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Optimization of Sugarcane Bagasse, Nutrient and Temperature on the Yield of

Penicillin V in Solid State Fermentation by Penicillium Chrysogenum

Article · February 2010

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International Journal of Biotechnology and Biochemistry
ISSN 0973-2691 Volume 6 Number 3 (2010) pp. 477–483
© Research India Publications
http://www.ripublication.com/ijbb.htm

Optimization of Sugarcane Bagasse, Nutrient and

Temperature on the Yield of Penicillin V in Solid
State Fermentation by Penicillium Chrysogenum

V. Vinoth Kumar*, C. Vigneshwaran, K. Vasantharaj, J. Pradheep Isaac

and B. Bharathiraja

Arunai Engineering College, Department of Biotechnology,

Thiruvanamalai – 606603, India
*Kumaraguru College of Technology, Department of Biotechnology,
Coimbatore -641006, India
Correspondence Author: Email: projects0001@gmail.com

Abstract

An advanced solid state fermentation (SSF) system (liquid medium absorbed

on an inert support) has been applied to Penicillium chrysogenum for
penicillin V production. Penicillium chrysogenum showed an increased
penicillin production was investigated during static cultivations with bagasse
and lactose as the growth-limiting substrate. Pre-treatment of bagasse with
cellulase enzyme produces high yield of penicillin V. The activity was
increased in 50% bagasse, compared to other concentration of bagasse.
Among the carbon source tested, great yield was obtained in lactose followed
by starch, glucose and low production was noticed in sucrose. Enzyme treated
sugarcane bagasse with lactose shows high penicillin activity in 19.2 g/kg.
Production was further increased to 22 g/Kg on sixth day of the fermentation
carried out at optimum temperature 26 oC. The low cost material of sugar
industries like bagasse was used as carbon source assists great reduction in the
cost of penicillin V production.

Key words: Penicillin V; growth; baggase; sugar; solid state fermentation.

Introduction
The advancement of SSF technology has permitted a higher degree of control over the
process and hence, the production of high value products like antibiotics and other
secondary metabolites [1]. Secondary metabolites are compounds with varied and
478 C. Vigneshwaran et al

sophisticated chemical structures, produced by strains of certain microbial species,

and by some plants. Although antibiotics are the best known secondary metabolites,
there are other such metabolites with an enormous range of biological activities,
hence acquiring actual or potential industrial importance. Studies in liquid culture
show that production of secondary metabolites starts when growth is limited by the
exhaustion of one key nutrient: carbon, nitrogen or phosphate source. For example,
penicillin biosynthesis by Penicillium chrysogenum starts when glucose is exhausted
from the culture medium and the fungus starts consuming lactose, a less readily
utilized sugar [2].
Secondary metabolites are generally reported to be produced in higher
concentration in solid culture, often in short times and without the need of aseptic
condition [3,4]. From the biological point of view, a greater knowledge of the nature
of secondary metabolism in solid medium is needed to obtain the system’s full
production potential. The novel SSF system contains liquid media absorbed on an
inert support (sugarcane bagasse) that present several advantages over classical SSF
system for basic studies. In addition, good growth and excellent production are also
obtained in this system [5, 6].
A careful analysis of the literature confirms the importance of initial moisture as a
key process variable in SSF. Hence, this parameter has to be optimized for secondary
metabolites production, as well as for primary metabolites, enzymes or biomass
production. In practice, other key process variables are usually optimized or
controlled in SSF: pre-treatment, particle size, temperature and initial pH, aeration,
inoculum size, and sometimes mixing [7]. The objective of the present study was to
determine the effect of pre-treatment of bagasse with cellulase enzyme, different
concentration of bagasse, different carbon sources, fermentation time and temperature
on penicillin production in SSF using Penicillium chrysogenum.

Materials and methods

Chemicals and microorganism
Highly cellulose content sugarcane bagasse was obtained from EID Parry private
limited, Cuddalore, India. Cellulase was procured from Sigma Chemicals, USA. The
high yield penicillin V producing strain Penicillium chrysogenum was obtained SPIC
pharmaceutical division, Cuddalore, India.

Preparation of bagasse and POAA

Sugarcane bagasse was drying at 60 to 80 oC in hot air oven for two days, after drying
grinding of bagasse using mixer grinder (KSS, Chennai) was done and grinded
powder were kept through sieve (200 micron) to obtain fine powder. That powder was
used in solid state fermentation. 10 g of Phenoxy acetic acid (POAA) was weighed
and adjusted to pH 6.3 by adding 50 ml of 2.5N NaOH solution and made upto 1000
ml.
Optimization of Sugarcane Bagasse, Nutrient 479

Pre-treatment of bagasse by enzyme method

For pre-treatment, 500 g of bagasse was added to 100 ml of cellulase enzyme and 100
ml of phosphor-citrate buffer (pH 7) that contain 0.2 ml of Tween 60. This mixer was
incubated in shaking water bath at 40 oC for 3 days. After 3 days samples were taken
and glucose was estimated by DNS method. Then this enzyme treated bagasse is used
in solid state fermentation method 1g bagasse released 0.4520 g glucose released is
determined remaining glucose should be added to fermentative medium

Preparation of seed and fermentative medium

Seed medium consists of (g/l) cotton seed meal 10; corn steep powder 20; sucrose 40;
ammonium sulphate 2; KH2PO4 0.5; CaCO3 5; (pH 6.3) and sterilised at 121 oC for 15
min. Ten ml of 106 spore suspensions were inoculated into the sterile medium and
incubated in rotatory shaker for 54 hrs at 230 rpm in 25 oC. Fermentative medium
contains (g/l) Lactose 120; cotton seed meal 27.5; (NH4)2SO4 12; KH2PO4 1.5; K2SO4
5; CaCO3 10; Phenoxy acetic acid 10 ; Corn oil 10 ml. 35 ml of above mentioned
media were kept in 250 ml wide mouth conical flasks were used for fermentation. 2
ml of seed medium was inoculated into fermentation medium and incubated for 168
hrs at 28 oC. (pH 6.3). For extraction, 50 ml buffer was added to the fermentation
medium and kept in gyratory shaker at 200 rpm for 2 hours. Extracted samples were
centrifuged at 10, 000 rpm for 10 min at 4 oC and supernatant were analyzed for
penicillin V production.

Optimization studies on penicillin V production

Five conical flasks which containing fermentation medium were taken, each
containing 25 %, 50 %, 75 %, 100 % of bagasse and 50 % enzyme treated. Various
carbon sources like lactose, glucose, sucrose, starch were added to the fermentation
medium containing 50 % bagasse. Different temperatures like 23, 26 and 30 oC were
used the production of penicillin V and monitored for seven days. Penicillin
concentration was determined by HPLC under the conditions described previously
[7]. Production was expressed in milligram of penicillin G per kilogram of dry
fermented material. Experiments were performed in duplicate and in at least two
separate experiments.

Results
On penicillin production, carbon source, nitrogen source and minerals were
essentially required. In this study, sugarcane bagasse was added to the fermentative
media with Penicillium chrysogenum and their effects were studied.

Effect of bagasse concentration on penicillin V production

Bagasse was good enough for penicillin production and the results were positive,
because Penicillium chrysogenum utilised bagasse and high activity (18 g/Kg) was
recorded in 50 % bagasse. Compare to normal, enzyme treated bagasse shows
maximal activity 19.2 g/Kg (Fig. 1). 100 % bagasse does not assist and produce low
yield (0.68 g/Kg), because no extra carbon sources in the medium.
480 C. Vigneshwaran et al

Culture conditions : pH 6.3; Temperature 28 oC; Incubation time 7 days; Inoculum

size 2 %; Inoculum age 2 days.

Figure 1: Effect of bagasse with lactose on penicillin V production.

Effect of carbon sources on penicillin V production

The carbon sources like lactose, glucose, sucrose and starch were added to bagasse
and enzyme treated. Sugar molecules with bagasse shows high activity of penicillin in
case of enzyme treated only. In case of sugars, high penicillin activity was recorded
with lactose only 18 g/Kg; whereas enzyme treated bagasse with lactose shows high
productivity than control. Lactose provides additional time to the fermentation, so
high yield of penicillin was produced (Fig.2). Low yield was noticed in sucrose with
bagasse 9.39 g/Kg and enzyme treated was 11.19 g/Kg. Sucrose shows certain
inhibitory effect and it does not provide necessary conditions to the fermentation.
Glucose and starch provides good yield, but not satisfactory with lactose. So it was
concluded that lactose give high yield in penicillin V production using Penicillium
chrysogenum and used for further studies.

*Shaded bar indicates enzyme treated bagasse and unshaded bar indicates control.
Culture conditions : pH 6.3; Temperature 28 oC; Incubation time 7 days; Inoculum
size 2 %; Inoculum age 2 days.

Figure 2: Effect of carbon sources on penicillin V production by solid state

fermentation.
Optimization of Sugarcane Bagasse, Nutrient 481

Effect of temperature and fermentation time on penicillin V production

Optimum temperature and fermentation time for the penicillin V production is 26 oC
and six days, respectively. High temperature (30 oC) produces more biomass and
decreases the moisture content and low yield (8.9 g/Kg) was obtained. Slow growth
would be observed at the lowest temperatures. One of the temperatures, in the vicinity
of 26 oC is best for the penicillin-forming phase and produces high activity (22.2
g/kg). Low temperature inhibit the mycelium forming phase and penicillin activity (11
g/Kg) was decreased (Fig.3).

Culture conditions : pH 6.3; Inoculum size 2 %; Inoculum age 2 days

Figure 3: Effect of temperature and fermentation time on Penicillin V production.

Discussion
SSF method is good enough for penicillin production because does not require shaker,
very low power and low cost materials were used. The low cost material of sugar
mills were used as carbon source when compared to other costly carbon source. The
activity was increased in enzyme treated and 50 % bagasse, compared to other
concentration of bagasse. Enough level of aeration was passed in 50 % bagasse and
showed good result. In case of 25 % bagasse, inhibition was found in semi solid
condition. 100 % and 75 % of bagasse contain high level of cellulose, Penicillium
chrysogenum was unable to utilize nutrient so low amount of yield was obtained.
When carbon source like lactose, glucose, starch and sucrose are added to the
fermentation medium with sugarcane bagasse, significant level of activity was
increased. Great effect was showed on lactose combination when compared another
carbon source. Compare to submerged fermentation, solid state fermentation shows
high production of penicillium. Sugarcane bagasse was very complex material and
high cellulose content, hence this is not able to take ultimately by Penicillium
chrysogenum. Sugarcane bagasse contains around 50 % of cellulose, 27.9 %
hemicellulose and 11.3 % of lignin content; all these cause decrease in penicillin
production. Cellulase enzyme convert the cellulose to glucose, easy uptake of glucose
by Penicillium chrysogenum and results in elevated yield of penicillin V was
482 C. Vigneshwaran et al

obtained. Sugarcane bagasse contain elevated amount of ash content (12-20 %) and
non nitrogenous acid, they also decreased the penicillin production. But in case of
pre-treatment of bagasse, good result was obtained.
In enzyme treated bagasse with lactose shows maximal penicillin activity (19.2
g/kg), whereas 50 % of bagasse concentration showed penicillin activity (18 g/kg).
Submerged fermentation was showed very low level of penicillin activity (0.6 g/kg)
and pure cellulase with additives did not induce penicillin production. Maximal
penicillin V production was illustrated in lactose and 50 % bagasse combination
followed by starch, glucose and sucrose. Mycelium grown at 30 oC used sugar more
slowly during the penicillin-forming phase, regardless of the temperature during that
phase. The optimal temperatures reported for maximum production of penicillin by
Penicillium chrysogenum have varied between 23 oC and 28 oC [8]. It was well
known that maximum penicillin production by Penicillium chrysogenum occurs only
for the mycelium-producing phase but different, conditions exist for the penicillin-
producing phase. These two phases should have the same temperature optimum seems
unlikely [9]. In our study, high penicillin production was obtained at 26 oC, but
biomass was satisfactory with 30 oC.

Conclusion
Penicillium chrysogenum mycelia grown in a bagasse with complex medium contain
lactose as a carbon source and maximum production of penicillin V was obtained on
6th day. Compare to raw sugarcane bagasse, cellulase treated bagasse shows great
potential in the production. 50 % bagasse concentration in the medium assists
maximal penicillin activity, further induction of yield was by addition of lactose.
Optimum temperature of 30 oC was best for the mycelium-producing phase; whereas
26 oC was best for the penicillin-forming phase.

Acknowledgement
The authors were expressing their thanks to SPIC, pharmaceutical division, Cuddalore
for providing high yield penicillin producing strain and valuable guidelines.

References
[1] Dominguez, M., Mejia, A., Revah, S., and Barrios Gonzalez, J., 2001,
“Optimization of bagasse, nutrients and initial moisture ratios on the yield of
penicillin in solid state fermentation,” World J microbiology and
biotechnology, 17, pp. 751-756.
[2] Barrios Gonzalez, J., Fernandez F.J., Tomasini A., Mejia, A., 2005,
“Secondary metabolites production by solid state fermentation,” Malays J
Microbiol, 1 (1), pp.1-6.
[3] Hesseltine, C.W., 1977, “Solid state fermentation,” Process biochemistry, 12,
pp. 29-32.
Optimization of Sugarcane Bagasse, Nutrient 483

[4] Barrios Gonzalez, J., Tomasini, A., Viniegra Gonzalez, G., and Lopez L.,
1988, “Penicillin production by solid state fermentation,” Biotechnology
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[5] Acuna-Argulles, M., Gutierrez-Rojas, M., Viniegre-Gonzalez, G and Favela-
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[6] Barrios Gonzalez, J., and Mejia, A., 1996, “Secondary metabolites production
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[7] Barrios Gonzalez, J., Gonzalez, H. and Mejia, A., 1993, “Effect of particle
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[8] Calam, C. T., Driver, N., and Bowers, R. H., 1951, “Studies in the production
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[9] Owen, S.P., and Johnson, M.J., 1955, “The effect of temperature changes on
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375-380.
 484 C. Vigneshwaran et al

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