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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

(Nuntnarumit et al., 2006). Besides all these medical appli-
Erythromycin is a polyketide antibiotic produced by cations, erythromycin is also used as prophylactic and
Saccharopolyspora erythraea mainly in submerged culture. curative therapeutic agent for many diseases of poultry
This antibiotic has many pharmaceutical applications in and farm animals (Prescott, 1997). Although erythromycin
the treatment of many diseases caused by gram-positive originally developed for its use in human and veterinary
bacteria and some gram-negative bacteria. Therefore, medicine, it has also many applications in the animal feed-
erythromycin is widely used clinically in respiratory infec- ing (Moffitt, 1998) and the cultivation of marine microal-
tious diseases (Hoyt and Robbins, 2001), in the treatment gae (Kim and Cerniglia, 2005; Campa-Córdova et al.,
of many acute infections caused by bacteria belonging to 2006). Nowadays, the industrial production of erythromy-
Staphylococci spp. and Neisseria spp. (Lesmana et al., cin is mainly carried out by Saccharopolyspora erythraea.
2001) and as an antimalarial in combination with other The production process of this antibiotic is mainly by
drugs to reduce the pathogen resistance (Nakornchai and means of submerged culture system of either free cells
Konthiang, 2006). Recently, erythromycins were also used (Martin and Bushell, 1996; Heydarian et al., 1999) or
immobilized cells (El-Enshasy and Beshay, 2000; Hamedi
*
Corresponding author. Tel.: +2 010 5177482; fax: +20 3 4593423. et al., 2005). Like other secondary metabolites produced
E-mail address: h.elenshasy@mucsat.sci.eng (H.A. El-Enshasy). 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- glucose. The SCM was obtained from the Sugar and Inte-
position and cultivation conditions as well. Concerning grated Chemicals Co. (El Hawamdya, Giza, Egypt). The
medium composition, the production of erythromycin is used SCM had the following composition: total carbohy-
highly regulated by type and concentration of the carbon drate, 55%, nitrogen content, 0.4%; CaO, 0.6%; MgO,
source (Martin and Demain, 1980; Escalante et al., 1982), 0.5% and ashes, 4%.
nitrogen source (Flores and Sánchez, 1989; Potvin and The cultivations were carried out in Erlenmeyer flasks of
Péringer, 1993), phosphate and trace elements (Singh 250 ml (working volume 50 ml). The flasks were placed on
et al., 1981). In general, carbon source is usually used in rel- a rotary incubator shaker at 200 rpm and 30 C for 144 h.
atively higher concentration compared to other medium After that time, the media were filtered and analyzed for
component and thus has high share in the raw material the determination of cell dry weight, antibiotic production
cost. Therefore, molasses, milk whey, starch, cellulose and final pH.
and other cheap complex carbon sources were used to
replace glucose in many production processes. A normal 2.3. Analysis
cane molasses usually has a water content of 17–25%, a
sugar content (sucrose, glucose and fructose) of 45–50% 2.3.1. Determination of cell dry weight
and polysaccharides (dextrin, pentosans, polyuronic acids) The cell dry weight of cells after cultivation was deter-
containing 25% (Najafpour and Shan, 2003). However, the mined gravimetrically. The fermentation broth was filtered
international price of sugar cane molasses is ranged through a dry preweighed filter paper (Whatman No. 1)
between $120–150 per metric ton. Therefore, it is widely and washed several times with distilled water. The washed
used as main component in the industrially used medium cells were dried in an oven at 100 C until a constant weight
for the production of different primary and secondary was obtained.
metabolites (Ikram-ul et al., 2004; Cazetta et al., 2005;
Banik et al., 2007; Cha et al., 2007). In the present work, 2.3.2. Determination of erythromycin
an attempt was done to optimize the cultivation medium The amount of the erythromycin produced in the fer-
through the replacement of carbon and nitrogen sources mentation broth was determined by means of biological
to cheaper one in order to reduce the cost of raw materials. method according to Bershtein et al. (1983). The antibiotic
The present study was also extended to investigate the assay medium (Difco) composed of (g/l): glucose, 10.0;
influence of other medium ingredients on the antibiotic peptone, 10.0; meat extract, 2.5; yeast extract, 5.0; NaCl,
yield. 10.0 and agar, 20.0. The erythromycin sensitive strain of
Bacillus subtilis NRRL B-543 was used the biological activ-
2. Methods ity determination. The diameters of the inhibition zones
obtained were measured and the amount of erythromycin
2.1. Microorganism and inoculum preparation produced was calculated from a biological standard curve
previously made using extra pure standard erythromycin
Saccharopolyspora erythraea NCIMB 8594 was used (Sigma, USA) as an authentic reference material.
throughout this study. This stain was obtained from the
National Collection of Industrial and Marine Bacteria, 3. Results and discussion
Aberdeen, Scotland, UK. This strain was maintained on
ISP-2 solid medium composed of (g/l): glucose, 4; yeast 3.1. Batch cultivation of saccharopolyspora erythraea in
extract, 4, malt extract, 10 and bacteriological agar, 20. industrial medium
The pH was adjusted to 7.0 before sterilization. The slants
were inoculated and incubated at 28 C for 10 days in order Fig. 1 shows the time profiles of cell growth, volumetric-
to obtain a heavy sporulated growth. After that time, and specific erythromycin production during a typical
spores (in a concentration of 1 · 108 spores/ml) were used batch cultivation of S. erythraea in industrial medium.
to inoculate the fermentation medium. The maximal value of volumetric erythromycin production
of about 450 mg/l was obtained after 144 h and kept more
2.2. Production medium and cultivation conditions or less constant for the rest of cultivation time. On the
other hand, the maximal cell growth of 10.2 g/l was
Unless otherwise mentioned, the initial industrially used obtained after 96 h and decreased gradually after that time.
medium for erythromycin production was composed of Concomitantly, significant increase in pH value was
(g/l): glucose, 20; corn steep liquor, 4.0; NH4(SO4)2, 3; observed during the phase of cell lysis. The decrease in cell
NaCl, 2.5; and CaCO3, 5. The pH was adjusted to 7.2 mass was occurred as a result of hyphal cell fragmentation
before sterilization. Medium sterilization was carried out due to the shear influence. Bushell et al. (1997) observed
by autoclaving at 121 C for 15 min. The carbon source also a significant fragmentation in S. erythraea in sub-
was autoclaved separately at 121 C for 10 min and added merged cultures even in low shear cultures such as non-baf-
to the medium prior inoculation. In case of molasses based fled shaken flasks. This was due to the fragility of S.
medium, sugar cane molasses (SCM) was used instead of erythraea hyphae especially when cells inter stationary
 H.A. El-Enshasy et al. / Bioresource Technology 99 (2008) 4263–4268 4265

Fig. 1. Cell growth and erythromycin production during batch cultivation

of S. erythraea in glucose based industrial medium. 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.
phase (Stocks and Thomas, 2001a). The increase in pH
may due to the release of ammonia in fermentation med-
in pure or in polymer forms were the best C-sources for
ium as a function of cell lysis and fragmentation (Stocks
erythromycin production. Since the cane molasses is
and Thomas, 2001b). The specific erythromycin production
cheaper than starch and more available than beet molasses,
(YP/X) reached about 48 mg/g after 144 h (the time of max-
as industrial by-product from sugar refining industries in
imal volumetric erythromycin production). The observed
Egypt, cane molasses in a concentration of (50 g/l) was
increase in the specific antibiotic production after that time
used in the subsequent experiments.
was due to the decrease in cell mass rather than the increase
in antibiotic production.
3.3. Effect of different inorganic nitrogen sources
3.2. Effect of different carbon sources
The effect of seven types of ammonium salts on the pro-
The suitability of different C-sources (monosaccharides, duction of antibiotic was investigated in this experiment.
disaccharides and complex carbon sources) for the produc- The original industrially used erythromycin production
tion of erythromycin was studies. As shown in Fig. 2, medium was containing both organic (CSL) and inorganic
among different monosaccharides tested both of maximal (ammonium sulphate) nitrogen sources. The aim of this
volumetric erythromycin production and cell growth was experiment was to study the effect of different types of
obtained in glucose culture and reached about 450 mg/l ammonium salts in CSL supplemented medium on the
and 9.6 g/l, respectively. On the other hand, in spite of a erythromycin production. The nitrogen content of each
good growth in galactose, fucose and arabinose cultures, source was equivalent to the amount of nitrogen in the ori-
the production of antibiotic was very low. Of different ginal ammonium sulphate concentration (3 g/l). The results
disaccharides tested, the maximal erythromycin production in Fig. 3 show that the addition of different ammonium
was obtained in sucrose culture followed by trehalose, salts instead of ammonium sulphate did not increase the
maltose b-lactose and a-lactose. On the other hand, the erythromycin production. Ammonium sulphate and
production of erythromycin in complex carbon source ammonium chloride were the best salts supporting erythro-
media, containing either starch or molasses, reached mycin production. The volumetric maximal antibiotic pro-
480 mg/l. These results indicate that, glucose and sucrose duction in both cultures was about 450 mg/l. Other
4266 H.A. El-Enshasy et al. / Bioresource Technology 99 (2008) 4263–4268

Fig. 4. Effect of different organic nitrogen sources on cell growth and

Fig. 3. Effect of different inorganic nitrogen sources on the cell growth erythromycin production by S. erythraea.
and erythromycin production by S. erythraea.
fore, it could be concluded that it is not preferable to sub-
ammonium salts like ammonium oxalate, ammonium stitute the inorganic nitrogen source in the medium with
nitrate and ammonium carbonate support better cell organic source. On the other hand, the price of ammonium
growth on the cost of antibiotic production. However, sulphate is not playing significant role in media formula-
the maximal yield of erythromycin production based on tion cost. Thus, the nitrogen source in the form of (4.0 g/
cell mass (YP/X) of about 55 mg/g was obtained in ammo- l CSL and 3 g/l ammonium sulphate) was used in the sub-
nium sulphate supplemented culture. sequent experiments.

3.4. Effect of different organic nitrogen sources 3.5. Effect of different C:N ratios between (cane molasses
and the best inorganic nitrogen source)
The aim of this experiment was to study the possibility
for the substitution of the inorganic nitrogen source in In general, the C:N ratio in the culture medium is a crit-
the production medium, ammonium sulphate, with organic ical factor for cell growth and metabolite production as
nitrogen source. The amount of nitrogen content in 4 g/l of well. Since the cell productivity on the best organic nitro-
CSL and 3 g/l ammonium sulphate in the medium was gen source (CSL) was lower than the inorganic nitrogen
replaced by an equivalent amount of nitrogen in the differ- source, the effect of different ratios between the C-source
ent organic nitrogen sources. Fig. 4 shows the different (cane molasses) and the inorganic N-source (ammonium
organic nitrogen sources used and the corresponding cell sulphate) in culture medium on erythromycin production
growth, erythromycin production after 144 h cultivation. was studied. As shown in Fig. 5, the medium without
The maximal volumetric erythromycin production was ammonium sulphate showed a very low erythromycin pro-
obtained in medium supplemented by CSL followed by duction of about 200 mg/l in all applied molasses concen-
milk whey and fodder yeast. The maximal volumetric anti- trations (Fig. 5a–c). At low molasses concentrations of
biotic production in the CSL culture was almost the same 40 g/l, the increase of ammonium sulphate concentra-
that obtained in ammonium sulphate culture in the previ- tion resulted in a significant increase in erythromycin pro-
ous experiment. On the other hand, the cell productivity duction up to 380 mg/l in 4 g/l supplemented cultures.
(YP/X) in CSL culture was about 40 mg/g which is lower In 50 g/l molasses cultures, the addition of ammonium
than the ammonium sulphate culture by about 38%. There- 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 Flores, M.E., Sánchez, S., 1989. Ammonium assimilating enzymes and
and other polyketide synthases, increasing the enzyme erythromycin formation in Saccharopolyspora erythraea. J. Gen.
Microbiol. 35, 203–211.
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ger, 1993; Khosla et al., 1999). On the other hand, the pres- opolyspora erythraea ATCC 11365. FEMS Microbiol. Lett. 139, 57–62.
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Ikram-ul, H., Ali, S., Qadeer, M.A., Iqbal, J., 2004. Citric acid production
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