium.
Additional file 1. Data from Fig. 2 including standard deviations.
GCT, JH, MW, IG, IB, EGN and TP performed the experimental and laboratory
work. GCT, JH, ASCS, YC, IG, IB, JMF, EGN and TP worked on the analysis and
interpretation of the data and contributed with valuable discussions. GCT, JH,
LA, IB, JMF, EGN and TP conceived the project, worked on the structure and
wrote the paper. All authors read and approved the final manuscript.
1 Antenne sud du laboratoire de chimie des Substances Naturelles et des
Sciences des Aliments (LCSNSA), EA 2212, Université de la Réunion, UFR des
Sciences et Technologies, 15 Avenue René Cassin, CS 92003, 97744 Saint‑Denis
Cedex 9, France. 2 Laboratoire de Physique et Ingénierie Mathématique pour
l’Energie et l’Environnement (PIMENT), EA 4518, Université de la Réunion,
UFR Sciences de l’Homme et de l’Environnement, 117 rue Général Ailleret,
97430 Le Tampon, France. 3 Groupement de Recherche Eau Sol Environne‑
ment (GRESE), EA 4330, Université de Limoges, Faculté des Sciences et Tech‑
niques, 123 Avenue A. Thomas, 87060 Limoges Cedex, France. 4 LISBP, UMR
INSA‑ CNRS &/INRA 792, 135 Avenue de Rangueil, 31077 Toulouse Cedex 4,
France. 5 Laboratoire de Biotechnologies Agroalimentaire et Environnementale
(LBAE), EA 4565, Université de Toulouse III, Institut Universitaire de Technolo‑
gie, 24 Rue d’Embaquès, 32000 Auch, France. 6 Present Address: Département
Hygiène Sécurité Environnement (HSE), Institut Universitaire de Technologie,
Université de La Réunion, 40 Avenue de Soweto, 97410 Saint‑Pierre, France.
The authors declare that they have no competing interests.
Availability of data and materials
Consent for publication
Ethics approval and consent to participate
This article does not contain any studies with human participants or animals
performed by any of the authors.
This work was financially supported by the Regional Council of La Reunion
(French overseas territory).
Springer Nature remains neutral with regard to jurisdictional claims in pub‑
lished maps and institutional affiliations.
1. Lima AM , Souza RRD . Use of sugar cane vinasse as substrate for biosurfactant production using Bacillus subtilis PC . Chem Eng Trans . 2014 ; 37 : 673 - 8 .
2. Bhattacharyya A , Pramanik A , Maji S , Haldar S , Mukhopadhyay U , Mukherjee J . Utilization of vinasse for production of poly‑3‑(hydroxybutyrate ‑ cohydroxyvalerate) by Haloferax mediterranei . AMB Express . 2012 ; 2 : 34 .
3. Tewari PK , Batra VS , Balakrishnan M. Water management initiatives in sugarcane molasses based distilleries in India . Resour Conserv Recycl . 2007 ; 52 : 351 - 67 .
4. Wilkie AC , Riedesel KJ , Owens JM . Stillage characterization and anaerobic treatment of ethanol stillage from conventional and cellulosic feedstocks . Biomass Bioenergy . 2000 ; 19 : 63 - 102 .
5. Fuess LT , Garcia ML . Implications of stillage land disposal: a critical review on the impacts of fertigation . J Environ Manag . 2014 ; 145 : 210 - 29 .
6. Baez‑Smith C. Anaerobic digestion of vinasse for the production of methane in the sugar cane distillery . In: SPRI Conference on Sugar Processing Research , Águas de São Pedro, S.P. , Brazil . 2006 .
7. Rajagopal V , Paramjit SM , Suresh KP , Yogeswar S , Nageshwar RDVK , Avinash N. Significance of vinasses waste management in agriculture and environmental quality-review . Afr J Agric Res . 2014 ; 9 : 2862 - 73 .
8. Acharya BK , Mohana S , Madamwar D. Anaerobic treatment of distillery spent wash-a study on upflow anaerobic fixed film bioreactor . Bioresour Technol . 2008 ; 99 : 4621 - 6 .
9. España‑ Gamboa E , Mijangos‑ Cortes J , Barahona‑Perez L , Dominguez‑Maldonado J , Hernández‑Zarate G , Alzate ‑ Gaviria L. Vinasses : characterization and treatments . Waste Manag Res . 2011 ; 29 : 1235 - 50 .
10. Mohana S , Acharya BK , Madamwar D. Distillery spent wash: treatment technologies and potential applications . J Hazard Mater . 2009 ; 163 : 12 - 25 .
11. Bustamante MA , Paredes C , Moral R , Moreno‑ Caselles J , Pérez‑Espinosa A , Pérez‑Murcia MD . Uses of winery and distillery effluents in agriculture: characterization of nutrient and hazardous components . Water Sci Technol . 2005 ; 51 : 145 - 51 .
12. Khairnar P , Chavan F , Diware VR . Generation of energy from distillery waste water . Int J Sci Spiritual Bus Technol . 2013 ; 2 : 30 - 5 .
13. Biswas AK , Mohanty M , Hati KM , Misra AK . Distillery effluents effect on soil organic carbon and aggregate stability of a Vertisol in India . Soil Tillage Res . 2009 ; 104 : 241 - 6 .
14. Ansari F. Environmental impact of distillery effluent on vertical soil horizon due to leaching effect: an experimental approach . Int J Chem Environ Eng . 2014 ; 5 : 223 .
15. Pant D , Adholeya A . Biological approaches for treatment of distillery wastewater: a review . Bioresour Technol . 2007 ; 98 : 2321 - 34 .
16. Kanimozhi R , Vasudevan N. An overview of wastewater treatment in distillery industry . Int J Environ Eng . 2010 ; 2 : 159 - 84 .
17. Palacios‑ Cabrera H , Taniwaki MH , Hashimoto JM , Menezes HC . Growth of Aspergillus ochraceus, A. carbonarius and A. niger on culture media at different water activities and temperatures . Braz J Microbiol . 2005 ; 36 : 24 - 8 .
18. Schrickx JM , Raedts MJH , Stouthamer AH , Vanverseveld HW . Organic acid production by Aspergillus niger in recycling culture analyzed by capillary electrophoresis . Anal Biochem . 1995 ; 231 : 175 - 81 .
19. Quintanilla D , Hagemann T , Hansen K , Gernaey KV . Fungal morphology in industrial enzyme production-modelling and monitoring . Adv Biochem Eng Biotechnol . 2015 ; 149 : 29 - 54 .
20. Schuster E , Dunn‑ Coleman N , Frisvad JC , Van Dijck PWM. On the safety of Aspergillus niger-a review . Appl Microbiol Biotechnol . 2002 ; 59 : 426 - 35 .
21. Oshoma CE , Imarhiagbe EE , Ikenebomeh MJ , Eigbaredon HE . Nitrogen supplements effect on amylase production by Aspergillus niger using cassava whey medium . Afr J Biotechnol . 2010 ; 9 : 682 - 6 .
22. Rosalem P , Tauk S , Santos MCN . Efeito da temperatura, pH, tempo de cultivo e nutrientes no crescimento de fungos imperfeitos em vinhaca . Rev microbiologia . 1985 ; 16 : 299 - 304 .
23. Silveira Ruegger MJ , Tauk‑ Tornisielo SM . Biomass production by filamentous fungi in sugar cane vinasse medium supplemented with molasses . Arq Biol Tecnol . 1996 ; 39 : 323 - 32 .
24. Sluiter A , Hames B , Scarlata C , Sluiter J , Templeton D. Determination of ash in biomass (No . NREL/TP‑510‑42622). National Renewable Energy Laboratory of U.S. Department of Energy, Golden, US. 2008 .
25. Janke L , Leite A , Nikolausz M , Schmidt T , Liebetrau J , Nelles M , Stinner W. Biogas production from sugarcane waste: assessment on kinetic challenges for process designing . Int J Mol Sci . 2015 ; 16 : 20685 - 703 .
26. Suutari M , Priha P , Laakso S. Temperature shifts in regulation of lipids accumulated by Lipomyces starkeyi . J Am Oil Chem Soc . 1993 ; 70 : 891 - 4 .
27. Simon S , Païro B , Villain M , D'Abzac P , Van Hullebusch E , Lens P , Guibaud G . Evaluation of size exclusion chromatography (SEC) for the characterization of extracellular polymeric substances (EPS) in anaerobic granular sludges . Bioresour Technol . 2009 ; 100 : 6258 - 68 .
28. Cropotova J , Popel S , Parshacova L , Colesnicenco A . Effect of 1‑ year storage time on total polyphenols and antioxidant activity of apple fillings . J Food Packag Sci Tech Technol . 2015 ; 4 : 44 - 9 .
29. Hoarau J , Caro Y , Petit T , Grondin I . Evaluation of direct wet transesterification methods on yeast and fungal biomass grown on sugarcane distillery spent wash . Chem Eng Process Technol . 2016 ; 2 : 1032 .
30. Sangave PC , Pandit AB . Enhancement in biodegradability of distillery wastewater using enzymatic pretreatment . J Environ Manag . 2006 ; 78 : 77 - 85 .
31. España‑ Gamboa EI , Mijangos‑ Cortés JO , Hernández‑Zárate G , Maldonado JAD , Alzate‑ Gaviria LM. Methane production by treating vinasses from hydrous ethanol using a modified UASB reactor . Biotechnol Biofuels . 2012 ; 5 : 82 .
32. Ferreira LFR , Aguiar MM , Messias TG , Pompeu GB , Lopez AMQ , Silva DP , Monteiro RT . Evaluation of sugarcane vinasse treated with Pleurotus sajorcaju utilizing aquatic organisms as toxicological indicators . Ecotoxicol Environ Saf . 2011 ; 74 : 132 - 7 .
33. Sheehan GJ , Greenfield PF . Utilization, treatment and disposal of distillery wastewater . Water Res . 1980 ; 14 : 257 - 77 .
34. Miranda MP , Benito GG , Cristobal NS , Nieto CH . Color elimination from molasses wastewater by Aspergillus niger . Bioresour Technol . 1996 ; 57 : 229 - 35 .
35. Patil PU , Kapadnis BP , Dhamankar VS . Decolorisation of synthetic melanoidin and biogas effluent by immobilised fungal isolate of Aspergillus niger UM2 . Int Sugar J . 2003 ; 105 : 10 - 3 .
36. Cavka A , Jönsson LJ . Comparison of the growth of filamentous fungi and yeasts in lignocellulose‑ derived media . Biocatal Agric Biotechnol . 2014 ; 3 : 197 - 204 .
37. Jin B , Yan XQ , Yu Q , van Leeuwen JH. A comprehensive pilot plant system for fungal biomass protein production and wastewater reclamation . Adv Environ Res . 2002 ; 6 : 179 - 89 .
38. Khosravi‑Darani K , Zoghi A . Comparison of pretreatment strategies of sugarcane baggase: experimental design for citric acid production . Bioresour Technol . 2008 ; 99 : 6986 - 93 .
39. Agarwal R , Lata S , Gupta M , Singh P . Removal of melanoidin present in distillery effluent as a major colorant: a review . J Environ Biol (India) . 2010 ; 31 : 521 - 8 .
40. Li WT , Chen SY , Xu ZX , Li Y , Shuang CD , Li AM . Characterization of dissolved organic matter in municipal wastewater using fluorescence PARAFAC analysis and chromatography multi‑ excitation/emission scan: a comparative study . Environ Sci Technol . 2014 ; 48 : 2603 - 9 .
41. Lakowicz JR , editor. Principles of fluorescence spectroscopy . Boston: Springer; 2006 .
42. Huang M , Li Y , Gu G . Chemical composition of organic matters in domestic wastewater . Desalination . 2010 ; 262 : 36 - 42 .
43. Bhatia D , Bourven I , Simon S , Bordas F , van Hullebusch ED , Rossano S , Lens PNL , Guibaud G . Fluorescence detection to determine proteins and humic‑like substances fingerprints of exopolymeric substances from bio ‑ logical sludges performed by size exclusion chromatography . Bioresour Technol . 2013 ; 131 : 159 - 65 .
44. Pokhrel D , Viraraghavan T. Treatment of pulp and paper mill wastewater-a review . Sci Total Environ . 2004 ; 333 : 37 - 58 .
45. Soobadar A. Agronomic and environmental impacts of application of coal/bagasse ash and vinasse to sugarcane fields in Mauritius (PhD thesis) . Université d'Avignon , Avignon, France. 2009 .
46. Bridgeman J , Baker A , Carliell‑Marquet C , Carstea E. Determination of changes in wastewater quality through a treatment works using fluorescence spectroscopy . Environ Technol . 2013 ; 34 : 3069 - 77 .
47. Zheng Y , Yu X , Zeng J , Chen S. Feasibility of filamentous fungi for biofuel production using hydrolysate from dilute sulfuric acid pretreatment of wheat straw . Biotechnol Biofuels . 2012 ; 5 : 50 .
48. Singh A . Lipid accumulation by a cellulolytic strain of Aspergillus niger . Experientia . 1992 ; 48 : 234 - 6 .
49. André A , Diamantopoulou P , Philippoussis A , Sarris D , Komaitis M , Papanikolaou S . Biotechnological conversions of bio‑ diesel derived waste glycerol into added‑ value compounds by higher fungi: production of biomass, single cell oil and oxalic acid . Ind Crops Prod . 2010 ; 31 : 407 - 16 .
50. Katre G , Joshi C , Khot M , Zinjarde S , RaviKumar A . Evaluation of single cell oil (SCO) from a tropical marine yeast Yarrowia lipolytica NCIM 3589 as a potential feedstock for biodiesel . AMB Express . 2012 ; 2 : 36 .
51. Hoekman SK , Broch A , Robbins C , Ceniceros E , Natarajan M. Review of biodiesel composition, properties, and specifications . Renew Sustain Energy Rev . 2012 ; 16 : 143 - 69 .
52. Ramírez‑ Verduzco LF , Rodríguez‑Rodríguez JE , del Rayo Jaramillo ‑ Jacob A. Predicting cetane number, kinematic viscosity, density and higher heating value of biodiesel from its fatty acid methyl ester composition . Fuel . 2012 ; 91 : 102 - 11 .
53. Ramos MJ , Fernández CM , Casas A , Rodríguez L , Pérez Á . Influence of fatty acid composition of raw materials on biodiesel properties . Bioresour Technol . 2009 ; 100 : 261 - 8 .
54. Su YC , Liu YA , Diaz‑ Tovar CA, Gani R . Selection of prediction methods for thermophysical properties for process modeling and product design of biodiesel manufacturing (thesis) . Virginia Tech . 2011 .