Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Utilization of algal sugars and glycerol for enhanced

cephalosporin C production by Acremonium chrysogenum
M35
J.H. Lee1, H.Y. Yoo1, X. Yang1, D.S. Kim1, J.H. Lee1, S.K. Lee1, S.O. Han2 and S.W. Kim1
1 Department of Chemical and Biological Engineering, Korea University, Seoul, Korea
2 Department of Biotechnology, Korea University, Seoul, Korea

Significance and Impact of the Study: Microalgae are the biomass containing various components, such
as carbohydrates, lipids, and amino acids. In this study, carbon sources contained in microalgae were
obtained by acid extraction, and cephalosporin C (CPC), a b-lactam antibiotic intermediate, was pro-
duced by using Acremonium chrysogenum M35. In addition, the increase of CPC production was not
distinct for A. chrysogenum M35 with algal sugars as the only carbon source; therefore, glycerol was
added, increasing the CPC production. Thus, cheap residues such as algal sugars form microalgal and
glycerol form biodiesel process could be used as the alternative sources for the production of various
products.

Keywords
algal sugars, cephalosporin C, fermentation,
microalgae, PlackettBurman design.
Correspondence
Seung Wook Kim, Department of Chemical
and Biological Engineering, Korea University,
145, Anam-Ro, Seongbuk-Gu, Seoul 136-
701, Korea.
E-mail: kimsw@korea.ac.kr
2016/1662: received 29 July 2016, revised 25
September 2016 and accepted 26 September
2016
doi:10.1111/lam.12684
Abstract
In our previous study, glycerol was utilized as an additional carbon source for
the production of cephalosporin C (CPC) by Acremonium chrysogenum M35.
In this study, algal sugars extracted from the third-generation biomass were
utilized in the CPC production for the first time. The CPC production
improved about twofold when using the algal sugars as the carbon source. The
complex medium including algal sugars and glycerol was utilized, and 7
3 g l 1
CPC production was achieved in a 250-ml shaking flask. To determine the
important variables for the CPC production, PlackettBurman design was
carried out and 6
18 g l 1 of CPC was estimated under the numerically
optimized conditions. Under the optimized conditions, the CPC production
was performed in a 5-l scale bioreactor, affording CPC production at a rate of
7
1 g l 1. Moreover, 6
7 g l 1 CPC was produced using crude glycerol as the
substrate.

using Acremonium chrysogenum. Feeding soybean oil and

Introduction
Although many human diseases caused by germs or
moulds have been cured, developing new antibiotics or
anti-bacterial materials is still necessary, because of con-
tinuous mutation of many micro-organisms. Cephalos-
porin has not only a powerful antibacterial function
inhibiting germs from growing throughout wide areas but
also has a high level of stability on b-lactamase. Now
penicillin antibiotics are gradually being replaced by
cephalosporin antibiotics (Kargosha et al. 2003).
As for other studies on cephalosporin C (CPC), Luo
et al. (2013) studied the CPC fermentation process by
glucose at a ratio of 1 : 0
7 improved the fermentation
performance (Luo et al. 2013), and Adinarayana et al.
(2003) obtained maximum yield by using 1 wt% starch
and 1 wt% yeast extract as the additives (Adinarayana
et al. 2003). Shin et al. (2011) and Cruz et al. (2001)
reported increased CPC yield through fed batch culture
(Cruz et al. 2001; Shin et al. 2011). Kim et al. (2006)
investigated the effects of fatty acids by adding rice oils,
oleic acids, and linoleic acids during the CPC fermenta-
tion (Kim et al. 2006).
Fatty acids or vegetable oils are being used in the CPC
production process of using A. chrysogenum, and their

66 Letters in Applied Microbiology 64, 66--72 2016 The Society for Applied Microbiology
J.H. Lee et al.
demand and prices are increasing every year because of
increasing cost of the overall production of biodiesel. In
addition, fatty acids and vegetable oils added for the pro-
duction of antibiotics are not totally consumed, thus
making isolation and purification more expensive (Revin
et al. 1991; Brakhage et al. 2004; Kwon and Yeom 2015).
Recently, technology using various sorts of bioconver-
sion of marine biomass has been actively developed. In
particular, there are many types of microalgae living on
the globe in diverse forms from single cells to multi-cells.
As they contain lipids, carbohydrate, and proteins, they
are being highlighted as the next-generation biomass
(Park et al. 2014; Jeong and Park 2015).
In this study, during the process of searching the car-
bon source that could substitute glucoses and vegetable
oils in the CPC production using A. chrysogenum,
microalgae was selected as the resource that could meet
the two conditions. Microalgae usually contain lipids;
therefore, many studies have been conducted on using
them for biodiesel production. However, lipids are
extracted along with a large amount of carbohydrate and
protein. In addition, in the biodiesel production, glycerol
is also produced as a by-product. By applying these com-
ponents to the A. chrysogenum cultivation medium and
the CPC fermentation medium, the results of the process
applying glucoses and vegetable oils were compared, and
their effects on the CPC production were investigated.
Results and discussion
Effect of algal sugars and glycerol on cephalosporin C
production
The analysis of the components of Chlorella pyrenoidosa
showed that it consisted of 9
94% carbohydrate, 56
61%
protein, 0
64% fibre, 4
55% lipid, and 7
86% ash. In par-
ticular, carbohydrate contains 55% glucose and 45%
galactose. More than 95% of the components contained
in microalgae was extracted. In this study, CPC produc-
tion was conducted by using A. chrysogenum M35, utiliz-
ing various carbon sources obtained from the acid
extraction. As shown in Fig. 1(a), 1% glucose (Glu), 1%
mixed sugars (MS): 0
5% glucose, 0
5% galactose, and
1% algal sugars (AS) were used as the carbon sources and
fermented for 96 h to produce CPC. As a result, when
complex sugars obtained from microalgae were applied,
CPC concentration increased approximately twofold.
Although glucoses have been added as a carbon source
for the production of CPC and various antibiotics, the
production of the final product and secondary metabo-
lites was inhibited because of the catabolite repression by
glucoses (Jekosch and K uck 2000). According to the
report of Ozcengiz and Demain (2013), A. chrysogenum
Letters in Applied Microbiology 64, 66--72 2016 The Society for Applied Microbiology
CPC production by using algal sugars and glycerol
showed a faster growth rate when glucoses, maltose, and
fructose were used than when galactose and sucrose. In
contrast, galactose and sucrose were effective for the CPC
production. In addition, in the presence of glucose, the
specific growth rate decreased and complex polysaccha-
rides affected the CPC production (Ozcengiz and Demain
2013). As microalgae contain complex polysaccharides
such as lipids and proteins as well as carbohydrates, they
are useful resources for the CPC production. Although
A. chrysogenum M35 consumes sugars in the growth
phase, sugars are not sufficient in the production phase.
Therefore, CPC concentration must be increased by add-
ing various sources (Behmer and Demain 1983). Fig-
ure 1(a) shows that the CPC concentration increased by
4- to 6-fold, when glycerol was added compared to when
only sugars were used. Typical morphological changes of
a 6-day culture in a 250-ml shake-flask were evaluated
over 96 h using a phase microscope equipped with a
camera. In the early stage of the main culture, filamen-
tous hyphae and a few swollen hyphal fragments were
observed. Differentiation of A. chrysogenum M35 was
clearly observed at 96 h, with many swollen hyphal frag-
ments and arthrospores (Fig. 1b). Figure 1(ac) shows
the differences in the cell growth and differentiation; 1%
Glu, 1% MS, and 1% AS were used as the carbon sources
in Fig. 1(ac), respectively. More swollen hyphal frag-
ments are observed in Fig. 1(c) than in Fig. 1(a or b)
using AS, and the CPC production also increased as
shown in Fig. 1(c). Figure 1(df) is the results of adding
6% glycerol to the culture media in Fig. 1(ac), respec-
tively. The added glycerol worked as a secondary carbon
source, exhibiting a synergistic effect on the differentia-
tion of A. chrysogenum M35. In particular, the morphol-
ogy in Fig. 1(f) indicates that the swollen hyphal
fragment was relatively large compared with those of
Fig. 1(d or e), accelerating its differentiation, thus
increasing the CPC production (Kim et al. 2007; Shin
et al. 2010).
Cephalosporin C could not be produced using
A. chrysogenum by the addition of glycerol because of the
metabolic inhibition of carbon source. According to the
report of Gatenbeck and Brunsberg (1968), glycerol inhi-
bits the production of b-lactam antibiotics such as CPC
suppresses the polymerization of initial antibiotics
(Gatenbeck and Brunsberg 1968). However, Fig. 2 shows
the specific CPC concentration when glycerol was added
as the carbon source to the medium, indicating that glyc-
erol did not cause metabolic inhibition and thus the CPC
concentration increased. Figure 2(a) shows the CPC con-
centration after 120 h of fermentation. The results
showed that 96 h of fermentation was the most effective
for the production of CPC. The CPC recovery and purifi-
cation for 0120 h were also used in the actual industrial
67
CPC production by using algal sugars and glycerol
(a) 10
8
CPC concentration (g l1)
0
a b c d
(b)
a d
b e
c f
process. In the industrial process, although the amount of
materials produced is important, as the production costs
increase over time, productivity also becomes an impor-
tant factor. Figure 2(b) shows the concentration and yield
of CPC when the glycerol concentration was in the range
68
J.H. Lee et al.
e f
Figure 1 (a) Effect of carbon source (glucose
(Glu), mixed sugars (MS): 0
5% glucose
+0
5% galactose, algal sugars (AS)) and
glycerol (Gly) on cephalosporin C production
by Acremonium chrysogenum M35. (b)
Typical changes in the morphology during the
cultivation of A. chrysogenum M35 after
4 days (a: 1% Glu, b: 1% MS, c: 1% AS, d:
1% Glu +6% Gly, e: 1% MS +6% Gly and f:
1% AS +6% Gly).
08%. When glycerol concentration was 6%, the CPC
concentration and yield was at the highest value of
7
3 g l 1 and 0
73 g(CPC) g(AS) 1, respectively. However,
when glycerol concentration was above 8%, carbon inhi-
bition occurred in the metabolic pathway where
Letters in Applied Microbiology 64, 66--72 2016 The Society for Applied Microbiology
J.H. Lee et al. CPC production by using algal sugars and glycerol

(a) 8 70 100 of variables A and G as the index for interpretation of the

Glycerol concentration (g l1)

model was improper. However, the variables A and G did
60

Dry cell weight (g l1)

Concentration (g l1)

80 not affect the overall model. The results of the ANOVA in

6 50
Table 1 show that F- and P-values of the overall model
40 60
4 were 15
8 and 0
0041, respectively, and considering their
30 40 statistical significance, they showed reliability at 2% sig-
2 20 nificance level (Kim et al. 2013). The coefficient of deter-
20 mination (R2) of the model for the CPC production was
10
0 0 0 0
9499, showing an excellent significance, and its coeffi-
0 24 48 72 96 120 cient of variation was 19
82%. As a result of performing
Fermentation time (h) variable optimization based on this model, the CPC con-

Specific CPC production (gCPC gDCW1)

centration predicted under the optimized conditions (B:
(b) 010
0
92 ml, D: 0
1 g, E: 0
8 g, F: pH 7 and H: 0
02 g) was
8
6
18 g l 1.
CPC concentration (g l1)

6 microalgae, fermentation was performed in a 5 l with 6%
4 oxygen is required for the CPC production due to the
2 002
0 000
0 2 4 6
Glycerol concentration (%)
Figure 2 Effect of (a) fermentation time (glucose con. (), galactose
con. (), cephalosporin C (CPC) con. (), glycerol con. () and DCW
(Dry Cell Weight) ()) and (b) glycerol concentration (CPC con. ()
and specific CPC production ()).
A. chrysogenum produces CPC. As a result, CPC produc-
tion decreased when glycerol at a concentration above 8%
was added. Thus, in order to produce a high concentra-
tion of CPC in the early stage of production, 6% glycerol
was added to the medium (Shin et al. 2010).
Determination of important variables for CPC
production by PlackettBurman design
In general, various studies have been performed on the
types and concentration of carbon sources to enhance the
CPC production. The effect of medium composition was
investigated by using PlackettBurma design. The eight-
variable two-level PlackettBurma method was used to
select the conditions affecting the CPC production, with
ammonium sulphate, corn steep liquor (CSL), KH2PO4,
K2HPO4, DL-methionine, pH, trace elements and CaCO3
being the independent variables and the CPC concentra-
tion being a dependent variable. As a result of analysing
the effects of the eight variables on the CPC production,
predicted values of variables A and G calculated by a sta-
tistical analysis method were above 0
05, even 0
1. In gen-
eral, if a predicted value is above 0
05, the hypothesis for
its statistical model is inappropriate, and thereby, the use
008
After the selection of carbon sources extracted from
006
glycerol and an optimal medium. As a large amount of
004
existence of the biosynthesis pathway of oxidation reac-
tion, oxygen partial pressure (pO2) was maintained in the
range of 2530% through supplying air and oxygen gases.
Figure 3 shows the results of the fermentation performed
8
for 120 h, and 7
1 g l 1 CPC was produced at the 96 h
(Lee et al. 2010).
Effect of crude glycerol for CPC production
In the previous study, the optimum concentration of pure
glycerol for CPC production was found to be 6%. Thus,
the concentration of crude glycerol was also set to be 6%.
In order to examine the effects of crude glycerol as a car-
bon source, shake-flask culture was conducted for 5 days
at 27C. Six per cent pure glycerol and crude glycerol
were added to main culture mediums to compare their
CPC production. The CPC concentration in the main cul-
ture medium containing 6% pure glycerol was 6
7 g l 1,
which was lower than in the medium containing crude
glycerol; however, the difference was insignificant. Thus,
crude glycerol can be used as an alternative carbon source
for the CPC production by A. chrysogenum M35 and can
be applied to the CPC production owing to its merits
such as a high-yield rate and cost-effective purification
process in the industrial aspect.
Materials and methods
Algal biomass and reagents
Algal biomass (C. pyrenoidosa) dried powder was
obtained from WUDI LV QI Bioengineering Co., Ltd.
(Shandong, China). Hydrochloric acid (37%, HCl) used
in the investigation of hydrolysis process was purchased
from SigmaAldrich (St. Louis, MO). All the chemicals

Letters in Applied Microbiology 64, 66--72 2016 The Society for Applied Microbiology 69
CPC production by using algal sugars and glycerol J.H. Lee et al.

Table 1 PlackettBurman experimental design matrix and response The acid concentration was made according to the
data for determination of important variables for cephalosporin C specific gravity using a gravimeter. Heat treatment was
production
performed in an autoclave for 15 min at 121C. After the
Coded factor acid extraction, the mixture was neutralized to pH 7 with
levels 50% NaOH.
Symbol Variables Unit 1 1
Cultivation of fungal strain and media
A A sulphate ml 0 0
2
B Corn steep liquor ml 0 1 Acremonium chrysogenum M35 is a UV-induced mutant
C KH2PO4 g 0 0
06 of ATCC 20339 and is capable of increasing CPC produc-
D K2HPO4 g 0 0
1
tion. The basal seed medium was formed following our
E DL-methionine g 0 0
1
F pH 5 7
previous report (Kim et al. 2014). The final contents of
G Trace elements ml 0 2 the main media for the 400-ml total volume were 20 ml
H CaCO3 g 0 0
1 CSL, 1
2 g KH2PO4, and 2 g K2HPO4 in 280 ml of dis-
tilled water and the pH was adjusted to 7
0. 2 g of DL-
Sum of Mean P-value
methionine was added selectively, followed by addition of
Source squares df square F value Prob > F
2 g of CaCO3 and 40 ml of 109 trace element solution.
Model 31
01692 6 5
169486 15
80246 0
0041 The seed and main cultures were incubated in a 250-ml
B-corn steep 5
091321 1 5
091321 15
56352 0
0109 Erlenmeyer flask and the working volume was 25 ml at
liquor
300 rpm and 27C for 120 h. Fermentation was per-
C-KH2PO4 4
516555 1 4
516555 13
80653 0
0138
D-K2HPO4 7
907326 1 7
907326 24
17169 0
0044
formed in a 5-l stirred-tank bioreactor (Kobiotech Co.,
E-DL-methionine 6
127093 1 6
127093 18
72974 0
0075 Ltd., Incheon, Korea) at 27C and 300 rpm. The operat-
F-pH 3
235662 1 3
235662 9
891009 0
0255 ing volume was 3
0 l, and the air flow rate was 1
2 vvm.
H-CaCO3 4
138959 1 4
138959 12
65227 0
0163

Statistical optimization of media composition

PlackettBurman design (PDB) was carried out to deter-
8 75 100
mine the important variables for the CPC production. Ele-
70 ven variables including eight main fermentation medium
Dry cell weight (g l1)
Concentration (g l1)

80
6 65 components and three dummy variables were designed for
60 60 12 run experiments. Nutritional variables are the compo-
pH

4 nents of the fermentation medium and consist of ammo-

55
2 50
45
0 40
0 24 48 72 96 120
Fermentation time (h)
Figure 3 Production of cephalosporin C (CPC) in a 5-l bioreactor cul-
ture of Acremonium chrysogenum M35 (CPC con. (), pH () and
DCW (Dry Cell Weight) ()).
used were of reagent grade. Crude glycerol was obtained
from Dansuk Industrial (Gyeonggi-do, Korea).
Dilute acid extraction of algal biomass
Algal sugars were obtained by dilute acid extraction from
C. pyrenoidosa and used for the CPC production as the
substrates of A. chrysogenum M35. To investigate the
optimum condition of acid extraction, the solid (algal
biomass)liquid (dilute acid) ratio and acid concentration
in a 20-ml vial were set as 100 g l 1 and 2%, respectively.
40
nium sulphate, CSL, KH2PO4, K2HPO4, DL-methionine,
20 pH, trace elements, CaCO3, and agitation speed. The factor
operating conditions were prepared at two levels: high level
0 (+1) and low level ( 1) (Table 1). The dummy variables
were used to estimate experimental errors in data analysis
(Pareek et al. 2011; Jung et al. 2015).
Image capturing
All the samples were examined using an optical micro-
scope (Samwon Scientific Ind. Co. Ltd., Seoul, Korea)
and IMAGE PRO 3.0 software (Media Cybernetics, Silver
Spring, MD). Images were digitally captured using a CCD
camera (Sony, Tokyo, Japan).
Analytical methods
The solid algal biomass was analysed to determine its
absolute composition (carbohydrate, fibre, protein, lipid,
and ash) based on the standard procedures of the
National Renewable Energy Laboratory (NREL, USA) and

70 Letters in Applied Microbiology 64, 66--72 2016 The Society for Applied Microbiology
J.H. Lee et al.
Official Methods of Analysis of AOAC INTERNATIONAL
(OMA) (Sanchez-Machado et al. 2004; Sluiter et al.
2012).
Cephalosporin C was measured by high-performance
liquid chromatography (HPLC) using a Bondapak C-18
reverse-phase column (Waters, Milford, CT, USA) and a
254 nm UV detector (Shimadzu, Japan). The mobile
phase consisted of 40% (v/v) acetonitrile applied at a flow
rate of 0
9 mL min 1. Cephalosporin C zinc salt (Sigma,
Saint Louis, MO, USA) was used as the standard.
The concentrations of sugars and glycerol were mea-
sured by HPLC using an Aminex HPX-87H column
(300 mm97
8 mm, Bio-Rad, Inc., Hercules, CA, USA)
and a refractive index detector (Shimadzu, Kyoto, Japan).
The temperature of the column and detector was main-
tained at 50C. The mobile phase was 0
005 N H2SO4 at
a flow rate of 0
8 ml min 1. Pure glycerol (Daejung
Chem., Gyeonggi-do, Korea) was used as the standard.
The dry cell weight of the mycelium was measured as
follows: 10 ml of culture broth was filtered through a
Whatman GF/C, washed twice with deionized water, and
then dried at 80C for 24 h.
Acknowledgements
This work was supported by the National Research Foun-
dation of Korea (NRF) grant funded by the Korea gov-
ernment (MSIP) (no. NRF-2014R1A2A2A01007321) and
the Industrial Strategic Technology Development Program
(10051513) funded by the Ministry of Trade, Industry &
Energy (MI, Korea).
Conflict of Interest
No conflict of interest declared.
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Letters in Applied Microbiology 64, 66--72 2016 The Society for Applied Microbiology