RESULT AND DISCUSSION 1 Isolation and Screening of Fungal Strain
Bogor, 21-22 October 2015
278 carried on in 100 ml Erlenmeyer flask, composed of 20 ml of malt extract medium 20 gL,
glucose 20 gL, peptone 1 gL, and supplemented with CuSO
4
.5H
2
O to give nominal metal ion concentration of 0.1, 1, and 2 mM. The pH of the medium was adjusted to 4.5.
2.4 Dry mycelium weight dmw After incubation, fungal mycelia obtained were removed from a medium by filtered using pre-
weighed filter paper, while the filtrate was used for decolorization and enzyme assay. The mycelia then was left to dry to constant weight at 105˚C. Dry mycelium weight dmw was
expressed in terms of g of biomass per liter of culture Erkurt et. al., 2007.
2.5 Decolorization assay The supernatant of filtered fungal mycelium were used for decolorization using
spectrophotometric examination. Dye decolorization were determined by measuring the absorbance change of RBBR at 592 nm with a UV-vis spectrophotometer Shimadzu 2010.
Decolorization efficiency R, was determined according to the following formula:
R = 1 – A observed A initial x 100
1 Where, A
initial
is the initial absorbance and A
observed
is observed absorbance Yanto et. al., 2014.
2.6 Enzyme assays The enzymatic activities laccase, MnP, and LiP secreted in the culture were determined using
method according to Minussi et. al. 2001 with a little modification. Laccase activity was measured by monitoring the oxidation of syringaldazine at 525 nm. One unit of laccase
activity was defined as the amount of enzyme necessary to oxidase one µmol of substrate in 1 minute. The reaction mixture for laccase assay contained 0.9 ml culture filtrate, 0.75 ml 0.1 M
sodium acid buffer, and 0.1 ml 0.5 mM syringaldazine. LiP activity was determined by the oxidation of veratrylic alcohol at 310 nm. One unit of LiP was defined as the amount of
enzyme that oxidised one µmol of verattrylic alcohol per minute per litre of culture filtrate. The reaction mixture for LiP assay was composed of 1 ml culture filtrate, 0.3 ml 2 mM H
2
O
2
, and 2 ml LiP buffer.
Manganese peroxidase activity were determined by the formation of Mn
3+
in sodium malonate buffer pH 4.5 in the presence of H
2
O
2
at 470 nm. One unit of MnP was defined as the amount of enzyme required to form 1 µmol of Mn
3+
in 1 minute at 25˚C Kariminiaae- Hamedaani et. al., 2007. The reaction mixture for MnP assay was consisted of 1 ml culture
filttrate, 1.75 ml 50 mM malonic buffer pH 4.5, 0.125 ml 20 Mm 2.6 dimethoxy phenol, 0.125 ml 20 mM MnSO
4
, and 0.3 ml 2 mM H
2
O
2
. Enzyme activity UL was calculated according to the following formula:
Enzyme activity = Abs ɛ x V
mixture
µl : 10
6
x 10
6
x 60 t : V
enzyme
µl : 10
3
2 Where,
ɛ for laccase, LiP, and MnP were 6500, 9300, and 49600, respectively.
3. RESULT AND DISCUSSION 3.1 Isolation and Screening of Fungal Strain
A total of seven fungi samples were successfully found from two location in Cibinong Science Center area. Three fungi samples were collected from RC Biomaterials area, namely Biom 1,
Bogor, 21-22 October 2015
279 Biom 2, and Biom 3, while four fungi samples were collected from Ecology Park Ecopark
area, namely Eco 1, Eco 2, Eco 3, and Eco 4 Figure 1.
Figure 1: Fungi samples collected in RC Biomaterials: a Biom 1 b Biom 2 c Biom 3; and EcoPark area d Eco 1, e Eco 2, f Eco 3, g Eco 4.
Figure 2: Biodecolorization of RBBR on solid media. a Uninoculated control, b Clear zone of Eco 3, c Clear zone of Biom 3. Bar = 1 cm
Figure 3: D7 strain a comparing with Eco 3 b and Biom 3 c isolate. Bar = 1 cm The screening method was done by using solid medium containing synthetic dye RBBR and
lignin. The ability of fungi sample to decolorize synthetic dyes was examined visually by the presence of clear zone or disappearance of color from agar media during incubation. The
result showed that two of seven fungi, Biom 3 and Eco 3, showed the ability to decolorize RBBR in agar media, while the other sample and uninoculated control showed no color
a b
c
d e
f g
a b
c
a b
c a
Bogor, 21-22 October 2015
280 removal Figure 2. The formation of clear zone on agar plate was measured daily during
incubation. Clear zone of fungi Eco 3 and Biom 3 at 4 days incubation reached out ±8 cm and ±6.85 in diametre, respectively. Based on morphological characteristics comparing with
Trametes D7 strain, Biom3 and Eco3 isolates showed characteristic of white rot fungi basidiomycetes macroscopically Figure 3. But further study in genomic identification
should be done to identifyd the species of screened isolates. Therefore, we selected Biom 3 isolate to be compared with D7 isolate for further decolorization study.
3.2 Fungi growth in liquid media
Fungi growth, enzyme production, and dye decolorization is effected by several factors such as media components, dye concentration, presence of heavy metals in culture medium, etc.
Kaushik Malik, 2009. Both of fungi, Biom 3 and D7, were able to grow in liquid medium containing RBBR 100 ppm during biodecolorization Figure 4. However, dry mycelium
weight dmw data reported that fungi D7 isolate grew more rapidly than Biom 3 isolate in all experiment. Growth of all isolates in medium containing RBBR showed that RBBR has no
toxic effect to fungi growth. This results similar with Erkurt et al. 2007 that studied RBBR as high as 100 mgl has no toxic effect for growth of white rot fungi C. versicolor and F. Trogii.
a b
c
Figure 4: Dry mycelium weight of fungi isolates in liquid medium during decolorization. a Liquid medium containing no carbon source, b medium supplemented glucose + peptone,
and c medium supplemented glucose + peptone + CuSO
4
0.1 mM. Effect of carbon source in the growth of fungi was asessed by adding glucose and peptone
into growth medium. As shown on Fig. 4a and b, maxium dmw of fungi isolate was detected in culture medium containing glucose and peptone. Fungi D7 isolate showed maximum of
dmw at 48 h 11.4 gl when it grown in medium containing glucose and peptone, compared with maximum of dmw 4.7 gl, 72 h when it grown in medium which lack of carbon source.
Similarly, Biom3 isolate also shown maximum of dmw 6.2 gl, 48 h in medium containing glucose and peptone, while maximum of dmw in medium without carbon source was only
2 4
6 8
10 12
24 48
72 D
ry m
yc el
iu m
w ei
gh t
gl
Incubation time h Biom 3
D7
2 4
6 8
10 12
24 48
72 D
ry m
yc el
liu m
w ei
gh t
gl
Incubation time h Biom 3
D7
2 4
6 8
10 12
24 48
72 D
ry m
yc el
liu m
w ei
gh t
gl
Incubation time h Biom 3
D7
Bogor, 21-22 October 2015
281 reached as weight as 3.0 gl at 72 h. This results showed that carbon source was important
factor for growth of fungi. Fungi as living cells required a nutrient, supply in dye decolorization, if the influent or dye lacks nutrient, as well as culture maintenances are needed
Fu Viraraghavan, 2001. Various concentration of Cu
2+
as CuSO
4
.5H
2
O; 0.1, 1.0, 2.0 mM added to the culture medium in order to evaluating the effect of copper ion to growth of fungal isolate. The study
showed that the growth of fungal isolates was only detected in medium containing 0.1 mM of CuSO
4
. Both of isolates showed no growth in medium containing 1.0 and 2.0 mM of CuSO
4
may indicated the toxic effect of CuSO
4
at higher concentration data not shown. Soni et. al. 2014 described that many researchers were concluded copper as more toxic than other
metals due to negative effect on transcription mechanism in cell. 3.3 Decolorization of RBBR by fungal strain in liquid media
Both of fungal isolates were found to capable of decolorizing RBBR in liquid media, which D7 isolate showed higher decolorization efficiency than Biom 3 isolate. In limited carbon
source medium, maximum decolorization efficiency of D7 isolate was 71.7 while Biom 3 isolate was only able to decolorize 23.4 RBRR. Maximum decolorization efficiency of both
isolates was detected after 72 h incubation Figure 5a. Addition of glucose and peptone to culture medium increased decolorization efficienccy of both isolates. As can be seen in figure
5b, maximum decolorization of D7 isolate 97.4 and Biom 3 isolate 48.0 were reached at 48 h incubation. It can be concluded that addition of carbon source glucose and peptone
enhanced RBBR decolorization efficiency of fungi isolate. Kapdan et al. 2000 was reported that the use of glucose as carbon source showed 100 decolorization of dye after 5 days of
incubation Kaushik Malik, 2009.
Figure 5: Decolorization efficiency of RBBR in liquid media, without the presence of glucose and peptone a, with glucose and peptone b, and with glucose, peptone, and 0.1 ppm
CuSO
4
a b
c
26,9 48,0
35,4 84,6
97,4 94,7
10 20
30 40
50 60
70 80
90 100
110
1 2
3
De co
lo riz
at io
n
Incubation time d Biom 3
D7
Bogor, 21-22 October 2015
282 Effect of copper ion on dye decolorization was investigated. The presence of copper ion in
the media affected decolorization of RBBR. In presence of 0.1 ppm CuSO
4
, decolorization efficiency by Biom 3 was increased from 26.9, 48.0, and 35.4 to 45.2 , 66.5, and
77.9 within 24, 48, and 72 hours incubation, respectively. Decolorization efficiency by D7 isolate was also increased from 84.6 to 85.3 after 24 hours incubation, but no increasing
decolorization efficiency was observed after 48 and 72 hours incubation Figure 5. However, when concentration of CuSO
4
were increased to 1 and 2 ppm, no decolorization was observed in medium of both isolates Table 1. As described before, the presence of Cu
2+
up to 1.0 and 2.0 mM in medium inhibited fungi growth, so it caused no removal of RBBR.
Effect of Cu
2+
on decolorization activity of white rot fungi was also reported by several studies. Murugesan et. al. 2009 reported that enhanced decolorization of RBBR by G. lucidum
was observed when culture medium supplemented with 1 mM Cu
2+ .
Moreover, increasing concentration of Cu
2+
up to 10 mM exhibited positive effects on decolorization. Pointing et. al. 2000showed that the presence of heavy metal could reduced dye decolorizing activity of
white rot fungi. The reports showed that the presence of any metal including Cu
2+
, even in the concentration of 0.1 mM, inhibited decolorization of Poly R by P. chrysosporium, but enhanced
decolorization activity by T. versicolor. Decolorization activity of T. versicolor was only inhibited if the culture medium supplemented with higher concentration of Cu
2+
. Table 1: Decolorization efficiency of RBBR by two fungi isolates in the presence of various
concentration of CuSO
4
Fungi Isolate
Time h Decolorization at different CuSO
4
concentration 0.1 ppm
1 ppm 2 ppm
Biom 3 24
45.2 48
66.5 72
77.9 D7
24 85.3
48 86.1
72 83.5
3.4 Enzyme activities in liquid culture during biodecolorization Activities of three enzimes Laccase, MnP, and LiP produced by fungal growth during
biodecolorization was evaluated using medium supplemented with glucose, peptone, and CuSO
4
0.1 mM Table 2. Based on the results, MnP was successfully assayed in both culture medium of Biom 3 and D7 isolate, while laccase was only produced by D7 isolate. No LiP was
detected in culture medium of both isolates. The highest activity of MnP as high as 351.4 Ul was obtained by D7 isolate after 24 hours of
incubation, while the highest activity of MnP produced by Biom 3 was only 57.8 Ul after 48 h of incubation. Increasing activity of MnP was showed by both fungi when dye was added to
the culture media. It can be seen as MnP activity of Biom 3 and D7 in control medium were only 16.3 and 325.3 Ul, but increased up to 50.2 and 351.4 Ul, respectively, after the
addition of dye RBBR. Laccase was only produced by D7 isolate. Different with MnP, laccase activity decreased after
the addition of RBBR. When comparing with control which showed laccase activity of 1,817.6 Ul, the highest laccase activity of D7 isolate was only reached 1,636.6 Ul after 24 h of
Bogor, 21-22 October 2015
283 incubation. But, in the case of D7 isolate, MnP and laccase activity have the same profile
during biodecolorization. Both of enzymes showed decreasing activity after 48 h of incubation before the activities reached up until 72 h of incubation.
Figure 6 showed the correlation between decolorization efficiency and MnP activity of both fungi Biom 3 and D7. From the figure, it can be seen that was a positive correlation between
increasing activity of MnP and decolorization activity. Decolorization activity of both fungi was increased when higher activity of MnP was obtained. It was suggested that decolorization
was mainly due to the action of ligninolytic enzyme secreted by fungus Sangeeta et. al., 2011. Table 2: Enzyme activities produced by fungi isolates during biodecolorization
Isolate Incubation
time h Enzyme Activity Ul
MnP Laccase
LiP
Biom 3 16.3
n.d n.d
24 50.2
n.d n.d
48 57.8
n.d n.d
72 27.5
n.d n.d
D7 325.3
1,817.6 24
351.4 1,636.6
n.d 48
174.3 602,3
n.d 72
277.0 783.2
n.d
= control without addition of RBBR n.d = not detected
Figure 6: Correlation between decolorization efficiency and MnP activity Interestingly, decolorization of Biom 3 isolate showed maximum activity which closed with
maximum decolorization of D7, eventhough Biom 3 isolate showed lower MnP activity than D7. Moreover as previously described above, D7 isolate produced two main ligninolytic
enzyme laccase and MnP in decolorization process which laccase showed higher activity than MnP Table 2. It was suggested that laccase was the main important ligninolytic enzime in
decolorization activity of D7 isolate, while MnP could play important role in dye decolorization of Biom 3 isolate.
Comparison of laccase production in culture medium during biodecolorization showed that higher laccase activity was found in medium limited carbon source after 72 hours of
incubation than in medium supplemented with carbon source Figure 7. It contrast with the
Bogor, 21-22 October 2015
284 dry mycelium weight and decolorization profile which the efficiency is higher if in the culture
medium was supplemented with glucose and peptone. Other reports by Sangeeta et. al. 2011 described that LiP and MnP activities of fungus increased 3 and 10 fold higher when media
containing carbon source was used for treating wastewater, but no reports for laccase. We still need further study to correlated between laccase activity and medium composition used,
because according to
D’Agostini et. al., 2011 fungi responses differed due to CN ratios and N source when laccase production is considered and there was a general trend that laccase
production reduces when CN ratios increases. The addition of copper ion showed positive effect on laccase activity. As shown in Figure 7.,
laccase activity were induced by addition of 0.1 mM CuSO
4
. The highest activity of laccase induced by Cu
2+
was 1,636.6 Ul at 24 h of incubation, while the highest activity of laccase without inducing Cu
2+
was only 158 Ul at 72 h of incubation. According to Hu et. al. 2009 copper atom was very important for laccase activity. It was because of laccase belongs to the
multinuclear copper-containing oxidases. Their experiment investigated that laccase activity was improved as the increasing of copper ion concentration of 0
– 0.6 mM.
Figure 7: Laccase activity by fungi D7 in different medium: without the presence of glucose and peptone , with glucose and peptone , and with glucose, peptone, and 0.1 ppm
CuSO
4