OPTIMIZATION OF CULTURAL CONDITIONS FOR THE PROPAGATION OF GLIOCLADIUM VIRIDE ZIC2063 AS POTENTIAL BIOSORBENT
Arifa Tahir1, Sidra Zahid1, Bushra Mateen1, Tasnim Farasat1, Tahira Mughal2
1. Environmental Science Department, Lahore College for Women University Lahore, Pakistan 2. Botany Department, Lahore College for Women University Lahore, Pakistan
Keywords: Gliocladium viride, Biosorbant, Culture
Abstract

In the present study, the propagation of a newly isolated Gliocladium viride ZIC2063 for maximum mycelium formation is checked. This is the first report on the optimization of Gliocladium viride ZIC2063 cultural conditions to get high mycelium concentrations that is to be used in further biosorption process. The mould mycelium was exploited as biosorbent. The present study was designed to optimize culture medium and other cultural conditions (pH, temperature, incubation time, inoculum age and size) for the growth of fungal culture as biosorbent. The amount of mycelium was doubled due to optimization. The Gliocladium viride ZIC2063, being high chromium resistant, thermostable and acid stable, can find application in the treatment of tanning effluent of leather industry and combating

Article Information

Identifiers and Pagination:
Year:2010
Volume:2
First Page:37
Last Page:46
Publisher Id:JAppPharm (2010 ). 2. 37-46
Article History:
Received:September 23, 2009
Accepted:December 17, 2009
Collection year:2009
First Published:January 13, 2010

INTRODUCTION
Fungi are well known for their metal binding abilities. Fungi exhibit marked tolerance towards metals and
other adverse conditions (Faryal et al., 2006). The metal binding capacities of fungi depends on the
presence of functional groups on cell wall (Luef et al., 1991). These functional groups can be blocked or
damaged by surrounding environments of fungi (Ewan and Pamphlett, 1996). One of studies investigated
that chemical composition of cell wall is very sensitive to growth conditions (Yang and Illman, 1999). This
is how the composition of growth medium and other cultural conditions effects the present study. The
cultural conditions were optimized to get high biomass concentration that can be used in further biosorption
experiments. The conditions in which microorganisms grow affect its cell surface phenotype which in turn
affects its biosorption potential (Gadd, 1990). Aspergillus niger grown in the presence of potassium
hexacyanoferate exhibited high biosorption (Luef et al., 1991). Gram-positive bacteria show increased
biosorption by adding nutrients in 2h of incubation, while Gram-negative bacteria didn’t show the same
trend (Gourdon et al., 1997). Phormidium laminosum, biosorption increased with addition of sufficient
nitrogen source (Sampedro et al., 1995). Detailed work was done on the optimization of pH, temperature,
inoculum size, agitation and carbon nitrogen sources on fungal culture. This is the first report on the
optimization of cultural conditions of Gliocladium viride ZIC2063 on potato dextrose medium . In this
study, an attempt has been made to formulate a suitable and cost-effective medium for the propagation of
Gliocladium viride ZIC2063 as biosorbent


MATERIAL AND METHODS
Organism
More than fifty fungal cultures from tanning unit effluent were isolated using Potato Dextrose Agar
medium. After screening the mould culture Gliocladium viride ZIC2063 was selected for further
investigation. The selected culture was transferred in glycerol at -20°C and also at 4°C.
Biomass Analysis
Fungal cell biomass was estimated gravimetrically by filtering the culture through a pre-weighed dry
Whatman No. 1 filter paper. The mycelium was thoroughly washed with distilled water and then weighed.
Propagation of biomass
To achieve maximum amount of Gliocladium viride ZIC2063, cultural conditions were optimized. Four
culture medias, Potato dextrose broth (M1), yeast peptone sucrose medium (M2), liquid medium (M3) and
Czapek Dox medium (M4) were used for the propagation of fungal growth (Table1). Selected medium was
further supplemented with different carbon sources (Sucrose, dextrose and Sodium acetate) and nitrogen
sources (Sodium nitrate, urea and yeast extract) to evaluate their effect on the growth of fungi. These
carbon and nitrogen sources were added at the concentration of 1%. In order to investigate the effect of
initial pH of growth medium the pH was varied from 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0. Phosphoric
acid (1M) was used to adjust the pH. Incubation temperature ranged from 20 oC to 35 oC for different time
periods i.e., 1, 2, 3 and 4 days to optimize incubation temperature as well as incubation time. Inoculum age
(3, 5 and 7 days old) and size (2%, 4%, 6%, 8% and 10%) were optimized. Role of agitation on mycelium
growth was also recorded.


RESULTS & DISCUSSION
Screening of culture Medium
Four different culture media (M1, M2, M3 and M4) were evaluated for the propagation of Gliocladium viride
ZIC2063 for mycelium formation (Table 1). Of all the media testedM1 (Potato Dextrose Broth) gave best
results (4.9029 g wet weight) and was used in further propagation process. While in M2, M3 and M4
medium the biomass growth was 3.6945 g wet weight, 3.0246 g wet weight and 2.8301 g wet weight
respectively.
Potato broth was supplemented with different carbon and nitrogen sources to study the effect on the growth
of fungi. It was found that type and concentration of carbon and nitrogen sources had a significant effect on
growth. Among carbon sources dextrose gave the maximum results (4.7813 g wet weight) (Table 2). All
nitrogen sources were found effective for fungal growth but Sodium Nitrate was found to be best nitrogen
source (4.9508 g wet weight) for the propagation of Gliocladium viride ZIC2063 (Table 3). Fungi use
nitrogen in the form of ammonium and nitrate nitrogen as nitrogen sources (Srivastava and Thakur, 2006).
Potatoes Dextrose Broth (PDB) is the most commonly used media for fungi (Nourisepehr et al., 2005;
Srivastava and Thakur, 2006; Congeevaram et al., 2007). These results are in agreement with previous
work (Srivastava and Thakur, 2006).
Effect of initial pH
It has identified that pH can change the charge of fungal cell surface and its attached functional groups
(Yan and Viraraghavan, 2003). It was observed that the initial pH of medium affected the growth rate
during log phase with a maximum biomass growth (3.8303 g wet weight) observed at pH 4.0. The biomass
growth was poor at pH above or below 4.0 as shown in Table 4.
Effect of incubation time
Gliocladium viride ZIC2063 was incubated for 24, 48, 72 and 96 h (Table 5). Fungal growth was found
maximum (4.6967 g wet weight) after 72 h of incubation. Thick mass of brown to black mycelial pellets
appeared after 72 h incubation. Further increase in the time of incubation did not cause considerable
increase in mycelium concentration.
Effect of incubation temperature
Incubation temperature was varied from 20, 25, 30 and 35 oC. The data of table 6 shows that maximum
mycelium growth (4.6847 g wet weight) was obtained at 30 oC. Decreasing temperature below 25 oC
decreased the fungal mycelium growth. Further increase in temperature above 30 oC also decreased the
mycelium concentration. Temperature of growth media is much important because growth is energy
dependent mechanism (Srivastava and Thakur, 2006). The optimal temperature for Gliocladium viride
ZIC2063 growth is 30 oC. Similar observations have also been reported by other researchers (Congeevaram
et al., 2007).
Effect of inoculum age & inoculum size (%)
Inoculum must be in healthy and active state to minimize the length of lag phase. Inoculum of different age
(3, 5 and 7 days) was used to check the effect of inoculum age on the propagation of Gliocladium viride
ZIC2063. It was observed that maximum mycelial pellet (4.8905g wet weight) was obtained with 5 days old
inoculum (Table 7). Inoculum size has profound effect on fungal morphology (Foster, 1949). Similarly to
investigate the effect of inoculum size the growth medium (M1) was incubated with 5 days old inoculum of
different inoculum sizes (2%, 4%, 6%, 8% and 10%) mycelium concentration for 2%, 4%, 6%, 8% and
10% inoculum was 3.7794 g, 3.7802 g, 3.7813 g, 4.0023 g and 4.0132 g wet weight respectively (Table 8).
Hence, in further experiments 8% inoculum size was used.
Effect of agitation
To see the effect of static and agitated conditions, the culture was incubated for 48 h at 122 rpm in orbital
shaker for agitated conditions and flasks were incubated at 30 oC in static conditions. It was interesting to
observe that agitation affects the biomass yield and it was about three times greater when after 48h shaking
the cultural conditions were shifted to static conditions. Kirk et al, 1986, Yang Illman, 1999 wee also
recorded the similar trend. Uniform agitation for 72 h gave 4.7813 g wet weight mycelial pellet and
biomass growth was very poor when it was incubated under static conditions (2.7194 g wet weight).
Maximum thick black mycelium (5.2802 g wet weight) was obtained, when after 48 h shaking the cultural
conditions were shifted to static conditions (Table 9).

Table – 1 Effect of different growth media (along with composition) on the propagation of
Gliocladium viride ZIC2063

Table – 2 Effect of carbon sources on the propagation of Gliocladium viride ZIC2063

Table – 3 Effect of Nitrogen sources on the propagation of Gliocladium viride ZIC2063

Table – 4 Effect of initial pH growth medium on the propagation of Gliocladium viride ZIC2063

Table – 5 Effect of incubation time on the propagation of Gliocladium viride ZIC2063

Table – 6 Effect of incubation temperature onto the propagation of Gliocladium viride ZIC2063

Table – 7 Effect of inoculum age on biomass production of Gliocladium viride ZIC2063

Table – 8 Effect of inoculum size (%) on biomass production of Gliocladium viride ZIC2063

Table – 9 Effect of agitation onto the propagation of Gliocladium viride ZIC2063


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Editor in Chief
Prof. Dr. Cornelia M. Keck (Philipps-Universität Marburg)
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Welcome to the research group of Prof. Dr. Cornelia M. Keck in Marburg. Cornelia M. Keck is a pharmacist and obtained her PhD in 2006 from the Freie Universität (FU) in Berlin. In 2009 she was appointed as Adjunct Professor for Pharmaceutical and Nutritional Nanotechnology at the University Putra Malaysia (UPM) and in 2011 she obtained her Venia legendi (Habilitation) at the Freie Universität Berlin and was appointed as a Professor for Pharmacology and Pharmaceutics at the University of Applied Sciences Kaiserslautern. Since 2016 she is Professor of Pharmaceutics and Biopharmaceutics at the Philipps-Universität Marburg. Her field of research is the development and characterization of innovative nanocarriers for improved delivery of poorly soluble actives for healthcare and cosmetics. Prof. Keck is executive board member of the German Association of Nanotechnology (Deutscher Verband Nanotechnologie), Vize-chairman of the unit „Dermocosmetics“ at the German Society of Dermopharmacy, active member in many pharmaceutical societies and member of the BfR Committee for Cosmetics at the Federal Institute for Risk Assessment (BfR).

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