upstream and downstream processing of lovastatin byaspergillus terreus

ORIGINAL PAPER

Upstream and Downstream Processing of Lovastatinby Aspergillus terreus

Hamid Mukhtar • Syeda Sidra Ijaz •

Ikram-ul-Haq

� Springer Science+Business Media New York 2014

Abstract The present study describes the enhanced pro-

duction and purification of lovastatin by Aspergillus terreus

in submerged batch fermentation. The enhancement of

lovastatin production from A. terreus was attempted by

random mutagenesis using ultraviolet radiations and

nitrous acid. UV mutants exhibited increased efficiency for

lovastatin production as compared with nitrous acid

mutants. Among all the mutants developed, A. terreus UV-

4 was found to be the hyper producer of lovastatin. This

mutant gave 3.5-fold higher lovastatin production than the

wild culture of A. terreus NRRL 265. Various cultural

conditions were also optimized for hyper-producing mutant

strain. 5 % glucose as carbon source, 1.5 % corn steep

liquor as nitrogen source, initial pH value of 6, 120 h of

incubation period, and 28 �C of incubation temperature

were found as best parameters for higher lovastatin pro-

duction in shake flasks. Production of lovastatin by wild

and mutant strains of A. terreus was also scaled up to

laboratory scale fermentor. The fermentation process was

conducted at 28 �C, 200 rpm agitation, and 1vvm air flow

rate without pH control. After the optimization of cultural

conditions in 250 ml Erlenmeyer flasks and scaling up to

laboratory scale fermentor, the mutant A. terreus UV-4

gave eightfold higher lovastatin production (3249.95 lg/ml)

than its production by wild strain in shake flasks. Purification

of lovastatin was carried out by solvent extraction method

which yielded 977.1 mg/l of lovastatin with 98.99 % chro-

matographic purity and 26.76 % recovery. The crystal

structure of lovastatin was determined using X-ray diffraction

analysis which is first ever reported.

Keywords Lipid lowering � Fermentation �Mutagenesis �Crystal analysis � Downstream processing

Introduction

Lovastatin (C24H36O5) is a potent drug that is used to lower

down the increased blood cholesterol level of humans and

animals and prevents them from the risks of hypercholes-

terolemia, atherosclerosis, and ischemic heart disease. It

actually belongs to a group of fungal secondary metabolites

known as statins which also include various other choles-

terol-lowering drugs such as pravastatin, simvastatin,

mevastatin, etc. It is a reversible competitive inhibitor of

the enzyme 3-hydroxy-3-methylglutaryl coenzyme A

(HMG CoA) reductase (mevalonate:NADP? oxidoreduc-

tase [EC 1.1.1.34]) which catalyzes a rate-limiting step in

the biosynthesis of cholesterol [13].

In addition to lowering down the serum’s high choles-

terol level, lovastatin has also many other applications such

as it reduces the prevalence of Alzheimer’s disease and

renal diseases [8]. It is a therapy of choice for cancer

treatment and bone fracture treatment [12]. It has also

immunosuppressive, anti-inflammatory, and immunomod-

ulatory properties [46]. So lovastatin could also be used in

immune-mediated neurological disorders. b-hydroxy acid

form of lovastatin is antifungal in nature and inhibits the

growth of Neurospora crassa [19]. It triggers an apoptosis-

like cell death process in the fungus Mucor racemosus and

inhibits the nuclear division in myxomycete Physarum

polycephalum by decreasing the activity of HMG-CoA

reductase [34].

Lovastatin and pravastatin are natural statins, simva-

statin is semi-synthetic while atorvastatin and fluvastatin

are synthetic statins. Natural statins can be produced

H. Mukhtar (&) � S. S. Ijaz � Ikram-ul-Haq

Institute of Industrial Biotechnology, GC University,

Lahore 54000, Pakistan

e-mail: [email protected]

123

Cell Biochem Biophys

DOI 10.1007/s12013-014-9914-7

through microbial fermentation. The first discovered statin,

mevastatin, was isolated from Penicillium citrinum and

Penicillium brevicompactum [5, 10]. Various species of

Monascus including Monascus rubber [9], M. paxi [28],

M. anka [42], and M. purpureus [46] also produce statins.

Among the genus Aspergillus, the most important species

for statin production are A. terreus [4, 14].), A. flavipes

[44], A. flavus, A. umbrosus, and A. parasiticus [35].

However, commercially important statin, lovastatin was

first isolated from Monascus rubber [9]. Now, it is pro-

duced by A. terreus and Monascus rubber [11] but

A. terreus remains the organism of choice for lovastatin

production.

Lovastatin productivity by A. terreus is greatly affected

by various carbon sources, rate of their utilization, culture

homogeneity, pH of the culture medium, rate of agitation,

and level of dissolved oxygen tension [43]. Rapidly

metabolizable substrates in the culture medium result in

uncontrolled filamentous growth which in turn increase the

viscosity and hinder oxygen transfer so lovastatin con-

centration is decreased [18]. Therefore, the optimization of

medium composition is very necessary. Among all the

culture nutrients, carbon and nitrogen sources are very

critical because these are directly associated with microbial

growth and metabolite production. Various carbon and

nitrogen sources have been used for lovastatin production

but yeast extract, corn steep liquor, lactose, glucose, and

glycerol are preferred sources for yielding higher titers of

lovastatin [24, 28].

Suitable fermentation conditions for higher titers of

lovastatin include pelleted growth,\10 days of incubation

in submerged fermentation at 28 �C, initial medium pH of

5.8–6.3, and [40 % of air saturation [18]. Type of inocu-

lum is also very important in the case of secondary

metabolite production. Extensive growth and significant

amount of statin can be produced using vegetative inocu-

lum [35]. However, more recent studies show that the

mode of inoculation either with fungal spores or hyphae

does not affect the behavior of fermentation [24].

The present study is aimed at the hyper production of

lovastatin from A. terreus through random mutagenesis and

optimization of submerged fermentation and downstrea-

ming of the product to get a high grade purity level.

Materials and Methods

Microorganism

A wild strain of A. terreus NRRL 265 was taken from the

culture bank of the Institute of Industrial Biotechnology,

GC University Lahore. The organism was transferred to the

PDA slants and was maintained by monthly transfers to

fresh slants and was stored at 4 �C.

Up Streaming

The fermentation experiments for the production of extra-

cellular lovastatin by A. terreus were carried out in shake

flasks and in lab scale fermentor of 5L working capacity.

Inoculum Preparation

Inoculum was prepared in 250-ml Erlenmeyer flask con-

taining 50 ml of inoculum medium consisting of (g/L)

Glucose, 12; corn steep liquor, 5.0; tomato paste, 40 and

10 ml trace element solution {(g/L) FeSO4�7H2O, 1.0;

MnSO4�4H2O, 1.0; CuCl2�2H2O, 0.025; CaCl2�2H2O, 0.1;

H3BO3, 0.056; (NH4)6 MoO4�H2O, 0.019; ZnSO4�7H2O,

0.2} [1]. The sterilized medium was inoculated with spores

of A. terreus taken from slant and was incubated in shaking

incubator at 30 �C and 200 rpm for 30 h.

Batch Process

a. Shake Flask Fermentation Erlenmeyer flasks (250 ml)

containing 50 ml of the fermentation medium consisting of

(g/L) Glucose, 90; corn steep liqour, 2; ammonium sulfate,

3.0; tomato paste, 5.0 and 10 ml salt solution (pH 6.0) were

used for this experiment. The flasks were cotton plugged,

autoclaved, cooled, and then inoculated with 5 % of the

vegetative inoculum as prepared earlier. The flasks were

placed in shaking incubator for 144 h at 30 �C and

200 rpm.

b. Fermentor Operation Batch process was scaled up to

lab scale fermentor (BioFlo 110, New Brunswick Scien-

tific, USA) for the enhanced production of lovastatin by

A. terreus wild and mutant strains. The glass vessel of 7.5 l

capacity was loaded with 4.5 l of culture medium. Fer-

mentor was sparged with the sterile air during continuous

agitation while vegetative inoculum (5 %) was directly

poured into the production vessel under aseptic conditions.

The initial settings for this experiment were: temperature

value of 30 �C, agitation rate of 160 rpm, and the aeration

rate of 1.0 vvm. Samples for lovastatin analysis were taken

at specific intervals.

Downstream Processing

The whole fermentation material was filtered through

preweighed Whattman filter paper #41. The retentate was

used for the dry cell mass (DCM) determination, and fil-

trate was used for lovastatin assay.


123

Lovastatin was analyzed by HPLC (Perkin Elmers, USA)

using the method of Samiee et al. [35]. The separation was

achieved using SpherSil C18 column (250 9 4.6 mm).

Acetonitril and Potassium phosphate buffer (0.02 M) at a

ratio of 65:35 (v/v) was used as mobile phase. Flow rate was

adjusted to 1.5 ml/min, and chromatograms were obtained at

235 nm by the UV detector.

Sample was prepared by filtering the fermentation broth

through a syringe filter of 0.22 lm pore size to remove all

the suspended small particles. This filtrate was mixed with

the mobile phase in 1:1 ratio in order to dilute it. Each

sample was degassed using water bath sonicator for 5 min.

The best way to determine lovastatin in fermentation

broth is to measure its mevinolinic acid form because this

form predominantly exists in fermentation broths [45].

Lactone forms can easily be converted into acid form in

water and plasma, but it takes place more efficiently in

alkaline solutions [32]. Conversion step is called hydrolysis

of the lovastatin during which pH is very important.

Hydrolysis of standard lovastatin in alkaline or acid med-

ium gives one or two HPLC peaks, respectively [2].

Purification of lovastatin from fermentation broth was

done by the method of [20] with some modifications. Three

steps of this method are as follows:

1. Conversion of hydroxy acid form of lovastatin into

lactone form.

2. Purification of lactone form of lovastatin.

3. Purification with isopropanol.

Memberane filtered (0.45 lm) fermentation broth

(100 g) was cooled to 20 �C, and pH was adjusted to 3.5

with orthophosphoric acid (85 %). Hydrophobic solvent

was added *1.4 times more than the weight of broth.

Mixture was incubated at 55 �C in shaking incubator for

18 h. Organic layer was separated, and 2.5 % sodium

bicarbonate was added (10 % volumes) to this organic

layer. This mixture was agitated for 5 min, and aqueous

layer was separated and discarded. Washing was also done

with distilled water in the same way. Organic layer was

concentrated by distillation under reduced pressure at

40 �C. Residue obtained after distillation was cooled at

-10 �C for 4 h and then dried at 35 �C under vacuum.

Crystals from the first step were dissolved in 200 ml of

dichloromethane. This mixture was stirred for 10 min at

room temperature. Mixture was filtered through 0.45 lm

nitrocellulose membrane filter. Insoluble suspended resin-

ous impurities were discarded, and filtrate was mixed with

200 ml of xylene. 2 g of activated carbon (charcoal) was

added into this mixture and heated at 55 �C for 30 min in

shaking incubator. Activated carbon was separated by fil-

tration of the mixture through Whatmann filter paper #42.

Filtrate was concentrated by distillation under reduced

pressure at 40 �C. Residue was cooled to 4 �C and then

dried at 35 �C under vacuum.

Crystals from second step were dissolved in 300 ml of

isopropanol. This mixture was heated at 70 �C for 30 min.

Mixture was cooled to 0 �C and then dried at 35 �C under

vacuum. Purified crystals were collected and were used for

purity determination and other analysis. Crystals of the

purified lovastatin were analyzed by high performance

liquid chromatography to measure the percentage purity

and X-rays diffraction by KAPA APEX II XRD.

Random Mutagenesis

Random mutagenesis of wild strain of A. terreus NRRL

265 was carried out by UV irradiation and nitrous acid for

enhancing the production potential of the wild strain.

For UV irradiation, one milliliter of the homogenous

conidial suspension was diluted 108 times (20–200 colo-

nies) with sterilized saline water, and density was adjusted

to 2 9 107 conidia/ml. 0.1 ml of this dilution was poured

on each plate separately, and the plates were exposed to

UV radiations for different time intervals (10–60 min)

under a UV lamp. The distance between UV source and

plates was adjusted at 20 cm for each trial. During and

after UV treatment, all plates were incubated in dark at

30 �C for 6 days.

For Nitrous acid Treatment, vigorously shaken conidial

suspension was centrifuged at 6000 rpm for 20 min to

harvest conidia. Conidia were washed with distilled water

and resuspended in 0.1 M sodium acetate buffer (pH 4.4).

NaNO2 was added to give final concentration of 0.1 M. All

the tubes were incubated at 37 �C in a water bath for

5–30 min. Samples were transferred to phosphate buffer

(pH 7.0) to stop the reaction. Whole procedure was also

done with control sample except the addition of NaNO2.

Treated spores were spreaded on plates and incubated at

30 �C for 6 days.

The mutants were randomly selected from the plates

having at least 90 % death rate, especially those which

showed morphological changes. Mutants were transferred

to fresh slants and incubated at 30 �C for 6 days. All the

selected mutants were tested for lovastatin production.

Statistical Analysis

Treatment effects were compared by the protected least

significant difference method and one-way ANOVA (Co-

stat, cs6204 W.exe) after Snedecor and Cochran [39].

Significance difference among the replicates has been

presented as Duncan’s multiple ranges in the form of

probability (\p[) values.


123

Results and Discussion

Mutagenesis using UV Irradiation

Under the selected conditions of treatment, 90 % of the

conidia were killed by about 8–10 min of irradiation. So,

seventeen survivors of A. terreus after UV irradiation

(10–15 min) were picked and screened for lovastatin pro-

duction. Eleven survivor strains of A. terreus showed vari-

able lovastatin production (Table 1). The lovastatin

production by these variants ranged between 66.57 ± 0.66

and 1,396.09 ± 0.08 lg/ml. Many of these survivors also

showed various morphological changes which include the

partial or total loss of color from conidia and also the var-

iation in color (Images 1–11). The mutant strain UV-4 gave

the maximum lovastatin production (1,396.09 ± 0.08 lg/ml)

with a DCM of 19.47 ± 0.11 mg/ml.

In present investigation, efforts have been made to

improve the wild culture of A. terreus for enhanced lova-

statin by UV mutagenesis. The complete death of the fungus

was observed after 20 min of UV exposure. It may be due to

the relationship between mutation rate and the amount of

dose. Production of mutants by this wavelength (260 nm)

was necessary because the wavelength which is most

effective in killing is highly suitable for the production of

mutations [15]. It is reported that random mutation and

selection strategies are important to obtain overproducing

isolates of A. terreus [45]. Random mutations using ultra-

violet radiations is a good choice to increase the production

of metabolites, e.g., production of itaconic acid has been

increased by inducing ultraviolet mutations in A. terreus

NRRL 265 [23]. Genetic changes in organisms by UV irra-

diation increase the yields of certain chemicals by the

organisms. Many other workers have also reported the UV

radiations for the development of mutations [15, 16, 33].

Mutagenesis using Nitrous Acid Treatment

Under the selected conditions of treatment, 90 % of the conidia

were killed by about 15 min of treatment. Fifty five survivors of

A. terreus developed through nitrous acid treatment

(10–25 min) were selected and screened for lovastatin pro-

duction. Thirty eight survivors showed no lovastatin produc-

tion, and the results of the other seventeen survivor strains of A.

terreus are shown in the Table 2. Range for lovastatin pro-

duction by these variants was 0.1 ± 0.03–881.42 ± 0.08

lg/ml. Many of these survivors also showed morphological

changes (variation in color of conidia and number of conidia)

just like the survivors after UV irradiations. Maximum lova-

statin production (881.42 ± 0.08 lg/ml) among nitrous acid-

treated mutants was given by NA-14 while the DCM was

22.27 ± 0.07 mg/ml.

Table 1 Screening of Aspergillus terreus NRRL 265 survivors for

lovastatin production after UV irradiation using shake flask

fermentation

Sr. No. Strain Duration

for UV

irradiation

(min)

Dry cell

mass

(mg/ml)

Lovastatin

(lg/ml)

1 Wild – 20.05 ± 0.05 404.52 ± 0.02

2 UV-1 15 19.78 ± 0.21 958.08 ± 2.09

3 UV-2 15 19.28 ± 0.10 99.96 ± 0.17

4 UV-3 15 15.61 ± 0.07 66.57 ± 0.66

5 UV-4 10 19.47 ± 0.11 1,396.09 ± 0.08

6 UV-5 10 18.23 ± 0.11 1,118.52 ± 0.41

7 UV-6 10 16.78 ± 0.04 375.99 ± 0.92

8 UV-7 10 12.81 ± 0.04 754.17 ± 0.15

9 UV-8 10 19.74 ± 0.13 963.52 ± 2.98

10 UV-9 10 23.45 ± 0.06 701.54 ± 0.32

11 UV-10 10 19.84 ± 0.18 835.48 ± 0.39

12 UV-11 10 19.20 ± 0.08 1,205.87 ± 0.42

Fermentation medium GCAT, Initial pH of the medium 6.0, Incu-

bation period 144 h, Incubation temperature 30 �C. ± indicates

standard deviation (±SD) among the three parallel replicates, which

differ significantly at p B 0.05

Table 2 Screening of Aspergillus terreus NRRL 265 survivors for

lovastatin production developed through nitrous acid using shake

flask fermentation

Sr.No. Strain Duration for

nitrous acid

treatment (min)

Dry cell

mass

(mg/ml)

Lovastatin

(lg/ml)

1 Wild – 20.05 ± 0.05 404.52 ± 0.02

2 NA-1 15 20.48 ± 0.25 29.64 ± 0.43

3 NA-2 20 21.52 ± 0.14 7.31 ± 0.45

4 NA-3 20 22.86 ± 0.11 407.28 ± 0.25

5 NA-4 20 29.32 ± 0.13 0.30 ± 0.26

6 NA-5 20 20.44 ± 0.09 0.1 ± 0.03

7 NA-6 20 14.74 ± 0.05 762.81 ± 0.24

8 NA-7 20 20.08 ± 0.08 61.27 ± 0.10

9 NA-8 15 12.61 ± 0.11 23.49 ± 0.08

10 NA-9 15 20.11 ± 0.13 19.38 ± 0.26

11 NA-10 15 21.20 ± 0.16 18.21 ± 0.13

12 NA-11 15 10.81 ± 0.07 25.68 ± 0.13

13 NA-12 25 21.39 ± 0.09 360.76 ± 0.05

14 NA-13 15 19.85 ± 0.12 790.75 ± 0.09

15 NA-14 15 22.27 ± 0.07 881.42 ± 0.08

16 NA-15 15 19.39 ± 0.07 114.89 ± 0.10

17 NA-16 15 18.30 ± 0.03 54.56 ± 0.18

18 NA-17 15 13.16 ± 0.05 698.30 ± 0.05

Fermentation medium GCAT, Initial pH of the medium 6.0, Incu-

bation period 144 h, Incubation temperature 30 �C. ± indicates


differ significantly at p B 0.05


123

Mutagenic action of nitrous acid was reported 20 years

ago when [38, 41] reported mutations in A. niger and

A. amstelodami using nitrous acid. It is a general

assumption that the mutagenic action results from the

alterations of nitrogenous bases in nucleic acid so nitrous

acid is also a good mutagenic agent because it cause the

deamination of nucleic acid bases [37]. Various scientists

have reported the positive mutations caused by nitrous acid

in various organisms [27, 38].

Effect of Carbon Source

Different carbon sources were screened for lovastatin produc-

tion by A. terreus mutant strain UV-4 (Fig. 1). The growth of

microorganism and production of lovastatin was considerable

in all the carbon sources tested, but the best results were

obtained with glucose where maximum lovastatin production

(1,396.09 ± 0.08 lg/ml) was obtained. Cell mass in the pre-

sence of glucose was less than maltose and lactose, but glucose

was selected as best carbon source for further experiments.

Different amounts (1–10 %) of glucose were added in the

fermentation flasks to find out its optimum concentration for

lovastatin production by high-producing mutant strain of

A. terreus UV-4 (Fig. 2). Lovastatin production was

increased by increasing the amount of glucose from 1 %, and

the highest production of lovastatin (1,414.08 ± 0.09 lg/

ml) was observed at 5 % level of glucose, but above this level

there was a gradual decline. So, 5 % (w/v) level of glucose

was selected for the preparation of the fermentation medium

during further experiments.

All the living organisms require carbon as macroelement

for cell growth and for balanced metabolism but need for

carbon sources varies from organism to organism. Carbon

and energy source which had been completely utilized

during growth would prove unsatisfactory for metabolite

production; conversely, a compound which is only partly

consumed during cell growth may be much more suitable

for subsequent secondary metabolite formation [7]. The

two carbon sources glucose and glycerol have also been

optimized as best carbon sources for high titers of lova-

statin by various workers, for example, 4.18-fold increased

lovastatin production was obtained by optimizing the fer-

mentation medium containing 20.0 g/L glucose as sole

carbon source [21, 45].

The amount of carbon source has also a marked effect

on the metabolism of microorganisms. Low yield of lov-

astatin in low amount of carbon source might be due to the

fact that less amount could not fulfill the needs of organism

to grow and then to produce maximum lovastatin. In

present work, 5 % (w/v) glucose was found as best amount

of carbon source for enhanced production of lovastatin.

Similarly, Szakacs et al. [43] and Lai et al. [21] have

optimized 2 % glucose as best carbon level for lovastatin

synthesis by A. terreus which is very near to our findings.

Less lovastatin production in higher amounts of glucose

may be due to the reason that there was enough residual

glucose in the medium and it had not starved the micro-

organism to enhance lovastatin production because the

starvation of essential nutrients favors the lovastatin pro-

duction [24].

0

5

10

15

20

25

0

200

400

600

800

1000

1200

1400

1600

Glu Mal Lac Suc Sta Gly

Dry

Cel

l Mas

s (m

g/m

l)

Am

ou

nt

of

lova

stat

in (

µg

/ml)

Carbon source

Amount of lovastatin (µg/ml) Dry cell mass (mg/ml)

Fig. 1 Effect of different carbon sources on lovastatin production by

high-producing mutant strain of A. terreus. Y-error bars indicate


differ significantly at p B 0.05. Incubation temperature = 30 �C,

Initial pH = 6.0, Incubation period = 144 h, Fermentation med-

ium = GCAT. Glu glucose; Mal maltose; Lac lactose; Suc sucrose;

Sta starch; Gly glycerol

0

5

10

15

20

25

0

200

400

600

800

1000

1200

1400

1600

1 2 3 4 5 6 7 8 9 10

Dry

Cel

l Mas

s (m

g/m

l)

Am

ou

nt

of

lova

stat

in (

µg

/ml)

Amount of glucose (%)


Fig. 2 Effect of amount of glucose on lovastatin production by high-

producing mutant strain of A. terreus. Y-error bars indicate standard

deviation (±SD) among the three parallel replicates, which differ

significantly at p B 0.05. Incubation temperature = 30 �C, Initial

pH = 6.0, Incubation period = 144 h, Fermentation medium = GCAT


123

Effect of Nitrogen Source

Different nitrogen sources including meat extract, corn

steep liquor, beef extract, yeast extract, and peptone were

investigated to find out their effects on the growth and

lovastatin production by A. terreus mutant strain UV-4

(Fig. 3). All of the nitrogen sources gave considerable

growth of mutant strain and lovastatin production except

peptone. However, the medium containing corn steep

liquor as nitrogen source supported higher production as

compared to others. For this reason, corn steep liquor was

selected as the most suitable nitrogen source and was used

in the further studies.

When the different amounts (0.5–5.5 %, v/v) of this

nitrogen source were used to study their effects on the

growth of microorganism and production of lovastatin, it

was observed that DCM and lovastatin production were

increased from 0.5 to 1.5 % (v/v) level of the corn steep

liquor. At 1.5 % level of nitrogen source, the maximum

DCM (19.26 ± 0.58 mg/ml) and maximum lovastatin

production (1,505. 67 ± 0.04 lg/ml) was obtained but

above that there was a decrease in both the growth and

lovastatin production (Fig. 4). Therefore, 1.5 % (v/v) level

of corn steep liquor was used in the further experiments.

Just like carbon sources, requirement for nitrogen

sources also varies from organism to organism. Literature

supports that complex nitrogen source in batch fermenta-

tion gives maximum titers of lovastatin ([6]; [29, 31]

because it enhances the enzyme activities for polyketide

synthesis. In addition to fulfill the basic nitrogen

requirements, corn steep liquor also provides various sol-

uble minerals and amino acids to the microorganism. An

increase of 4.18-fold in lovastatin production was achieved

after optimization of medium containing 10 g/L corn steep

liquor [21]. Various other researchers also found the corn

steep liquor as best nitrogen source [3, 31].

Effect of Initial pH of Medium

The effect of initial pH (3.0–9.0) of the fermentation

medium on the growth of microorganism and the produc-

tion of lovastatin was studied (Fig. 5). It was found that the

growth of microorganism in the basic side of pH was much

less with no production of lovastatin at pH 9.0 but in the

acidic side of pH, there was gradual decrease in DCM from

6.0 to 3.0. When the pH value was kept at 6.0, there was

maximum DCM (19.01 ± 0.01 mg/ml) and maximum

lovastatin production (1,581.86 ± 0.07 lg/ml) so this pH

value was selected for further work.

Suitable pH of the fermentation medium for high lova-

statin production by A. terreus is ranged between 5.8 and

6.3 [19]. Many other researches have also optimized the

similar pH values during their work [31, 35, 36]. Deviation

of initial pH value of the medium from optimum value

results in the activation of some enzymes which decom-

pose the molecular structure of lovastatin [22]. Also, Bi-

zukojc and Ledakowicz [4] reported that the production of

lovastatin at pH of 7.6–7.8 successfully repressed the for-

mation of by-products in batch and fed batch experiments.

0

5

10

15

20

25

0

200

400

600

800

1000

1200

1400

1600

1800

ME CSL BE YE Pep

Dry

Cel

l Mas

s (m

g/m

l)

Am

ou

nt

of

lova

stat

in (

µg

/ml)

Nitrogen source


Fig. 3 Effect of nitrogen source on lovastatin production by high-




pH = 6.0, Incubation period = 144 h, Fermentation medium = G-

CAT. ME meat extract; CSL corn steep liquor; BE beef extract; YE

yeats extract; Pep peptone

0

5

10

15

20

25

0

200

400

600

800

1000

1200

1400

1600

1800

0.5 1.5 2.5 3.5 4.5 5.5

Dry

Cel

l Mas

s (m

g/m

l)

Am

ou

nt

of

lova

stat

in (

µg

/ml)

Amount of CSL


Fig. 4 Effect of amount of corn steep liquor on lovastatin production by

high-producing mutant strain of A. terreus. Y-error bars indicate standard



pH = 6.0, incubation period = 144 h, Fermentation medium = GCAT


123

Effect of Incubation Period

Results for the effect of incubation period (24–192 h) on the

growth of A. terreus and production of lovastatin are shown in

the Fig. 6. The growth of microorganism was started to

increase gradually as the fermentation batch started. After

24 h of incubation, very small amount of DCM was obtained

with least lovastatin production. The maximum DCM, i.e.,

19.36 ± 0.07 mg/ml and highest lovastatin production

(1,699.87 ± 0.08 lg/ml) was obtained when the fermenta-

tion flasks were incubated for 120 h. There was a decline in the

biomass and yield of lovastatin, as the incubation period was

increased above 120 h. So, the time period of 120 h was

selected as best incubation period for the production of lova-

statin by A. terreus UV-4.

Lovastatin is a secondary metabolite so its production is

related to the stationary phase of growth [26]. Maximum

production of lovastatin after 120 h of incubation may be

due to the fact that at that time the conditions resulted in

the secondary growth of the microorganism. After that, the

decrease in the production of lovastatin can be attributed to

feedback inhibition by the already produced lovastatin [21,

25]. Batch fermentation for lovastatin production under

suitable fermentation conditions usually lasts for not more

than 10 days [19]. Lai et al. [22] enhanced the lovastatin

production by 38 % after 10 days of incubation while

Szakacs et al. [43] obtained best yield of lovastatin in

7 days which is near to our optimized incubation time.

Effect of Incubation Temperature

Figure 7 shows the effect of different incubation temper-

atures (26–34 �C) on the growth of microorganism and

lovastatin production. At 28 �C, maximum amount of

DCM (20.82 ± 0.16 mg/ml) and maximum lovastatin

production (1,957.75 ± 0.09 lg/ml) was recorded but

above 28 �C, there was a decline in both the growth of

microorganism and lovastatin production, and at 34 �C

there was a considerable decline in the production of

0

5

10

15

20

25

0

200

400

600

800

1000

1200

1400

1600

1800

3 4 5 6 7 8 9

Dry

Cel

l Mas

s (m

g/m

l)

Am

ou

nt

of

lova

stat

in (

µg

/ml)

pH of medium


Fig. 5 Effect of pH of medium on lovastatin production by high



significantly at p B 0.05. Incubation temperature = 30 �C, Incuba-

tion period = 144 h, Fermentation medium = GCAT, Selected car-

bon source = glucose (5 %), Selected nitrogen source = Corn steep

liquor (1.5 %)

0

5

10

15

20

25

0

200

400

600

800

1000

1200

1400

1600

1800

2000

24 48 72 96 120 144 168 192

Dry

Cel

l Mas

s (m

g/m

l)

Am

ou

nt

of

lova

stat

in (

µg

/ml)

Incubation period (hrs)


Fig. 6 Effect of incubation period on lovastatin production by high-


deviation (±SD) among the three parallel replicates, which differ signif-

icantly at p B 0.05. Incubation temperature = 30 �C, Medium pH = 6.0,

Fermentation medium = GCAT, Selected carbon source = glucose (5 %),

Selected nitrogen source = Corn steep liquor (1.5 %)

0

5

10

15

20

25

0

500

1000

1500

2000

2500

26 28 30 32 34

Dry

Cel

l Mas

s (m

g/m

l)

Am

ou

nt

of

lova

stat

in (

µg

/ml)

Incubation temperature (oC)


Fig. 7 Effect of incubation temperature on lovastatin production by

high-producing mutant strain of A. terreus. Y-error bars indicate


differ significantly at p B 0.05. Medium pH = 6.0, Incubation

period = 120 h, Fermentation medium = GCAT, Selected carbon

source = glucose (5 %), Selected nitrogen source = Corn steep

liquor (1.5 %)


123

lovastatin. So, the optimum temperature for the growth of

microorganism and lovastatin production was found as

28 �C.

Batch fermentations for high lovastatin production are

typically maintained at *28 �C with other optimized condi-

tions [19]. The wild strain gave best production at 30 �C but

the optimum temperature was slightly shifted toward lower

side in case of the selected high-producing mutant strain.

Temperature value of 25 �C has been recommended as best

incubation temperature for high lovastatin production [17]

while Szakacs et al. [43] carried out good A. terreus fermen-

tation for lovastatin production at 30 �C. Even temperature

shifting from 28 to 23 �C enhanced the lovastatin production

to 25 % [22]. Lesser production of lovastatin at higher tem-

perature might be due to the fact that high temperature retards

the metabolic processes of the microorganism by denaturing

enzymes, transport carriers, and other proteins, e.g., temper-

ature elevation to 35 �C inhibited the lovastatin production by

A. terreus [30].

Batch Fermentation in Bioreactor

The shake flask experiments were upscaled to a 7 l labo-

ratory fermentor. A 2.9-fold increase in level of lovastatin

production was obtained 66 h earlier in lab scale fermentor

as compared to its production in shake fermentation flasks

(Fig. 8). After 78 h, there was a decline and even no pro-

duction of lovastatin was observed after 126 h of incuba-

tion. Total DCM after 144 h was 32.44 mg/ml.

The situation was slightly changed when fermentation

was carried out with high-producing mutant strain of

A. terreus (UV-4). A 2.8-fold increase in level of lovastatin

production was achieved 6 h earlier than its production by

wild strain in same way. The maximum lovastatin pro-

duction was 3,249.95 lg/ml (Chromatogram 4) at 72 h of

incubation (Fig. 9) which was actually twofold increased

production than its production in shake fermentation flasks.

After 72 h, there was a decline in lovastatin productivity,

and no production was found at 102 h of incubation. Total

DCM was 40.17 mg/ml after 102 h of incubation.

It is considered to be a great challenge to transfer a

fermentation process from shake flasks to bioreactors

because of the geometric variations in both systems and

other substantial differences in related parameters such as

aeration, agitation, temperature control, pH control, dis-

solved oxygen, etc. However, fermentor is a good choice to

increase the production of secondary metabolites. Also

scaling up from fermentation flasks to small scale fer-

mentors is more predictable than scaling up from fermen-

tation flasks to large scale fermentors [48]. Secondary

metabolite production could be increased just by improving

the oxygen supply in 2 l fermentor [14]. Similarly, 38 %

increased lovastatin production and 25 % decrease in bio-

mass production were achieved using a 5 l fermentor [22].

During the microbial fermentation, proper mixing of the

both is very important which could be done by the agitation

and aeration, but these must be optimum to prevent shear

stress and cell disruption [48]. In the present run of fer-

mentor, agitation was maintained at 200 rpm for enhanced

production of lovastatin. Lai et al. [22] optimized the

agitation rate of 225 rpm for lovastatin production in 5 l

fermentor. They explained that higher agitation caused

hyphal breakage and so lovastatin production decreased

and under lower agitation it was totally diminished because

of oxygen limitation.

Researchers have proposed the decreased aeration rate

for high lovastatin production because there is only one

oxidation step in lovastatin biosynthesis so increased level

of oxygen is not required. Various experiments confirm this

theory [4] in which good lovastatin titres have been

obtained at aeration rate of 0.6 L/min. In the present run of

fermentor, aeration rate was maintained at 1vvm which is

not a high rate and is also reported to give high lovastatin

titres [26]. Because lovastatin is non-growth related prod-

uct, just by reaching the stationary phase no higher air flow

rates are required. pH of the medium was maintained at 6.0

and during the fermentation it was not controlled. It has

already been reported that pH should not be controlled in

the lovastatin production because the pH control systems

cause the little decrease in lovastatin formation [22].

During the fermentation, the pH of the medium changes

but these changes range between 6.2 and 7.0 [42], so there

is no need to adjust the pH [22].

Purification of Lovastatin

The method used in present study for the purification of

lovastatin was found to be very promising and yielded

977.1 mg/l of off white crystalline lovastatin powder with

pH o

f th

e br

oth

Am

ount

of

lova

stat

in (

µg/m

l)

Time (hrs)

Amount of lovastatin (µg/ml) pH

Fig. 8 Lovastatin production by wild strain of A. terreus in lab scale

fermentor. Fermentation medium GCAT, Initial pH of the medium

6.0, Incubation period 144 h, Incubation temperature 30 �C


123

98.99 % chromatographic purity and recovery yield of

26.76 % (Table 3).

XRD Analysis of lovastatin crystals

Single crystal X-ray study of the purified lovastatin has

provided the detailed description about the 3D structure

and its complex in crystalline state. The final x-ray crys-

tallographic models (Figs. 10, 11) revealed the structure

and absolute stereochemistry of lovastatin. The related

geometrical data supplied in the CIF format is as follows: IUPAC Name

[(1S,3R,7S,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy-6-oxooxan-2-

yl]ethyl]-3, 7-dimethyl-1,2,3, 7,8,8a-hexahydronaphthalen-

1-yl] (2S)-2-methylbutanoate.

Crystal Data

Molecular formula = C24H36O5.

Molecular weight = 404.54 g/mol.

Crystal type = Orthorhombic.

Prism color = off white.

Shape = Needle.

Size = 0.14 9 0.07 9 0.05 mm.

Crystal space group name = P2(1)2(1)2(1).

Dimensions.

a = 5.9700 (5) A.

b = 17.3218 (15) A.

c = 22.1520 (18) A.

b = 90.00�

Volume (V) = 2,290.8 (3) A3.

Formula units (Z) = 46.

Density diffraction (Dx) = 1.435 Mg m-3.

F(000) = 1,012.

Absorption coefficient mu = 0.129.

Radiations = Mo Ka.

Radiations wavelength (k) = 0.71073 A.

Measurement reflections = 1,208.

Minimum theta measurement (h) = 2.53o.

Maximum theta measurement (h) = 19.09o.

Measurement temperature (T) = 296 K.

x y z

x ? 1/2 -y z ? 1/2

x y ? 1/2 -z ? 1/2

x ? � -y ? 1/2 -z

pH o

f th

e br

oth

Am

ount

of

lova

stat

in (

µg/m

l)

Time (hrs)

Amount of lovastatin (µg/ml) pH

Fig. 9 Lovastatin production by high-producing mutant of A. terreus

in lab scale fermentor. Fermentation medium GCAT, Initial pH of the

medium 6.0, Incubation period 102 h, Incubation temperature 28 �C

Fig. 10 Ortep diagram showing molecular structure of lovastatin

Table 3 Purity of lovastatin at each step of purification

Purification step Purity

(%)

Conversion of hydroxy acid form of lovastatin into

lactone form

91

Purification of lactone form of lovastatin 97

Purification with isopropanol 98.99


123

Conclusion

The enhanced production and purification of lovastatin has

been successfully achieved during this study. Random

mutagenesis of A. terreus NRRL 265 produced a stable

mutant which had resulted in 3.5-fold increased lovastatin

production than wild strain. After that the optimization of

cultural conditions in shake flasks and scaling up to a lab

scale fermentor resulted in further twofold increased lov-

astatin production. The overall increase in lovastatin

production through present research work was eightfold

(Fig. 12) starting from the wild strain.

Purification of lovastatin has been successfully carried

out by solvent extraction method which was confirmed by

HPLC and XRD analysis. Further improvement can be

made by optimizing the environmental conditions for the

scale up production of lovastatin in pilot scale fermentor.

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upstream and downstream processing of lovastatin byaspergillus terreus

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