Biodiesel-derived Crude Glycerol for the Fungal
Production of Lovastatin
Bernadeth S. Antonio1, 2, Hafiz M. Abd R.1, 3, Sophie Solchenbach1,
Alejandro Montoya1, Analiza P. Rollon2, Maria Auxilia T. Siringan4, Ali Abbas1*
1
School of Chemical and Biomolecular Engineering, The University of Sydney, Sydney, Australia
2
Department of Chemical Engineering, University of the Philippines, Philippines
3
Food Science Department, Universiti Putra Malaysia, Serdang, Malaysia
4
National Science Research Institute, Philippines
* E-mail: [email protected]
ABSTRACT
Aspergillus terreus ATCC 20542 is an important fungus for
lovastatin production, a cholesterol-lowering drug. In this
research, we attempted to produce lovastatin using the
fungus, with crude glycerol as an inexpensive carbon
source and a biodiesel industry-derived waste product.
The effect of varying concentrations of crude glycerol (10
g/L, 30 g/L and 50 g/L), incubation periods (2, 4, 6, 8, 10
and 12 days), nitrogen sources (ammonium sulfate, yeast
extract and soya waste) and nitrogen source
concentrations (4 g/L, 8 g/L and 12 g/L) were tested on
lovastatin production and biomass yield of A. terreus
ATCC 20542. Pure glycerol with a concentration of 30 g/L
along with 4 g/L of yeast extract was used in control
experiments. The highest lovastatin concentration, which
was 6.7 mg/L, was obtained in the culture amended with
10 g/L crude glycerol concentration. This value is 60%
higher than the value obtained in the culture with 50 g/L
crude glycerol (4.2 mg/L). In the presence of 4 g/L soya
waste, the 7-day old fungal culture produced 5.9 mg/L,
which was about 300% higher than the yield in yeast
extract-amended culture. These results suggest good
potential for valorisation of biodiesel-derived glycerol via
biochemical routes to value-added statin drugs.
I.
INTRODUCTION
Lovastatin is commonly used in medicine to lower blood
cholesterol, specifically the low density lipoprotein (LDL) or
simply known as bad cholesterol. Cholesterol is acquired by
our body through the food we intake but about two thirds is
produced in our liver. High cholesterol causes 4.4 million
deaths per year as reported by the World Health Organization
(2002). Because of the increasing number of diseases
associated with hypercholesterolaemia (i.e., elevated amount
of cholesterol in the blood) there is a need for cholesterollowering drugs, such as lovastatin.
With the high cost of lovastatin, averaging US$1.35 per 40 mg
lovastatin1, and with substantial generation of excess crude
glycerol attributed to the increasing demand for biodiesel,
conversion of crude glycerol to high value product like
lovastatin appears to be promising. Compared with pure
glycerol which costs US$1.11 per kg2, crude glycerol, at
US$0.11 per kg3, is also a cheaper carbon source for
biotechnological applications that are economically feasible.
Crude glycerol has been used as carbon source in many
industrial fermentations (Chatzifragkou, 2011). Biodieselderived crude glycerol as sole carbon source for microbial
cultivations has been found to be successful (Papanikolaou et
al., 2008).
Aspergillus terreus ATCC 20542 is a filamentous fungus
known for good production of lovastatin, a natural statin used
as cholesterol lowering drug (Lopez et al., 2003). A. terreus
ATCC 20542 was reported to produce lovastatin and has the
capacity to utilize lactose, pure glycerol fructose and glucose
(Lai et al., 2005; Lopez et al., 2003). Notably, Aspergillus
terreus ATCC 20542 has the capacity to survive harsh
environmental conditions, thus, this fungus is suitable to be
cultured in crude glycerol which contains impurities
(Thompson, 2004). Carbon and nitrogen sources were reported
to be the major factors in production of lovastatin. Long Shan
et al., (2004) noted that addition of methionine increased
lovastatin concentration by 20%. They also reported that
methionine was the best source of amino acid and methyl
groups for lovastatin production. Dong et al., (2005) reported
that soya wastes contain methionine in the form of crude
protein which is 23.8 % of the soya wastes. This paper
presents our recent study exploring the production of
1
2
3
www.pharmacychecker.com
www.biotechonologyforfuels.com
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lovastatin using biodiesel-derived crude glycerol as the carbon
source and soya wastes as the nitrogen source.
II.
EXPERIMENTALS
Glycerol Substrates
Pure and crude glycerol obtained from Biodiesel Producers
Limited, Australia were used as substrates for Aspergillus
terreus ATCC 20542. The biodiesel is a mixture of tallow and
used cooking oil. The crude glycerol was dark brown in color
with a pH of 6.5. The glycerol content of the crude glycerol
samples was 45% v/v as analyzed in this study. Impurities like
free fatty acids, methanol and salts were the major
contaminants of the crude glycerol (Thompson, 2004).
and soya wastes at varying concentrations, 4 g/L, 8 g/L and 12
g/L. The control culture contained 4 g/L yeast extract. Soya
waste was prepared by soaking the soya bean seeds in water
for 3 hours which were then dehulled, ground and boiled. The
soya emulsion was filtered by stainless steel filter. The soy
residue was considered as the soya waste. Soya wastes were
dried at 80oC for 8 hours and were pulverized (Dong et al.,
2005).
To evaluate the effects of the carbon and nitrogen sources on
the growth and lovastatin production, the following were
determined: dry biomass, concentration of residual glycerol
and lovastatin yield.
Production of Lovastatin by Aspergillus terreus ATCC
20542
Aspergillus terreus ATCC 20542 (American Type Culture
Collection) was inoculated onto Potato Dextrose Agar (PDA)
plates and incubated at 30 +1oC for 7 days. To prepare the
inoculum, 0.001% (v/v) Tween 80 in sterile deionized water
was added onto the culture and spores were dislodged using an
inoculating loop.
Dry Biomass Determination
Growth of A. terreus ATCC20542 in pure and crude glycerol
was assessed by determining the dry weight of the biomass.
To harvest the fungal biomass, the culture was aseptically
filtered using Whatman no. 2 filter paper inside a biological
safety cabinet. The collected biomass was washed twice with
distilled water and dried at 80oC for 24 hours or until constant
weight was achieved (Lai et al., 2004).
The fungal spore suspension containing 108 spores/ml, based
on direct spore count using a haemocytometer, was prepared
for all the experiments. To prepare the fungal inoculum, 100
µL spore suspension was inoculated in 50 ml pre-culture
medium. The pre-culture medium consists of the following in
(g/l): KH2PO4, 1.0 g, MgSO4·7H2O, 0.8 g, NaCl2 0.8 g and
ZnSO4·7H2O 0.001 g and yeast extract 8 g and 10 g lactose
(Lopez et. al., 2003). The medium was sterilized at 121oC for
15 minutes.
Analytical Method
Glycerol concentration was analyzed using the free glycerol
calorimetric detection kit (Sigma-Aldrich) at 575 nm using a
spectrophotometer as recommended by the supplier (SigmaAldrich). Production of lovastatin was assessed using High
Performance Liquid Chromatography or HPLC (Agilent,
USA) with the following parameters: 238 nm as detection
wavelength column temperature at 30˚C, chamber at 4˚C,
effluent flow rate at 1 ml/min. The analysis used XBD Eclipse
Zorbax C-18 column. The sample injection volume was 10
µL. The eluent was a mixture of 95% acetonitrile and 0.1%
phosphoric acid. Pharmaceutical grade lovastatin was used as
a standard as suggested by López et al. (2003).
The pre-culture set-up was then incubated in an orbital shaker,
at 185±5 rpm and 28±1˚C for 24 hours prior to lovastatin
production. The pre-culture was then filtered and inoculated
into a fermentation medium containing the same ingredients of
the pre-culture medium but with 4 g/l yeast extract and
varying crude glycerol concentrations. All the experiments
were performed at 28oC based on the studies of Casas et al.
(2003) and Siamak et al. (2003), in which pure glycerol,
lactose and fructose were used as carbon sources in lovastatin
production. The pH of the culture medium was not considered
as a major factor. Based on Lai and co-authors (2005), pH
regulation during fungal fermentation using biodiesel-derived
crude glycerol is not necessary.
To determine the optimum glycerol concentration for
lovastatin production, batch fungal cultures using the same
fermentation medium and conditions were grown at different
crude glycerol concentrations (10 g/l, 30 g/l and 50 g/l).
To determine the optimum nitrogen source and its
concentration, A. terreus ATCC 20542 was grown in preculture medium with optimum crude glycerol concentration.
Crude-glycerol amended cultures were grown in different
nitrogen sources, namely, yeast extract, ammonium sulphate
III.
RESULTS AND DISCUSSIONS
Optimization of Crude Glycerol Concentration with Preculture
Different concentrations of crude glycerol (10 g/L, 30 g/L and
50 g/L) were used to determine the effects of concentration of
glycerol substrates on lovastatin production and growth of
fungus. Pure glycerol with initial concentration of 30 g/L was
used as control.
Figure 1 shows the lovastatin produced in a culture with
varying concentration of crude glycerol as a function of
incubation period. For comparison purposes, the lovastatin
produced in a culture with pure glycerol is also shown. The
production of lovastatin is evident starting at day 4 at glycerol
concentrations of 10 and 30 g/L. The rate of lovastatin
production decreases using crude glycerol compared with the
control. At day 12, the lovastatin concentration (17.2 mg/L) in
the culture with 30 g/L pure glycerol was almost 270% higher
than the lovastatin concentration (6.7mg/L) in the culture with
30 g/L crude glycerol. This indicates that impurities in the
result since more nutrients would stimulate more growth while
limited or lower levels of nutrients would result to less
biomass. The increase in the biomass concentration as a
function of crude glycerol concentration, was likely due to the
increasing carbon to nitrogen (C:N) ratio. It has been reported
that a high C:N ratio under limited nitrogen medium results in
a greater biomass concentration (Lopez et.al., 2003).
Figure 1. Evolution of the lovastatin concentration during first twelve days
incubation period at 28+1oC and 185 + 5 rpm using different concentrations of
crude glycerol.
crude glycerol adversely affect the production of lovastatin.
This is more apparent in cultures amended with higher crude
glycerol concentrations. For example, the production of
lovastatin was observed only after 10 days of incubation at 50
g/L of crude glycerol. A relatively lower lovastatin yield of
4.2 g/L was noted after 12 days of fermentation.
Comparing the lovastatin concentration in the culture using
different concentrations of crude glycerol, lovastatin
concentration decreased as the concentration of crude glycerol
increased as shown (Figure 1). The highest lovastatin
concentration, which was 6.7 mg/L, was obtained in the
culture with 10 g/L crude glycerol concentration in which the
lovastatin yield was also the highest (0.54 mg lovastatin/gram
glycerol). This value is 60% higher than the value obtained in
culture with 50 g/L crude glycerol (4.2 mg/L).
The production of lovastatin continued to increase up to the
termination of the fermentation process in all cases, which was
at day 12. Lovastatin concentrations were highest at the 12th
day of fermentation in all cultures grown in different glycerol
concentrations. This was due to limited nutrients in which the
excess carbon was channelled to producing secondary
metabolite (lovastatin) rather than cell material synthesis for
growth (Barry and Wanniwright. 1997).
Figure 2 shows the biomass produced as a function of the
incubation period. The biomass concentration increased as the
crude glycerol concentration increased. At day 12, the highest
dry biomass concentration (16.1 g/L) was obtained in the
culture with 50 g/L crude glycerol concentration, while the
lowest biomass concentration (6.1 g/L) was obtained in the
culture with 10 g/L of crude glycerol. This was the expected
Figure 2. Biomass concentration during the first twelve days of incubation
period at 28+1 oC, 185+5 rpm using different concentrations of crude
glycerol; C:N ratio: 9 (10 g/L crude glycerol), 27 (30 g/L crude glycerol) and
45 (50 g/L crude glycerol).
The resultant biomass concentration of 12.2 g/L in the culture
with 30 g/L pure glycerol was 9.06% higher than with 30 g/L
crude glycerol 11.1 g/L. The values obtained after 12 days of
fermentation are however comparable, suggesting that the
growth of the fungus was not drastically inhibited by
impurities in crude glycerol such as free fatty acids and salts.
Figure 3 shows the residual glycerol during the first twelve
days incubation period. Glycerol consumption was fastest at
10 g/L crude glycerol concentration. With lower substrate
level, the crude glycerol at 10 g/L was consumed immediately
by Aspergillus terreus ATCC 20542 after 8 days of
fermentation. For cultures with higher crude glycerol (30 and
50 g/L) certain levels of glycerol were still available after day
8. This showed that impurities in the crude glycerol did not
inhibit the glycerol consumption by the fungus and that
impurities in the glycerol did not compete with glycerol as
carbon source in the fermentation.
that lovastatin productivity is higher when growth is inhibited
in a carbon limited environment.
TABLE I . Lovastatin concentration during the first twelve days
incubation period at 28+1oC, 185+5 rpm using 10 g/L crude glycerol
amended with different concentrations of ammonium sulphate.
Nitrogen Source
Figure 3. Residual glycerol during the first twelve days incubation period at
28+1oC, 185+5 rpm using different concentrations of crude glycerol.
Optimization of Lovastatin Production using Different
Nitrogen Sources and 10 g/L Crude Glycerol
The effects of different nitrogen sources on biomass growth
and lovastatin production by Aspergillus terreus ATCC 20542
were studied. Ammonium sulphate and yeast extract were
used as inorganic and organic nitrogen sources, respectively.
Table 1 shows the lovastatin concentration during the first
twelve days incubation period using ammonium sulphate as
nitrogen source. The culture amended with yeast extract had
higher lovastatin concentration and higher biomass
concentration than culture with ammonium sulphate. A 43%
increase in biomass concentration and 14% increase in
lovastatin concentration in the culture amended with yeast
extract may be attributed to the rich composition of amino
acids, carbohydrates, minerals and vitamins such as the B
complex which are present in yeast extract. Bizukojc et al.
(2008) reported that enhancement of lovastatin production was
attributed to addition of vitamin B groups in culture media
particularly the nicotinamide, calcium D-panthothenate and
pyridoxine, which are related to high requirement of reduced
NAD and CoA at the PKS stage lovastatin metabolism. The
results confirmed that yeast extract is a better nitrogen source
compared to ammonium sulphate.
Table 1 also shows that among different concentrations of
ammonium sulphate, the lovastatin concentration decreased as
the concentration of ammonium sulphate increased. The
highest lovastatin production was achieved in the culture with
4 g/L of ammonium sulphate which was 2.6 mg/L. This was in
constant agreement with the findings of Hajjaj et al. (2001)
Ammonium Sulfate, 4 g/L
Ammonium Sulfate, 8 g/L
Ammonium Sulfate, 12 g/L
Lovastatin Concentration
(mg/L)
2.6
1.3
0.2
Control (yeast extract, 4g/L)
2.9
Table 2 shows biomass concentration during the first
twelve days incubation period. The biomass concentration
increased as the concentration of ammonium sulphate in the
culture increased. Among different concentrations of
ammonium sulphate, the highest dry biomass concentration
(3.0 g/L) was obtained in the culture with 12 g/L ammonium
sulphate and the lowest dry biomass concentration (2.2 g/L)
was obtained in the culture with 4 g/L ammonium sulphate.
Apparently, higher biomass growth was attributed to higher
initial nitrogen concentration under the limited carbon
concentration.
TABLE II. Biomass concentration during the first twelve days
incubation period at 28+1oC, 185+5 rpm using 10 g/L crude glycerol amended
with different concentrations of ammonium sulphate.
Nitrogen Source
Ammonium Sulfate, 4 g/L
Ammonium Sulfate, 8 g/L
Ammonium Sulfate, 12 g/L
Control (yeast extract, 4g/L)
Biomass Concentration
(g/L)
2.2
2.8
3.0
4.3
Lovastatin Production using Soya Wastes as Nitrogen
Source
Industrial waste such as soya waste was tested as alternative
cheap source of nitrogen for lovastatin production. Table 3
shows the lovastatin concentration using crude glycerol
amended with soya waste as nitrogen source. Higher lovastatin
concentration was obtained in the culture with soya waste than
in the culture with yeast extract. Aspergillus terreus ATCC
20542 produced 5.9 mg/L after 7 days of fermentation using 4
g/L soya waste. This was about 2.70 times higher than the
lovastatin concentration in the culture with yeast extract which
is 2.2 mg/L. The higher lovastatin production can be attributed
to the amino acid present in the soya wastes as crude protein.
Among the relevant amino acids is the methionine, which is
the source of methyl group in lovastatin production. A typical
chemical composition of soya wastes showed that 23.8 % is
crude protein (Dong et al., 2005).
Table 3 shows lovastatin concentration and biomass
concentration using crude glyglerol and soya waste and yeast
extract as nitrogen sources. Yeast extract as nitrogen source
favored the growth of A. terreus ATCC 20542 indicated by
higher biomass obtained in the culture with yeast extract, that
is, 39% greater than that of soya waste supplemented culture.
However, lovastatin concentration was lower in the culture
with yeast extract by about three fold. Higher lovastatin
concentration was observed in soya waste-amended culture
which can be attributed to the methionine present in the soya
waste.
TABLE III. Lovastatin concentration and biomass concentration during the
first 7-day incubation period at 28+1 oC, 185+5 rpm with using 10 g/L crude
glycerol amended with soya waste as nitrogen source.
Nitrogen Source
Lovastatin
Concentration
(mg/L)
Biomass
Concentration
(g/L)
5.9
2.8
2.2
3.8
Soya waste, 4 g/L
Control (yeast
extract, 4 g/L)
Optimization of Soya Wastes as Nitrogen Source
Table 4 shows the lovastatin concentration and biomass using
different concentrations of soya waste and 10 g/L crude
glycerol. The lovastatin concentration decreased as the
concentration of the soya increased. Highest lovastatin
concentration of 12.1 mg/L was achieved when the lowest
concentration of soya wastes (4 g/L) was used. This indicates
that lovastatin production was inhibited by growth as earlier
discussed. Using different concentrations of soya waste as
nitrogen source, the highest concentration of soya wastes (12
g/L) gave the highest biomass concentration (10.1 g/L).
Increasing biomass concentration was probably due to
increasing initial concentration of nitrogen particularly in the
form of methionine, in soya wastes.
CONCLUSIONS
Aspergillus terreus ATCC 20542 was shown to produce
lovastatin using crude glycerol as carbon source which shows
the potential of crude glycerol for biotechnological
applications. While lovastatin yield with crude glycerolamended culture was lower than that of culture grown with
pure glycerol, using crude glycerol, an industrial waste, could
establish a more cost-effective lovastatin production. Crude
glycerol is an inexpensive alternative substrate for lovastatin
production by A. terreus ATCC 20542. The impurities in the
crude glycerol appeared not to inhibit the growth of the fungus
but negatively affected the production of lovastatin. It appears
that lovastatin concentration decreased as the concentration of
crude glycerol increased. Based on this study, A. terreus
ATCC 20542 produced the highest lovastatin yield (6.7 mg/L)
in the lowest crude glycerol concentration which is 10 g/L. As
with secondary metabolites, lovastatin production occurs at its
highest rate at the stationary phase, when glycerol has been
depleted.
Increasing concentration of nitrogen (i.e., ammonium sulphate
and soya wastes) at constant crude glycerol concentration
(10g/L) appeared to decrease lovastatin production and to
increase biomass growth. This observation indicates that
lovastatin production is a non-growth associated process as in
the case of production of most secondary metabolite.
The soya waste, an industrial waste, is a promising cheaper
alternative source of nitrogen. Lovastatin concentration was
about 300% higher in the culture with 4 g/L soya waste
compared to the culture with yeast extract.
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TABLE IV. Lovastatin concentration and biomass concentration during the
first 7-day incubation period at 28+1oC, 185+5 rpm using 10 g/L crude
glycerol amended with different concentrations of soya waste as nitrogen
source.
Nitrogen Source
Soya waste, 4 g/L
Soya waste, 8 g/L
Soya waste, 12 g/L
Lovastatin
Concentration
(mg/L)
Biomass
Concentration
(g/L)
12.1
4.45
4.35
6.78
1.87
10.1
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