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Bioresource Technology 99 (2008) 4263–4268

Improvement of erythromycin production by Saccharopolyspora

erythraea in molasses based medium through cultivation
medium optimization
a,*
H.A. El-Enshasy , N.A. Mohamed b, M.A. Farid b, A.I. El-Diwany b

a
Bioprocess Development Department, GEBRI, Mubarak City for Scientific Research, New Burg Al Arab, 21934 Alexandria, Egypt
b
Natural and Microbial Products Department, National Research Centre, Dokki, Cairo, Egypt

Received 14 February 2007; received in revised form 24 June 2007; accepted 25 August 2007
Available online 23 October 2007

Abstract

In the present work, erythromycin production was carried out in submerged culture using Saccharopolyspora erythraea. Different
experiments were conducted to optimize the cultivation medium through the change of carbon and nitrogen sources to cheaper one
in order to reduce the cost of medium and to utilize sugar cane molasses as one of major sugar industry by-products in Egypt. It
was found that the addition of sugar cane molasses a sole carbon source at a concentration of 60 g/l accompanied by corn steep liquor
(as organic N-source) in combination with ammonium sulphate (as inorganic N-source) gave the maximal erythromycin production. The
antibiotic production in this medium reached about 600 mg/l which is about 33% higher than the value obtained in glucose based med-
ium. On the other hand, the addition of n-propanol in concentration of 1% (v/v) increased the antibiotic production reaching about
720 mg/l after 144 h. Concluding, the new medium formulation based on cheap carbon source, sugar cane molasses, was a good alter-
native solution for the production of erythromycin economically.
 2007 Elsevier Ltd. All rights reserved.

Keywords: Erythromycin; Saccharopolyspora erythraea; Cultivation conditions; Agricultural wastes

1. Introduction for treatment of feeding intolerance in preterm infants

Erythromycin is a polyketide antibiotic produced by
Saccharopolyspora erythraea mainly in submerged culture.
This antibiotic has many pharmaceutical applications in
the treatment of many diseases caused by gram-positive
bacteria and some gram-negative bacteria. Therefore,
erythromycin is widely used clinically in respiratory infec-
tious diseases (Hoyt and Robbins, 2001), in the treatment
of many acute infections caused by bacteria belonging to
Staphylococci spp. and Neisseria spp. (Lesmana et al.,
2001) and as an antimalarial in combination with other
drugs to reduce the pathogen resistance (Nakornchai and
Konthiang, 2006). Recently, erythromycins were also used
*
Corresponding author. Tel.: +2 010 5177482; fax: +20 3 4593423.
E-mail address: h.elenshasy@mucsat.sci.eng (H.A. El-Enshasy).
(Nuntnarumit et al., 2006). Besides all these medical appli-
cations, erythromycin is also used as prophylactic and
curative therapeutic agent for many diseases of poultry
and farm animals (Prescott, 1997). Although erythromycin
originally developed for its use in human and veterinary
medicine, it has also many applications in the animal feed-
ing (Moffitt, 1998) and the cultivation of marine microal-
gae (Kim and Cerniglia, 2005; Campa-Córdova et al.,
2006). Nowadays, the industrial production of erythromy-
cin is mainly carried out by Saccharopolyspora erythraea.
The production process of this antibiotic is mainly by
means of submerged culture system of either free cells
(Martin and Bushell, 1996; Heydarian et al., 1999) or
immobilized cells (El-Enshasy and Beshay, 2000; Hamedi
et al., 2005). Like other secondary metabolites produced
by aerobic actinomycetes, the process of biosynthesis is

0960-8524/$ - see front matter  2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2007.08.050
4264 H.A. El-Enshasy et al. / Bioresource Technology 99 (2008) 4263–4268
complex process and highly influenced by the medium com-
position and cultivation conditions as well. Concerning
medium composition, the production of erythromycin is
highly regulated by type and concentration of the carbon
source (Martin and Demain, 1980; Escalante et al., 1982),
nitrogen source (Flores and Sánchez, 1989; Potvin and
Péringer, 1993), phosphate and trace elements (Singh
et al., 1981). In general, carbon source is usually used in rel-
atively higher concentration compared to other medium
component and thus has high share in the raw material
cost. Therefore, molasses, milk whey, starch, cellulose
and other cheap complex carbon sources were used to
replace glucose in many production processes. A normal
cane molasses usually has a water content of 17–25%, a
sugar content (sucrose, glucose and fructose) of 45–50%
and polysaccharides (dextrin, pentosans, polyuronic acids)
containing 25% (Najafpour and Shan, 2003). However, the
international price of sugar cane molasses is ranged
between $120–150 per metric ton. Therefore, it is widely
used as main component in the industrially used medium
for the production of different primary and secondary
metabolites (Ikram-ul et al., 2004; Cazetta et al., 2005;
Banik et al., 2007; Cha et al., 2007). In the present work,
an attempt was done to optimize the cultivation medium
through the replacement of carbon and nitrogen sources
to cheaper one in order to reduce the cost of raw materials.
The present study was also extended to investigate the
influence of other medium ingredients on the antibiotic
yield.
2. Methods
2.1. Microorganism and inoculum preparation
Saccharopolyspora erythraea NCIMB 8594 was used
throughout this study. This stain was obtained from the
National Collection of Industrial and Marine Bacteria,
Aberdeen, Scotland, UK. This strain was maintained on
ISP-2 solid medium composed of (g/l): glucose, 4; yeast
extract, 4, malt extract, 10 and bacteriological agar, 20.
The pH was adjusted to 7.0 before sterilization. The slants
were inoculated and incubated at 28 C for 10 days in order
to obtain a heavy sporulated growth. After that time,
spores (in a concentration of 1 · 108 spores/ml) were used
to inoculate the fermentation medium.
2.2. Production medium and cultivation conditions
Unless otherwise mentioned, the initial industrially used
medium for erythromycin production was composed of
(g/l): glucose, 20; corn steep liquor, 4.0; NH4(SO4)2, 3;
NaCl, 2.5; and CaCO3, 5. The pH was adjusted to 7.2
before sterilization. Medium sterilization was carried out
by autoclaving at 121 C for 15 min. The carbon source
was autoclaved separately at 121 C for 10 min and added
to the medium prior inoculation. In case of molasses based
medium, sugar cane molasses (SCM) was used instead of
glucose. The SCM was obtained from the Sugar and Inte-
grated Chemicals Co. (El Hawamdya, Giza, Egypt). The
used SCM had the following composition: total carbohy-
drate, 55%, nitrogen content, 0.4%; CaO, 0.6%; MgO,
0.5% and ashes, 4%.
The cultivations were carried out in Erlenmeyer flasks of
250 ml (working volume 50 ml). The flasks were placed on
a rotary incubator shaker at 200 rpm and 30 C for 144 h.
After that time, the media were filtered and analyzed for
the determination of cell dry weight, antibiotic production
and final pH.
2.3. Analysis
2.3.1. Determination of cell dry weight
The cell dry weight of cells after cultivation was deter-
mined gravimetrically. The fermentation broth was filtered
through a dry preweighed filter paper (Whatman No. 1)
and washed several times with distilled water. The washed
cells were dried in an oven at 100 C until a constant weight
was obtained.
2.3.2. Determination of erythromycin
The amount of the erythromycin produced in the fer-
mentation broth was determined by means of biological
method according to Bershtein et al. (1983). The antibiotic
assay medium (Difco) composed of (g/l): glucose, 10.0;
peptone, 10.0; meat extract, 2.5; yeast extract, 5.0; NaCl,
10.0 and agar, 20.0. The erythromycin sensitive strain of
Bacillus subtilis NRRL B-543 was used the biological activ-
ity determination. The diameters of the inhibition zones
obtained were measured and the amount of erythromycin
produced was calculated from a biological standard curve
previously made using extra pure standard erythromycin
(Sigma, USA) as an authentic reference material.
3. Results and discussion
3.1. Batch cultivation of saccharopolyspora erythraea in
industrial medium
Fig. 1 shows the time profiles of cell growth, volumetric-
and specific erythromycin production during a typical
batch cultivation of S. erythraea in industrial medium.
The maximal value of volumetric erythromycin production
of about 450 mg/l was obtained after 144 h and kept more
or less constant for the rest of cultivation time. On the
other hand, the maximal cell growth of 10.2 g/l was
obtained after 96 h and decreased gradually after that time.
Concomitantly, significant increase in pH value was
observed during the phase of cell lysis. The decrease in cell
mass was occurred as a result of hyphal cell fragmentation
due to the shear influence. Bushell et al. (1997) observed
also a significant fragmentation in S. erythraea in sub-
merged cultures even in low shear cultures such as non-baf-
fled shaken flasks. This was due to the fragility of S.
erythraea hyphae especially when cells inter stationary
 H.A. El-Enshasy et al. / Bioresource Technology 99 (2008) 4263–4268
Fig. 1. Cell growth and erythromycin production during batch cultivation
of S. erythraea in glucose based industrial medium.
phase (Stocks and Thomas, 2001a). The increase in pH
may due to the release of ammonia in fermentation med-
ium as a function of cell lysis and fragmentation (Stocks
and Thomas, 2001b). The specific erythromycin production
(YP/X) reached about 48 mg/g after 144 h (the time of max-
imal volumetric erythromycin production). The observed
increase in the specific antibiotic production after that time
was due to the decrease in cell mass rather than the increase
in antibiotic production.
3.2. Effect of different carbon sources
The suitability of different C-sources (monosaccharides,
disaccharides and complex carbon sources) for the produc-
tion of erythromycin was studies. As shown in Fig. 2,
among different monosaccharides tested both of maximal
volumetric erythromycin production and cell growth was
obtained in glucose culture and reached about 450 mg/l
and 9.6 g/l, respectively. On the other hand, in spite of a
good growth in galactose, fucose and arabinose cultures,
the production of antibiotic was very low. Of different
disaccharides tested, the maximal erythromycin production
was obtained in sucrose culture followed by trehalose,
maltose b-lactose and a-lactose. On the other hand, the
production of erythromycin in complex carbon source
media, containing either starch or molasses, reached
480 mg/l. These results indicate that, glucose and sucrose
4265
Fig. 2. Effect of different C-sources on the cell growth and erythromycin
production by S. erythraea. Results were obtained after 144 h cultivations
in batch culture.
in pure or in polymer forms were the best C-sources for
erythromycin production. Since the cane molasses is
cheaper than starch and more available than beet molasses,
as industrial by-product from sugar refining industries in
Egypt, cane molasses in a concentration of (50 g/l) was
used in the subsequent experiments.
3.3. Effect of different inorganic nitrogen sources
The effect of seven types of ammonium salts on the pro-
duction of antibiotic was investigated in this experiment.
The original industrially used erythromycin production
medium was containing both organic (CSL) and inorganic
(ammonium sulphate) nitrogen sources. The aim of this
experiment was to study the effect of different types of
ammonium salts in CSL supplemented medium on the
erythromycin production. The nitrogen content of each
source was equivalent to the amount of nitrogen in the ori-
ginal ammonium sulphate concentration (3 g/l). The results
in Fig. 3 show that the addition of different ammonium
salts instead of ammonium sulphate did not increase the
erythromycin production. Ammonium sulphate and
ammonium chloride were the best salts supporting erythro-
mycin production. The volumetric maximal antibiotic pro-
duction in both cultures was about 450 mg/l. Other
4266 H.A. El-Enshasy et al. / Bioresource Technology 99 (2008) 4263–4268
Fig. 3. Effect of different inorganic nitrogen sources on the cell growth
and erythromycin production by S. erythraea.
ammonium salts like ammonium oxalate, ammonium
nitrate and ammonium carbonate support better cell
growth on the cost of antibiotic production. However,
the maximal yield of erythromycin production based on
cell mass (YP/X) of about 55 mg/g was obtained in ammo-
nium sulphate supplemented culture.
3.4. Effect of different organic nitrogen sources
The aim of this experiment was to study the possibility
for the substitution of the inorganic nitrogen source in
the production medium, ammonium sulphate, with organic
nitrogen source. The amount of nitrogen content in 4 g/l of
CSL and 3 g/l ammonium sulphate in the medium was
replaced by an equivalent amount of nitrogen in the differ-
ent organic nitrogen sources. Fig. 4 shows the different
organic nitrogen sources used and the corresponding cell
growth, erythromycin production after 144 h cultivation.
The maximal volumetric erythromycin production was
obtained in medium supplemented by CSL followed by
milk whey and fodder yeast. The maximal volumetric anti-
biotic production in the CSL culture was almost the same
that obtained in ammonium sulphate culture in the previ-
ous experiment. On the other hand, the cell productivity
(YP/X) in CSL culture was about 40 mg/g which is lower
than the ammonium sulphate culture by about 38%. There-
Fig. 4. Effect of different organic nitrogen sources on cell growth and
erythromycin production by S. erythraea.
fore, it could be concluded that it is not preferable to sub-
stitute the inorganic nitrogen source in the medium with
organic source. On the other hand, the price of ammonium
sulphate is not playing significant role in media formula-
tion cost. Thus, the nitrogen source in the form of (4.0 g/
l CSL and 3 g/l ammonium sulphate) was used in the sub-
sequent experiments.
3.5. Effect of different C:N ratios between (cane molasses
and the best inorganic nitrogen source)
In general, the C:N ratio in the culture medium is a crit-
ical factor for cell growth and metabolite production as
well. Since the cell productivity on the best organic nitro-
gen source (CSL) was lower than the inorganic nitrogen
source, the effect of different ratios between the C-source
(cane molasses) and the inorganic N-source (ammonium
sulphate) in culture medium on erythromycin production
was studied. As shown in Fig. 5, the medium without
ammonium sulphate showed a very low erythromycin pro-
duction of about 200 mg/l in all applied molasses concen-
trations (Fig. 5a–c). At low molasses concentrations of
40 g/l, the increase of ammonium sulphate concentra-
tion resulted in a significant increase in erythromycin pro-
duction up to 380 mg/l in 4 g/l supplemented cultures.
In 50 g/l molasses cultures, the addition of ammonium
sulphate slightly increases the erythromycin production
 H.A. El-Enshasy et al. / Bioresource Technology 99 (2008) 4263–4268 4267

Fig. 5. Cell growth and erythromycin production in media composed of different concentrations of ammonium sulphate and molasses.

and the maximal production of about 550 mg/l was propanol on the production of erythromycin was reported
obtained in culture containing 4 g/l ammonium sulphate. by Bezborodov and Galynkin (1970). Marini and Teatini
Upon increasing the molasses concentration up to 60 g/l, (1973) reported also that the addition of propanol increases
the maximal erythromycin production of about 600 mg/l the biosynthesis of erythromycin without affecting the
was obtained in 2 g/l ammonium sulphate supplemented growth. The increase in erythromycin production may
culture. This culture gave also the maximal yield coefficient
of about 75 mg/g (Fig. 5g). However, the repression effect
of high ammonium salt concentrations on the erythromy-
cin production was also observed by other authors (Flores
and Sánchez, 1985; Potvin and Péringer, 1994; Flores,
1996). The phenomenon of catabolic nitrogen repression
of ammonium sulphate has been reported to affect many
catabolic enzymes, including histidinase and urease which
play a significant role in erythromycin biosynthesis as well
as on the transcription of ery genes (Reeve and Baumberg,
1998). Thus, medium containing 60 g/l molasses and 2 g/l
ammonium sulphate was used in subsequent experiment.

3.6. Different n-propanol concentration

Since the short chain alcohol may increase the erythro-

mycin production, the effect of n-propanol concentration
was investigated in the range 0–3.0% v/v. The highest
erythromycin concentration of 720 mg/l was achieved with
1.0% v/v n-propanol supplemented culture. On the other
hand, cell growth was almost the same in all n-propanol
supplemented cultures. Thus, the increase in erythromycin
Fig. 6. Production of erythromycin by S. erythraea in different production
concentration in n-propanol supplemented culture was due
media.(d), Industiral medium with glucoes as main C-source, (m),
to the increase in cell productivity where the value of YP/X medium with cane molasses as main carbon source, (), medium with
in n-propanol cultures was higher compared to normal optimized ratio between ammonium sulphate and cane molasses concen-
medium. However, the positive effect of propionate and tration, (), finally optimized medium with n-propanol supplementation.
4268 H.A. El-Enshasy et al. / Bioresource Technology 99 (2008) 4263–4268
attributed to the induction of propionyl-CoA carboxylase
and other polyketide synthases, increasing the enzyme
activity and erythromycin biosynthesis (Potvin and Périn-
ger, 1993; Khosla et al., 1999). On the other hand, the pres-
ence of n-propanol in culture medium may also affect the
membrane permeability of cells leading to a better mass
transfer (Moragues et al., 1994).
3.7. Production of erythromycin in different media
The comparison between different erythromycin produc-
tion media used in this study is represented in Fig. 6. As
shown, the maximal production of erythromycin of about
800 mg/l was obtained in medium containing cane molas-
ses as main carbon source and supplemented with n-propa-
nol. However, this value was about 80% higher than the
antibiotic concentration obtained in the initial industrial
media under the same cultivation conditions. Thus, the
substitution of glucose by cane molasses accompanied with
a reduction of ammonium sulphate concentration and sup-
plementation of medium with n-propanol resulted in an
increase in erythromycin production as well as a reduction
in the erythromycin production cost.
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