Embed Size (px)
Citation preview
79
CHAPTER 4
MATERIALS AND METHODS
4.1 Screening and isolation of Laccase producing fungi
4.1.1 General
The glass wares used were of Borosil® make. The chemicals used were
of Hi-media, Sigma and Merck make. All the chemicals and reagents were of
analytical grade or extra pure grade.
4.1.2 Sterilization of Glass wares
The glass wares (petri plates, pipettes, conical flasks etc.,) were
sterilized by standard procedure i.e. by immersing in chromic acid solution
followed by washing in clean water then by autoclaving (at 121°C and 15lb
pressure for 15 minutes) , hot air oven( at 160°C for 2 hours) etc.,
4.1.3 Preparation of culture media and reagents
The culture media was prepared by carefully weighing the contents
accurately using electronic balance (Shimadzu, Japan) and were dissolved in
sterile distilled water and the same procedure was followed for reagent
preparation also. The culture media was sterilized by autoclaving.
80
4.1.4 Screening of Laccase producing fungi
4.1.4.1 Collection of samples
The Fungal samples for the study were collected from Saw mill wastes-
which included the wood dust, wood pieces of various sizes, bark and other
plant portions. The samples were collected in sterile plastic bags and were
sealed and brought to the lab aseptically for further processing.
4.1.4.2 Processing of samples
The samples were homogenized and sieved (2 mm mesh) and 5 g of
samples were added into 250 ml flasks containing 20 ml sterilized
physiological sodium chloride solution and glass beads and kept on a rotary
shaker for 30 min for full distribution. The prepared suspensions were used for
inoculating onto the plates [161].
4.1.5 Culture media used
Malt Extract Agar (MEA) was used for the initial isolation and Potato
Dextrose Agar (PDA) for the maintenance of culture.
Malt Extract Agar is commonly recommended media for the detection,
isolation and enumeration of yeasts and moulds. The Composition of MEA is
given in the Table 4.1.
Sabouraud’s Dextrose Agar (SDA) is a universal complex medium for
cultivation and isolation of yeasts and moulds. The composition of SDA is
given in Table 4.2.
81
Table 4.1: Composition of malt extract agar
Ingredients g / L
Malt extract 30.0Mycological peptone 5.0Agar 20.0Final pH ( at 25°C) 5.4±0.2
Table 4.2: Sabouraud’s Dextrose Agar composition
Ingredients g / L
Dextrose 20.0Peptone 10.0Agar 20.0Final pH ( at 25°C) 5.6±0
The above media were prepared as per the standard procedure and were used.
4.1.6 Indicator compounds
The following indicator components were included in the culture media
for the identification and screening of Laccase producing fungi: ABTS (2,2%-
azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) (0.05% w/v),
Syringaldazine, Guaiacol (0.01% w/v),Gallic acid (0.5% w/v) and Tannic acid
(0.5% w/v).All these indicator solution were sterilized by filtration. The
indicators and the respective colour they produce on contact with laccase
enzyme are given in Table 4.3. The petriplates having culture media and
indicator compounds were inoculated with the isolated fungal species and then
the plates were incubated at 28°C for 2 weeks. The production of laccase
enzyme is indicated by the formation of coloured halo around the fungal
82
colonies [372]. The isolate which shows positive result were used for further
studies.
Table 4.3: Indicators and the Colour it produces in Positive result
Sl.No. Indicator Positive Reaction
1 ABTS Green coloured zones around colony2 Syringaldazine Reddish halo around colony3 Guaicol Dark brown coloured zone around colony4 Tannic acid Dark brown coloured zone around colony5 Gallic acid Dark brown coloured zone around colony
4.1.7 Identification of fungal isolates
The fungal isolates were identified by their morphological and colonial
characteristics. The confirmation was done with the following laboratories:
1.CAS Botany, University of Madras, Chennai-25,TN, 2. Marina Labs,
Nungambakkam, Chennai, TN, 3. Microlabs of industrial Research, Arcot,
Vellore Dt. TN, 4. Agarkhar Research Institute (ARI), Pune. For the molecular
identification the samples were submitted to Agarkhar Research Institute
(ARI), Pune.
4.2 Screening of culture media for laccase enzyme production
The following culture media were used for the study: 1.Semi synthetic
media and 2.Agro waste based media.
4.2.1 Semi synthetic media
Two semi synthetic culture media with the following composition were
used for the study: 1. Yeast extract peptone dextrose-Copper sulphate (YPD-
83
Cu) medium and 2. Glucose Peptone Broth (GPB) media. Table 4.4 and 4.5
gives the composition of both the media.
Table 4.4: YPD-Cucomposition
Ingredient Quantity
Glucose 20 g/lPeptone 5 g/lYeast extract 2 g/lCopper sulphate 100mg/l
Table 4.5: GPB composition
Ingredient Quantity(in g/l)
Glucose 10.0Peptone 3.0KH
2PO
4 0.6ZnSO
4 0.001K2HPO
4 0.4FeSO
4 0.0005MnSO
4 0.05MgSO
4 0.5CuSO
4 0.01
4.2.1.1 Inoculum preparation & inoculation
The isolated fungi, which were maintained in the MEA slants, were
transferred to SDA plates and were incubated at room temperature for 2
weeks. The well grown fungi were used for inoculating YPD-Cu and GPB
84
media. The fungal mat on SDA plates were cut with 8mm cork borer and with
the help of inoculation loop and 5 discs was transferred to YPD-Cu and GPB
flasks aseptically.
4.2.1.2 Culture conditions
The pH of the above culture media was adjusted to 6.5 and the flasks
were incubated at 30°CTemperature in shaking incubator with 150 rpm and
in a dark for 4 weeks.
4.2.1.3 Laccase assay
Culture medium was filtered with Whatman filter paper (0.42 µm pore
size) and the filtrate was centrifuged at 8,000 rpm for 10 min. and the
supernatant was used for enzyme assay. Laccase activity was assayed
spectrophotometrically by measuring the oxidation of ABTS at 420 nm at
30°C. The assay mixture in a total volume of 1 ml contained 0.1 ml cell-free
supernatants at various dilutions and 1 mM ABTS in 100 mM citrate buffer
(pH 3.4). One unit of enzyme activity was defined as the amount of enzyme
required to oxidize 1 µmol ABTS per minute [7].
4.2.1.4 Time scale for laccase enzyme production
The time taken by the 5 isolated fungi to produce laccase enzyme was
tested by inoculating them in YPD-Cu and GPB flasks and incubating the
flasks for 4 weeks at room temperature and in dark. The samples were
periodically drawn at the interval of one day and were assayed for laccase
activity by the procedure of Bourbonnais and Paice [7].
85
4.2.2 Agro waste based media
The following agro wastes were used for the initial screening: rice bran,
wheat bran, sugarcane bagasse, cotton seed, green gram husk, ground nut
shell, and sapota seeds. All of them were locally procured and were sterilized
at 121°C and 15lb pressure for 20 minutes and were used for the study.
4.2.2.1 Media Preparation
The media was prepared by adding 2% of each of the Agro wastes to
the Mineral Salt (MS) medium. The media was then sterilizing at 121°C and
15lb pressure for 20 minutes. This agro wastes mineral salt (AWMS) media
was used for the study. The composition of MS media is given in Table 4.6.
Table 4.6: Composition of AWMS medium
COMPONENT QUANTITY( g/l)
Agro waste 20.0
K2HPO
4 1.0(NH
4)SO
4 5.0NaCl 1.0MgSO
4.7H
2O 0.5
FeSO4.7H
2O 0.1
ZnSO4.7H2O 0.1
CuSO4.5H
2O 0.1
MnSO4.7H
2O 0.1
86
The 250 ml conical flasks with 100 ml of the above AWMS media and
were inoculated with well grown fungal discs from PDA plates and the flasks
were incubated in dark at a pH of 6.5 and incubated at 30°C temperature in
shaking incubator with 150 rpmfor 4 weeks and the enzyme extraction and
assay were done as per Bourbonnais and Paice [7] procedure. The best agro
waste which supports the maximal laccase assay were selected and used for
the optimization study.
4.3 Optimization of laccase enzyme production
4.3.1 Introduction
Selection of nutrients such as carbon, nitrogen and other nutrients is
one of the most critical stages in an efficient and economic process
development for enzyme production. The methodologies used for screening
the nutrients fall into two categories; 1.Classical Method and 2. Statistical
Method.
4.3.2 Optimization by classical method
This method is also called one-factor-at a time – method. It involves
keeping one variable fixed and varying other variables. It is time consuming
and laborious and does not include interactive effects among the variables.
4.3.2.1 Optimization of semi synthetic culture media
The yeast extract peptone dextrose (YPD) medium, containing glucose
20 g/l, peptone 5 g/l, and yeast extract 2 g/l, supplemented with 100mg
copper sulphate was used as the basal medium (YPD–Cu) for optimization of
87
laccase enzyme production study and the fungal strain used wasTrametes
versicolor.
4.3.2.1.1 Selection of carbon sources
RAPID HICARBOHYDRATE TEST KIT (HIMEDIA) was used for
the screening of carbon sources. It detects the ability of microorganisms to test
the carbohydrate utilization; about 35 different carbohydrates can be tested in
a single test. It is based on the pH change due to substrate utilization during
incubation. The procedure involves the steps as in Figure 4.1.
Based on the positive result, Glucose, Sucrose, Lactose, Mannitol and
Maltose were selected as carbon source.
Fungi (Trametes versicolor)↓
Inoculated in SDB↓
Incubated until O. D. reaches 0.5 at 620nm ↓
Kit opened aseptically in LAF↓
50µl of the inoculum should be added to each well ↓
Incubated at 30°C for one week↓
Results are interoperated by observing colour change and comparing with uninoculated control
↓Carbohydrates selected for further studies
Figure 4.1: Flow chart for carbohydrate test selection procedure
88
4.3.2.1.2 Effect of various carbon source on laccase enzyme production
Glucose, Sucrose, Maltose, Lactose and Mannitol were added to YPD-
Cu medium at the concentration of 2%. The flasks were inoculated with well
grown Trametes versicolor discs from PDA plates and the flasks were
incubated in dark at a pH of 6.5 and incubated at 30°C temperature in shaking
incubator with 150 rpmfor 4 weeks and the enzyme extraction and assay were
done as per Bourbonnais and Paice [7] procedure.
4.3.2.1.2.1 Effect of varying concentrations of glucose on laccase enzyme
production
Five separate YPD-Cu flasks with increasing concentrations of glucose
viz., 0.5% to 2.5% were taken and were inoculated with Trametes versicolor
culture and were incubated in dark at a pH of 6.5 and incubated at 30°C
temperature in shaking incubator with 150 rpm for 4 weeks and the enzyme
extraction and assay were done as per Bourbonnais and Paice [7] procedure.
4.3.2.1.3Effect of various nitrogen sources on laccase enzyme production
Peptone, Urea, Ammonium sulphate, Tryptone and ammonium chloride
were added to YPD-Cu media at the concentration of 0.5% and the flasks were
inoculated with well grown Trametes versicolor discs from PDA plates and
the flasks were incubated in dark at a pH of 6.5 and incubated at 30°C
temperature in shaking incubator with 150 rpmfor 4 weeks and the enzyme
extraction and assay were done as per Bourbonnais and Paice [7] procedure.
89
4.3.2.1.3.1 Effect of varying concentrations of peptone on laccase enzyme
production
Five separate YPD-Cu flasks with increasing concentrations of peptone
viz., 0.1% to 0.5% were taken and were inoculated with Trametes versicolor
culture and were incubated in dark at a pH of 6.5 and incubated at 30°C
temperature in shaking incubator with 150 rpm for 4 weeks and the enzyme
extraction and assay were done as per Bourbonnais and Paice [7] procedure.
4.3.2.1.4 Effect of different incubation temperatures on laccase enzyme
production
The YPD-Cu flasks were inoculated with the test fungi Trametes
versicolor and the flasks were incubated at different temperatures viz., 25°C,
30°C, 35°C, 40°C and 45°C. The flasks were incubated in shaking incubator
with 150 rpm for 4 weeks and the enzyme extraction and assay were done as
per Bourbonnais and Paice [7] procedure.
4.3.2.1.5 Effect of different pH on laccase enzyme production
The effect of various pH (viz., 5.0, 5.5, 6.0, 6.5 and 7.0) on laccase
production in YPD-Cu media was done by inoculating the YPD-Cu flasks
(with the above pH) with test fungi Trametes versicolor and incubated the
flasks at 30°C and in shaking incubator with 150 rpm for 4 weeks and the
enzyme extraction and assay were done as per Bourbonnais and Paice [7]
procedure.
90
4.3.2.1.6Effect of shaking speed on laccase enzyme production
The effect of shaking speed on laccase enzyme production was studied
by inoculating the YPD-Cu flasks with Trametes versicolor fungi and
incubating them at 30°C and in shaking incubator (with 80, 100,120,140, 150
and 160rpm’s) for 4 weeks, with the pH of the medium being 6.0. Laccase
enzyme assay was done as per Bourbonnais and Paice [7] procedure.
4.3.2.2 Optimization of Agro waste based media
4.3.2.2. 1 Effect of varying concentration of Achras sapota (Sapota) seeds
on fungal growth and laccase production
The effect of various concentrations, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%
and 3.0%, of Achras sapota (Sapota) seed powders were studied by
incorporating the various concentrations of dried seed powder in the MS
media and are called Sapota Seed Broth (SSB). The SSB flasks were
inoculated with Trametes versicolor fungi and incubated at 30°C with the pH
of the medium being 6.0 and in shaking incubator with 150 rpm for 4 weeks.
Laccase enzyme assay was done as per Bourbonnais and Paice [7] procedure.
The concentration which supports highest laccase production was used for
further studies.
4.3.2.2.2 Effect of temperature on laccase enzyme production
The effect of various temperature ranges on laccase enzyme production
in SSB was studied by inoculating the test fungi Trametes versicolor and the
incubating the flasks at different temperatures viz., 25°C, 30°C, 35°C, 40°C
and 45°C. The flasks were incubated in shaking incubator with 150 rpm for
91
2weeks and the enzyme extraction and assay were done as per Bourbonnais
and Paice [7] procedure.
4.3.2.2.3 Effect of pH on laccase enzyme production
The SSB media was prepared with different pH values like 5.0,5.5,
6.0,6.5 and 7.0 and were inoculated with the fungi Trametes versicolor and the
flasks were incubated at 30°C and in shaking incubator with 150 rpm for 2
weeks and the enzyme extraction and assay were done as per Bourbonnais and
Paice [7] procedure.
4.3.2.2.4 Effect of shaking speed on laccase enzyme production
The SSB flasks were inoculated with the fungi Trametes
versicolorincubated at the temperature of 30°C and pH of 6.0 and in the orbital
shaker incubator maintained at varying rpm’s (revolution per minute) viz.,
80,100,120,140, 150 and 160 rpm’s The flasks were incubated at 30°C for 2
weeks and the enzyme extraction and assay were done as per Bourbonnais and
Paice [7] procedure.
4.3.2.3 Effect of Vermiwash on laccase enzyme production
The vermiwash is the liquid collected during the vermi composting of
organic wastes and is rich in various nutrients. The vermiwash is normally
used as organic fertilizer for various crops. The vermiwash was obtained from
the local vermi-composting yard was used.
The effect of vermi wash on laccase production in different media
along with the effect of varying proportions of vermi wash on the same was
studied as follows:The vermi wash was incorporated in YPD medium (YPD-
92
VW) and SSB medium (SSB-VW) in varying concentrations like 25%,
50%,75% and 100% replacing the water accordingly so that the total fluid in
the medium remains 100% i.e. if 25% vermi wash was used then the water
incorporated in the medium was 75% and so on . The pH of above media
(YPD & YPD-VW and SSB & SSB-VW) was adjusted to 5.5 and 6.0 before
autoclaving. Flask experiments were performed in 250 ml Erlenmeyer flasks
containing 100 ml & YPD-VW and SSB & SSB-VW medium separately and
were inoculated with Trametes versicolor and were incubated at 30°C and 150
rpmon a rotary shaker for 2 weeks. The aerobic condition in the system was
maintained by putting non-absorbent cotton to the mouth of the flask. All
experiments were conducted aseptically. The laccase enzyme assay was done
by Bourbonnais and Paice [7] procedure.
4.3.2.3.1 Comparison of laccase enzyme production in vermiwash (50%)
supplemented YPD-Cu and SSB
A comparison between vermiwash (50%concentration) supplemented
YPD-Cu and SSB for the production of laccase enzyme by Trametes
versicolor was carried out by taking 100ml of respective culture media in
250ml Erlenmeyer flasks and were inoculated with Trametes versicolor and
were incubated at 30°C and 150 rpm on a rotary shaker for 2 weeks. The
laccase enzyme assay was done by Bourbonnais and Paice [7] procedure.
4.3.2.3.2 Comparison of laccase enzyme production by the submerged and
solid state fermentation using SSB
The production of laccase enzyme was carried out in SSB media which
was optimized as in as in above procedure. The production was carried out in
both submerged and solid state fermentation mode. The submerged
93
fermentation was carried out in conical flasks having SSB with the
composition as in Table 4.6 with 50 ml of water and 50 ml of vermiwash and
was inoculated with Trametes versicolor and were incubated at 30°C and 150
rpm on a rotary shaker for 2 weeks. Similarly, solid state fermentation was
carried out by taking the SSB media (Table 4.6) in a 250ml conical flask and
was maintained with 70% of moisture (vermiwash 50% and 20% of water)
and was inoculated with Trametes versicolor and were incubated at 30°C and
150 rpm on a rotary shaker for 2 weeks. The laccase enzyme assay was done
by Bourbonnais and Paice [7] procedure.
4.3.3Statistical optimization of laccase enzyme production
The application of statistical methodologies in fermentation process
development has numerous advantages in terms of rapid and reliable short
listing of nutrients, understanding the interactions among nutrients at varying
concentrations and tremendous reduction in total number of experiments
resulting in less time consumption, glassware, chemicals and man power.
Plackett- Burman design [373] is a two level fractional factorial design
and allows screening of up to ‘n–1’ variables in just ‘n’ number of
experiments. In this design, generally a multiple of four i.e., 4, 8, 12, 16, 20,
…., 4n experiments are required to screen 3, 7, 11, 15, 19, ….., 4n–1
components respectively, where ‘n’ is an integer. This design is employed for
production of various metabolites and enzymes in submerged and solid state
fermentation. Yield of any microbial product can be improved by optimization
of medium components that are required in fermentation processes;
application of statistical methodologies in fermentation process development
can result in improved yield of the product, reduced process variability, closer
confirmation of the output response (product yield/ productivity) to normal
and target requirements, reduced development time with overall costs.It
94
investigates the responses of variables to the changes in a set of design and
helps to find out the optimal conditions for response and also to find out the
interactive effects of variables on response.The analysis is performed with
MINITAB16.0.
4.3.3.1 Plackett- -Burman design
A Plackett- -Burman design with 15 factors (one way), resulting in 20
runs, performed in duplicate, was used to determine the media components in
the laccase production. Each variable is variables for a desired response
represented at two levels namely, “high and low”. This design assumes that
there are no factor interactions between the different media constituents, x1, in
the range of variables under consideration. A linear approach is considered to
be sufficient for screening.
Y = b0 + Σbi xi (i = 1, ................, k) (4.1)
where, Y is the estimated target functions β0 and β1 are the regression
coefficients of the model.
The PB design is a factorial design and the main effect (the contrast
coefficient) of such design may be calculated as the differences between the
averages of the measurements made at high level (+1) and at low level (-1) as
described in Table4.7.
After determining the most significant factors for laccase enzyme
production, their concentration optimizations were made by using Response-
surface- methodology (RSM). It is a combination of experimental designs and
statistical techniques for the empirical model building and optimization. By
conducting experiments and applying regression analysis, a model of
95
theresponse to some independent inputs variables can be obtained. Based on
the model response, anear optimal point can then be deduced. Response
surface methodology also quantifies therelationship between the controllable
input parameters and the Response surface obtained [374].
If all variables are assumed to be measurable, the response surface can
be expressed as follows:
Y = f (x1, x2, x3,….., xk) (4.2)
WhereY is the response and x1 is the variables of action called factors.
The goal is to optimize the response variable Y. It is assumed that the
independent variables are continuous and controllable by experiment with
negligible errors. It is required to find a suitable approximation for the true
functional relationship between independent variables and the response
surface. Usually a second order model is utilized in response surface
methodology [16]
� = �� + ∑ ������ + ∑ ���2��
� ∑ ∑ �������+∈��
���� (4.3)
where x1, x2 ,…… xk are the input factors which influence the response
Y; β0, βij (i=1,2,….k), βij(i=1,2….,k; j=1,2,….k) are unknown parameters and ε
is the random error. The β coefficients, which should be determined in the
second order model, are obtained by the least square method.
In this study, a 20 run Plackett-Burman design was applied to evaluate
15 factors (variables). Each variable was examined at two levels: -1 for low
level and +1 for high level. The variables and their corresponding levels used
in this experiment are shown in the following Table 4.7. The values were
96
chosen based on the previous study. The design for Plackett-Burman analysis
is given in the Table 4.8.
Table 4.7: Variables used in the study and their high and low levels
SYMBOL VARIABLES HIGH(+) LOW(-)
A SugarcaneBagasse 2 1
B Cotton seed 2 1
C Sapota seed powder 2 1
D Beef extract 1 0.5E Yeast Extract 1 0.5F Peptone 1 0.5G Glucose 1 0.5H Maltose 1 0.5I CuCl2 0.002 0.001J CuSO4 0.002 0.001K MgSO4 0.005 0.001L ZnSO4 0.005 0.001M KH2PO4 0.002 0.001N FeSo4 0.002 0.001O NaCl 0.002 0.001
All 20 experiments were conducted in duplicate, and the average values
of laccase yields were tabulated, as observed response. The predicted values
were calculated by using the regression coefficient model equation
97
Table 4.8: Base design for 20 run Plackett-Burman experiment.
Run
OrderA B C D E F G H I J K L M N O
1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1
2 1 1 -1 1 1 -1 -1 -1 -1 1 -1 1 -1 1 1
3 1 1 1 -1 -1 1 1 -1 1 1 -1 -1 -1 -1 1
4 1 -1 1 1 -1 -1 -1 -1 1 -1 1 -1 1 1 1
5 1 1 -1 -1 1 1 -1 1 1 -1 -1 -1 -1 1 -1
6 1 -1 -1 -1 -1 1 -1 1 -1 1 1 1 1 -1 -1
7 1 -1 -1 1 1 -1 1 1 -1 -1 -1 -1 1 -1 1
8 1 -1 1 1 1 1 -1 -1 1 1 -1 1 1 -1 -1
9 -1 1 1 -1 1 1 -1 -1 -1 -1 1 -1 1 -1 1
10 -1 -1 1 1 -1 1 1 -1 -1 -1 -1 1 -1 1 -1
11 -1 1 -1 1 1 1 1 -1 -1 1 1 -1 1 1 -1
12 -1 1 -1 1 -1 1 1 1 1 -1 -1 1 1 -1 1
13 1 -1 1 -1 1 1 1 1 -1 -1 1 1 -1 1 1
14 -1 -1 1 -1 1 -1 1 1 1 1 -1 -1 1 1 -1
15 -1 -1 -1 1 -1 1 -1 1 1 1 1 -1 -1 1 1
16 -1 1 1 1 1 -1 -1 1 1 -1 1 1 -1 -1 -1
17 1 1 -1 -1 -1 -1 1 -1 1 -1 1 1 1 1 -1
18 -1 1 1 -1 -1 -1 -1 1 -1 1 -1 1 1 1 1
19 -1 -1 -1 -1 1 -1 1 -1 1 1 1 1 -1 -1 1
20 1 1 1 1 -1 -1 1 1 -1 1 1 -1 -1 -1 -1
98
The goodness of fit of the model was checked by the regression
coefficient (R2). In this case, the value of the regression coefficient (R2 =
0.901) indicates that only 9.9% of the total variations are not explained by the
model. A higher value of the correlation coefficient (R = 90.1%) signifies an
excellent correlation between the independent variables and the response
[375].
The significance of each coefficient was determined by student's t-test
and p values. The larger the magnitude of thet-value and the smaller the
pvalue, the more significant is the corresponding coefficient [376,377].
From the above experimental results, the factors which have maximum
T value where selected for the next level of optimization using Box-Benhken
method.
4.3.3.2 Box-Behnken experimental design
In the present study Box-Behnken experimental design was chosen for
findings out the relationship between the response function (laccase activity)
and the variables (Sapota Seed Powder,Yeast Extract ,Glucose,CuSo4 and
ZnSo4) designated as X1, X2, X3, X4 and X5 (Table 4.9). There are several
reports on use of Box–Behnken experimental design for production of
metabolites in submerged fermentation but not much explored in case of solid
state fermentation hence a systematic study was made on optimization of
laccase production in SSF.
99
Table 4.9: Selected variables and their codes
As central
composite designs, Box–Behnken designs are response surface methods used
to examine the relationship between one or more response variables and a set
of quantitative experimental parameters [319]. Response surface methods are
often used once preliminary screening has been carried out; using factorial
designs such as Plackett-Burman (PB) to determine which factors significantly
affect the response.
The significant variables were identified by the analysis of the Placket-
Burman experiments and their levels were further optimized for enhanced
laccase production by employing Box- Behnken design [378]. Each selected
variable was analyzed at three levels-low, medium and high coded as -1, 0 and
+1 in a total of 46 runs (Table 4.10 and 4.11).
Box-Behnken design requires an experiment number according to N=k2
+k +cp, where k is the factor number and cpis the rotatable number of the
central point [379]. Box-Behnken is aspherical, revolving design viewed as a
cube, it consists of central point and the middle points ofthe edges. However,
it can also be viewed as consisting of the three interlocking 22 factorialdesigns
and a central point [379]. For the five level factorial Box-Behnken
experimental designs, a total of 46 experimental runs, shown in Table 4.10, are
needed.
Variable CodeSapota Seed Powder AYeast Extract BGlucose CCuSO4 DZnSO4 E
100
Table 4.10: 3 levels of the variables taken for optimization by BB
design
Variables3 levels of variables
-1 0 1
A 0.5 1 1.5
B 0.25 0.5 0.75
C 0.5 1 1.5
D 0.001 0.002 0.003
E 0.001 0.002 0.003
The model is of the following form [379]:
y = β0+ β1X1+ β2X2+ β3X3+ β4X4+ β5X5+ β11X21+ β22X2
2+ β33X23 +
β44X2 4+ β55X2
5+ β12X1X2+ β13X1X3+ β15X1X5 + β23X2X3+
β24X2X4+ β25X2X5+ β34X3X4+β35X3X5+ β45X4X5. (4.4)
where ‘y ‘predicted response; β0 mode constants; X1, X2, X3, X4 and X5
independent variables; β1, β2, β3, β4 and β5 are linear coefficients ; β12; β13; β15;
β23; β24; β25 ;β35; β35and β45 are cross product coefficients, and β11β22β33β44 and
β55 are the quadratic coefficients [379].
The simplest possible model (quadratic second order model) can be
used to explain themathematical relationship between the controllable
variables and response. This design is preferred because relatively few
experimental combinations of the variables are adequate to estimate
potentially complex response functions.
101
Table 4.11: 46 runs of selected 5 factors for the Box- Behnken design
RunOrder A B C D E1 0 1 -1 0 02 0 1 0 0 13 -1 1 0 0 04 -1 0 0 0 15 -1 0 0 0 -16 1 0 1 0 07 0 -1 0 -1 08 0 1 0 0 -19 0 0 1 1 0
10 1 0 0 -1 011 0 -1 0 0 112 0 0 -1 -1 013 0 0 0 -1 -114 -1 -1 0 0 015 -1 0 -1 0 016 0 0 -1 1 017 0 0 -1 0 -118 1 0 0 1 019 0 0 1 0 120 0 0 0 1 121 0 0 0 0 022 0 0 1 -1 023 0 -1 0 0 -124 -1 0 0 -1 025 1 0 -1 0 026 0 0 0 0 0
102
Table 4.11 continued…………..
27 0 0 0 0 0
28 0 1 1 0 0
29 -1 0 0 1 0
30 0 -1 1 0 0
31 1 0 0 0 1
32 0 0 1 0 -1
33 0 0 0 1 -1
34 0 0 0 -1 1
35 -1 0 1 0 0
36 0 -1 -1 0 0
37 1 1 0 0 0
38 0 0 0 0 0
39 0 0 -1 0 1
40 0 1 0 -1 0
41 0 0 0 0 0
42 0 1 0 1 0
43 0 0 0 0 0
44 1 0 0 0 -1
45 1 -1 0 0 0
46 0 -1 0 1 0
103
The regression equation obtained after analysis of variance gives the
levels of laccase as a function of different concentration of variables.
4.3.3.3 Optimization of process parameters by Response-to-surface
methodology (RSM)
Box-Benhken experimental design
Selection of physico-chemical parameters for maximum laccase
productions, the one-at-a time strategy of improving fermentation media and
physical conditions were successfully applied for the production of laccase
enzyme. However, this one at time techniques of optimization have some
major flaws. Due to these draw backs the use of simultaneous optimization
using experimental design has become more common and when using
experimentation design, full factorial, partial factorial or central composite,
where the techniques of choice is one of the most popular methods of
optimizations of culture medium is response surface methodology [380]. Here
the Box–Behnken experimental design was use for evaluating and optimizing
the concentration of most significant factor for laccase production in SSF. In
order to search for efficient laccase production the optimum combinations of
major components of the medium, for efficient laccase production,
experiments were performed according to the Box-Behnken design plan
(Table 4.12). Five nutrients had been identified as the most significant for
promoting enzyme yields, which established that optima could be found
within the ranges of parameters, studied the mathematical models, relating the
production of laccase with the independent process variables. The quadratic
regression equation obtained for dependent variables. The standard error (SE)
of the variables was the square root of variance and the significance level (p –
value) of each variable was calculated by using Student’s t – test (Exi is the
effect of the tested variable).
104
The results were analyzed statistically using second order polynomial
equation and the response surface graph for them were generated and from
that the hold values for each variable was calculated and a new experiment
was performed with that hold values and the response was calculated. The
experiment performed with the newly generated values for the variable
resulted in the increased production of laccase enzyme.
4.3.3.4 Interaction among the nutrients
The 3D response surface and the 2D contour plot are the graphical
representation of the regression equation. The main goal of response surface is
to efficiently hunt for the optimum value of the variables such that the
response is maximized. Response surface curve were made for variation in the
yields of laccase production as a function of concentration of two nutrients and
other nutrient being at their constant levels. From the response surface plot, it
is very easy and convenient to understand the interactions among the nutrients
and also to locate their optimum concentration.
4.3.3.5 Comparison of laccase enzyme production under optimized and
un-optimized conditions in SSB media
The comparison of laccase enzyme production by Trametes versicolor
in optimized and un-optimized SSB culture media was carried out. 100 ml of
optimized SSB and un-optimized SSB media was taken in a 250ml conical
flask and was maintained with 70% of moisture (vermiwash 50% and 20% of
water) and was inoculated with Trametes versicolor and were incubated at
30°C and 150 rpm on a rotary shaker for 2 weeks. The laccase enzyme assay
was done by Bourbonnais and Paice [7] procedure. The composition of un-
optimized and optimized SSB media is given in Table 4.12 and 4.13.
105
Table 4.12: SSB media composition (un-optimized)
COMPONENT QUANTITY( g/l)
Sapota seed 20.0K2HPO4 1.0(NH4)SO4 5.0NaCl 1.0MgSO4.7H2O 0.5FeSO4.7H2O 0.1ZnSO4.7H2O 0.1CuSO4.5H2O 0.1MnSO4.7H2O 0.1Moisture(vermiwash)
50%
Table 4.13: SSB media composition (optimized)
SYMBOL COMPONENT g/100ml
A Sapota Seed Powder 1.97
B Yeast Extract 0.9C Glucose 1.1D CuSO4 0.025E ZnSO4 0.008
4.4 Production of Laccase Enzyme
4.4.1Production of Laccase enzyme by submerged fermentation
The culture media with the composition (Table 4.13) was used for the
study.100 ml of optimized SSB media was taken in a 250ml conical flask and
was added with vermiwash 50ml and 50ml of water and was inoculated with
Trametes versicolor and were incubated at 30°C and 150 rpm on a rotary
106
shaker for 2 weeks. After fermentation the flask was added with 100ml
Acetate buffer (pH5.5) and was kept in rotary shaker for 2 hrs at 150 rpm,
after which the flask was kept in refrigerator for overnight and then the
enzyme assay was done by Bourbonnais and Paice [7].
4.4.2 Production of Laccase enzyme by solid state fermentation
The culture media with the composition (Table 4.13) was used for the
study. 100 ml of optimized SSB media was taken in a 250ml conical flask and
was maintained with 70% of moisture (vermiwash 50% and 20% of water) and
was inoculated with Trametes versicolor and were incubated at 30°C and 150
rpm on a rotary shaker for 2 weeks. The laccase enzyme assay was done by
Bourbonnais and Paice [7] procedure. After fermentation the flask was added
with 100ml Acetate buffer (pH5.5) and was kept in rotary shaker for 2 hrs at
150 rpm, after which the flask was kept in refrigerator for overnight and then
the enzyme assay was done by Bourbonnais and Paice [7].
4.5 Purification and Characterization of Laccase Enzyme
4.5.1 Ammonium sulphate precipitation
The culture fluid was filtered through a Whatman No. 1 filter paper and
centrifuged at 10,000 ×g for 10 min. Ammonium sulfate was added to the
supernatant to give 80% saturation, and the precipitated proteins were
collected by centrifugation at 10,000 ×g for 30 min. The precipitate was then
dissolved in an appropriate volumeof a 50 mM sodium acetate buffer (pH 6),
dialyzed overnight against the same buffer, and concentrated by ultrafiltration
using an YM10 membrane (Amicon Corp., U.S.A.). Next, the concentrated
proteins were applied to an ion-exchange DEAE-Sepharose FF column
(2.5×30 cm; Amersham Biosciences, Sweden) previously equilibrated with a
107
50 mM sodium acetate buffer (pH 6.0), and the bound proteins eluted using a
linear gradient of 0 to 0.5M NaCl in the same buffer at a flow rate of 2.5
ml/min. The fractions containing laccase activity were pooled and
concentrated in an Amicon stirred cell using a YM-10 membrane. The
fractions containing laccase activity were collected, concentrated, and used as
the purified enzyme preparation.The protein was determined according to the
method of Bradford [381].
4.5.2 Native PAGE
The Native PAGE was done by the following procedure: 5% [stacking
gel (6.8 ml H2O, 1.7 ml 30% acrylamide, 1.25 ml 1M Tris(6.8 pH), 0.1ml
freshly prepared APS, 0.01ml TEMED] 10% resolving gel [4.0 ml H2O, 3.34
ml 30% acrylamide, 2.5 ml 1.5 M Tris (8.8pH), 0.4ml 10% freshly prepared
APS] were prepared. Freshly prepared running buffer (25mM Tris, 192mM
glycine (8.3 pH) was used. Samples was loaded in the well and
electroporessed at 200V for 30 min. To detect the laccase activity, the gel was
incubated at 35°C for 30 min Following this the gel was rinsed with sodium
acetate buffer 0.05 M (pH 5.0) for 10 min and subsequently submerged in
2mM Guaiacol and the laccase active band was highlighted in dark brown.
4.5.3 Molecular mass determination
The molecular mass of the enzyme was estimated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE was
performed with 12% polyacrylamide gels using the method described by
Laemmli [292]. The molecular mass markers used were phosphorylase b (108
kDa), bovine serum albumin (98 kDa), ovalbumin (54 kDa), carbonic
anhydrase (33 kDa), and a soybean trypsin inhibitor (29 kDa). Following this
the gel was rinsed with sodium acetate buffer 0.05 M (pH 5.0) for 10 min and
108
subsequently submerged in 2mM Guaiacol and the laccase active band was
highlighted in dark brown.
4.5.4 pH and temperature maxima for laccase production
The optimum pH for the laccase was estimated using ABTS as the
substrate in a 100 mM sodium citrate buffer (pH 2.5-6.0) and 100 mM sodium
phosphate buffer (pH 6.5-8.0). The effect of pH on the enzyme stability was
measured after 1 h of incubation at various pH’s at 25°C. The optimum
temperature for the laccase was determined by measuring the enzyme activity
at various temperatures ranging from 20°C to 90°C in a 100 mM sodium
acetate buffer (pH 5.0). The effect of temperature on the enzyme stability was
investigated by incubating the enzyme solution for 1 h in a 100 mM sodium
acetate buffer (pH 3.0) at various temperatures. After incubation, the
remaining activity was determined.
4.6 Applications of Laccase Enzyme
4.6.1 Bio- decolourization of Azo dyes
4.6.1.1 Screening of Azo dye decolourizing fungi
The azo dye (acid orange-7) decolourizing ability of the 5 fungal strains
(Phanerochaete chrysosporium, Trichoderma harzianum, Trametes hirsuta,
Corioles versicolor and Aspergillus fumigatus – which were isolated from saw
mill wastes, purified, identified and characterized) were initially done in the
plate assay consisting of azo dye incorporated in SDA. Following incubation
at 30°C for 3 weeks, the decolourization was detected visually by comparing
109
with the control plates which had the azo dye but not the fungi. The best
decolourizing fungi was selected and used for further studies.
4.6.1.2 Bio-decolourization in solid media
SDA media was used for the experiment.The following concentrations
of Acid orange-7 0.01, 0.05, 0.1,0.5,0.76 and 1.0 grams /100ml of media were
added to SDA and following sterilization was poured into the sterile petri
plates and after solidification was inoculated with a well grown culture of
8mm plug of P.chrysosporium. The plates were incubated at 28°C for 3 weeks
and the growth and decolourization was observed periodically.
4.6.1.3 Bio-decolourization in liquid media
SDB was prepared and added with 0.01, 0.05, 0.1, 0.5, 0.76 and 1.0
grams /100ml of media concentrations of Acid orange -7. The conical flasks
having the media and dye, was inoculated with 5 plugs of P.chrysosporium
from a well grown SDA plate. The flasks were incubated at 28°C for 3 weeks
and the growth and decolourization was observed periodically.
For analysis of decolourization, the 0th day absorbance value and final
day absorbance were obtained after inoculation. The percentage of degradation
was calculated using the formula,
Initial absorbance –Final absorbance
-------------------------------------------- X100
Initial absorbance
---------- (4.5)
110
4.6.1.4 Bio-decolourization in mineral salt media
To study the ability P.chrysosporium to use Azo dye as sole source of
carbon and nitrogen Mineral Salt Medium (MS Medium) was used. The
composition of MS medium is (g/l): K2HPO4 1.0 g.,(NH4)SO4 5.0g.,Nacl1.0g.,
MgSO4.7H2O 0.5g., FeSO4.7H2O0.1 g., ZnSO4.7H2O 0.1g., CuSO4.5H2O
0.1g., MnSO4.7H2O 0.1g., in 1000 ml distilled water. The pH of the
medium was 7.2. MS media was inoculated with 0.01% 0f Acid orange -7 and
was inoculated with 5 plugs of P.chrysosporium from a well grown SDA
plate. The flask was incubated at 28°C for 3 weeks and the growth and
decolourization was observed periodically and percentage of decolourization
was calculated as in 4.6.1.3.
4.6.1.5 Optimization of Acid orange- 7 decolourization
The optimization study involved the selection of suitable carbon &
nitrogen sources and optimum temperature, pH and shaking speed on the
decolourization of acid orange -7 by P.chrysosporium.
4.6.1.5.1 Effect of various carbon sources on dye decolourization
The effect of following Carbon sources on the growth and dye
decolourization P.chrysosporium was studied: Glucose, Mannitol, Maltose,
Mannose, Sucrose and Xylose. 100ml of MS media (with 1% of carbon
sources) was taken in 250 ml conical flasks separately and wereadded with
0.01% 0f Acid orange-7 and was inoculated with 5 plugs of P.chrysosporium
from a well grown SDA plate. The flask was incubated at 28°C for 3 weeks
and the growth and decolourization was observed periodically and percentage
of decolourization was calculated as in 4.6.1.3.
111
4.6.1.5.2 Effect of various nitrogen sources on dye decolourization
The effect of following Nitrogen sources on the growth and dye
decolourization P.chrysosporium was studied:Tryptone, Peptone, Yeast
extract and Beef extract. 100ml of MS media (with 0.5% of Nitrogen sources)
was taken in 250 ml conical flask separately and was added with 0.01% 0f
Acid orange-7 and was inoculated with 5 plugs of P.chrysosporium from a
well grown SDA plate. The flask was incubated at 28°C for 3 weeks and the
growth and decolourization was observed periodically and percentage of
decolourization was calculated as in 4.6.1.3.
4.6.1.5.3 Effect of temperature on dye decolourization
The effect of following temperatures on the growth and dye
decolourization P.chrysosporium was studied: 25°C, 30°C, 35°C, 40°C and
45°C. 100ml of MS media (with 1% of glucose as carbon sources and 0.5%
peptone as nitrogen source) was taken in five separate 250 ml conical flasks
separately and were added with 0.01% 0f Acid orange-7 and were inoculated
with 5 plugs of P.chrysosporium from a well grown SDA plate. The flask
were incubated at 25°C, 30°C, 35°C, 40°C and 45°C for 3 weeks and the
growth and decolourization was observed periodically and percentage of
decolourization was calculated as in 4.6.1.3.
4.6.1.5.4 Effect of pH on dye decolourization
The effect of following pH on the growth and dye and dye
decolourization P.chrysosporium was studied: 5.5, 6.0, 6.5, 7.0 and 7.5.
100ml of MS media (with 1% of glucose as carbon sources and 0.5% peptone
as nitrogen source) was taken in five separate 250 ml conical flasks separately
and the pH was adjusted to 5.5, 6.0, 6.5, 7.0 and 7.5 were added with 0.01% 0f
112
Acid orange-7 and were inoculated with 5 plugs of P.chrysosporium from a
well grown SDA plate. The flasks were incubated at 28°C for 3 weeks and the
growth and decolourization was observed periodically and percentage of
decolourization was calculated as in 4.6.1.3.
4.6.1.5.5 Effect of shaking speed on dye decolourization
The effect of various shaking speed on dye decolourization was carried
out by incubating the dye containing flask inoculated with fungi at varying
shaking speeds viz., 80, 100,120,140 and 150 rpmindividually. 100ml of MS
media (with 1% of glucose as carbon sources and 0.5% peptone as nitrogen
source) was taken in five separate 250 ml conical flasks separately and were
added with 0.01% 0f Acid orange -7 and were inoculated with 5 plugs of
P.chrysosporium from a well grown SDA plate. The flasks were incubated at
28°C for 3 weeks at 80, 100,120,140, 150 and 160 shaking speed (rpm) and
the growth and decolourization was observed periodically and percentage of
decolourization was calculated as in 4.6.1.3.
4.6.1.5.6 Dye decolourization at optimized conditions
The effectiveness of dye decolourization, in comparison with un-
optimized conditions, was carried out by performing the experiment under
normal (un-optimized conditions) and optimized conditions individually.
100ml of MS media (with 1% of glucose as carbon sources and 0.5% peptone
as nitrogen source) was taken in a 250 ml conical flask (with a pH of 5.5) and
were added with 0.01% 0f Acid orange -7 and were inoculated with 5 plugs of
P.chrysosporium from a well grown SDA plate. The flasks were incubated at
30°C for 3 weeks at 150 shaking speed (rpm) and the growth and
decolourization was observed periodically and percentage of decolourization
was calculated as in 4.6.1.3.To compare the efficiency of optimization on dye
113
decolourization, a comparison was made by studying the decolourization
under optimized and un-optimized conditions.
4.6.1.6 Tests for absence of Re-colourization
To check the spontaneous re-colourization of azo dyes, following
decolourization experiment, the decolourized samples were kept for 8 weeks
to check the re-colourization.
4.6.1.7 Biodecolourization of Acid orange-7 by immobilized laccase
enzyme
The laccase enzyme was added to molten 2% sodium alginate solution
and mixed well. The slurry was extruded as drops into 2% calcium chloride
solution at room temperatures in aseptic condition were maintained. The beads
thus formed were hardened in the calcium chloride solution for an hour at 4°C
temperature. 25 ml of 0.01% acid orange-7 was taken in a 100ml conical flask
and was added with 10 beads of immobilized laccase enzyme and was
incubated at room temperature for 1 hour and was analyzed decolourization as
in 4.6.1.3.
4.6.1.7.1 Comparison of Acid orange-7 decolourization by free and
immobilized laccase enzyme
A comparison of bio-decolourization was made between free and
immobilized laccase enzyme. 25 ml of 0.01% acid orange-7 was taken in two
separate 100ml conical flask and one flask was added with 10 beads of
immobilized laccase enzyme and the other one was added with one unit of free
laccase enzyme (obtained and purified from Trametes versicolor) were
114
incubated at room temperature for 1 hour and was analyzed decolourization as
in 4.6.1.3.
4.6.1.7.2 Effect of temperature and pH on the free and immobilized
laccase enzyme for the decolourization of acid orange-7
The decolourization of acid orange-7 with immobilized laccase enzyme
was carried out under varying temperatures (25°C,30°C,35°C,40°C and 45°C)
and pH (5.5,6.0,6.5,7.0 and 7.5).
To study the effect of pH on dye decolourization, 100ml of 0.01% of
Acid orange-7 was taken in five separate 250 ml conical flasks separately and
the pH’s was adjusted to 5.5, 6.0, 6.5, 7.0 and 7.5 and were added with 10
beads of immobilized laccase enzyme. The flasks were incubated at 28°C for
one hour and the decolourization was observed periodically and percentage of
decolourization was calculated as above.
To study the effect of temperature on dye decolourization, 100ml of
0.01% of Acid orange-7 was taken in five separate 250 ml conical flasks
separately and the pH’s was adjusted to 5.5 and were added with 10 beads of
immobilized laccase enzyme.The flasks were incubated at 25°C, 30°C, 35°C,
40°C and 45°C for one hour and the decolourization was observed periodically
and percentage of decolourization was calculated as in 4.6.1.3.
4.6.1.8 Stability of immobilized enzyme
In order to check the stability of immobilized laccase enzyme, repeated
cycles of decolourization was carried out in batch wise manner. 100ml of
0.01% of Acid orange-7 was taken in a 250 ml conical flask and the pHs was
115
adjusted to 5.5 and was added with 10 beads of immobilized laccase enzyme
and were incubated for one hour at 30°C and the decolourization was observed
periodically and percentage of decolourization was calculated as in 4.6.1.3.
4.6.2 Bio-delignification of Eucalyptus sp.
4.6.2.1 Screening of organisms for the Bio-delignification of Eucalyptussp.
White rot fungi like Phanerochaete chrysosporium, Trichoderma
harzianum, Trametes hirsuta, Corioles versicolor and Aspergillus fumigatus
were used for this study.
Inoculum was produced by initially growing fungi on 2% malt extract
agar (MEA) plates. From these plates, plugs overgrown with mycelium were
used to start a pre-inoculum in 100 ml of SSB. The pre-inoculum was
incubated in stationary culture for seven days at 28°C before being
homogenized. Part of the homogenized culture (20 ml) was inoculated into
200 ml SSB in 500 ml conical flasks. Cultures were incubated at 30°C on a
rotary shaker at 100 rpm for 2 weeks, after which they were again
homogenized to produce inocula for wood chips.
Eucalyptus sp. was procured from timber depots and was dried in
sunlight. They were then chopped into small pieces. These small pieces were
used for the delignification study. The chopped woods were taken in conical
flask (20g/flask) and were autoclaved for 20 min at 121°C and 15lb pressure.
The flasks were inoculated with the inocula prepared as above. Then these
flasks were incubated at 30°C for 3 weeks. A control treatment consisted of an
additional 20 ml of the medium above, which was added to the wood instead
of inoculum.
116
4.6.2.2 Tests for delignification
The lignin content of the wood was estimated by AB Method. Lignin
was determined by the acetyl bromide method adapted from Kirsten
Brinkmannet al.,[382]. For this purpose, 400 mg of dry plant powder were
suspended in 10 ml of 80% Methanol (MeOH), stirred for 1 hr at room
temperature, and centrifuged at 5500g (20 min). The pellet was subjected to
the following washing steps: 1X(1 M NaCl, 0.5% Triton X-100), 2 x distilled
water, 2 x 100% MeOH, and 2 x 100% acetone (30 min each). The resulting
AB pellet was used for lignin analysis by the acetyl bromide method [382].
Aliquots of about 2.5 mg of hydrolyzed AB pellet (3 replicates) were mixed
with 250 µl of 25% acetyl bromide (v/v in glacial acetic acid) and incubated
for 30 min at 70± ◦C. Samples were rapidly cooled on ice, mixed with 250 µl
of 2 N NaOH, and centrifuged for 5 min at 15,000g. An aliquot of the
supernatant (250 µl) was mixed with 5 µl of 15 N NH4OH and 2495 µl of
glacial acetic acid. The absorbance of the solution was determined at 280 nm.
Calibration curves were generated by subjecting increasing amounts of 0.5 to
2.5 mg of commercial lignin (alkaline spruce lignin, Aldrich) to the same
procedure.
4.6.2.3 Tests for lignolytic enzymes
Manganese peroxidase activity was determined by using Castillo
procedure [383]. One ml reaction mixture contained 0.7mM 3-methyl 2-benzo
thiazolinone hydrazone (MBTH), 0.99mM 3-dimethylamino benzoic acid
(DMBA), 0.34mMMnsO4, 100mM sodium lactate/succinate buffer (pH5.0)
and 100-200 µL culture fluid. The reaction was initiated by the addition of
0.05mM H2O2. Absorbance was measured at 590nm using UV-Vis
spectrophotometer after 1min.
117
Lignin peroxidase (EC 1.11.1.14) activity was evaluated by UV spectrometry
of the veratryl aldehyde produced (ε 310= 9300 M−1cm−1) during veratryl
alcohol oxidation. The reactive mixture contained 375 µL sodium tartrate
buffer 0.33 M pH 3.0, 125 µL veratryl alcohol 4 mM, 50 µL hydrogen
peroxide 10 mM, 450 µL distilled water and 250 µL culture medium for a
final volume of 1250 µL [166].
Laccase activity was assayed spectrophotometrically by measuring the
oxidation of ABTS at 420 nm at 30°C. The assay mixture in a total volume of
1 ml contained 0.1 ml cell-free supernatants at various dilutions and 1 mM
ABTS in 100 mM citrate buffer (pH 3.4). One unit of enzyme activity was
defined as the amount of enzyme required to oxidize 1 µmol ABTS per minute
[7].
4.6.2.4 Effect of physical and chemical parameters on the delignification
The effect of various physical (temperature and shaking speed) and
chemical (carbon and nitrogen source) parameters on the delignification
activity was also carried out.
4.6.2.5 Effect of temperature on delignification
The effect of various incubation temperatures on delignification was
studied. Conical flasks containing wood chips were inoculated with
Phanerochaete chrysosporium and were incubated at 25°C, 30°C, 35°C, 40°C
and 45°C temperatures. The flasks were tested for delignification after 2
weeks by the procedure of Kirsten Brinkmannet al.,[382].
118
4.6.2.6Effect of shaking (rpm)speed on delignification
Conical flasks containing wood chips were inoculated with
Phanerochaete chrysosporium and were incubated at room temperatures and
were kept in shaking incubator with these varying rpm’s (80, 100, 120,
140,150 and 160rpm). The flasks were tested for delignification after 2 weeks
by the procedure of Kirsten Brinkmannet al., [382].
4.6.2.7 Effect of glucose concentration on delignification
Conical flasks containing wood chips were added with varying
concentrations of Glucose (0.5, 1.0, 1.5, 2.0 and 2.5 g per 100 ml) and were
inoculated with Phanerochaete chrysosporium and were incubated at room
temperatures in shaking incubator with 150 rpm. The flaks were tested for
delignification after 2 weeks by the procedure of Kirsten Brinkmannet al.,
[382].
4.6.2.8 Effect of peptone on delignification
Conical flasks containing wood chips were added with varying
concentrations of Peptone (0.1, 0.2, 0.3, 0.4 and 0.5 g/100ml) and were
inoculated with Phanerochaete chrysosporium and were incubated at room
temperatures in shaking incubator with 150 rpm. The flasks were tested for
delignification after 2 weeks by the procedure of Kirsten Brinkmannet al.,
[382].
4.6.2.9 Optimization of the delignification process
The delignification process was studied under optimized conditions in
order to check the effectiveness of the optimum conditions in delignification.
119
Conical flasks containing wood chips were supplemented with 2% glucose and
0.5% peptone and then were incubated at 30°C in a shaking incubator with
150rpm speed. The flasks were tested for delignification after 2 weeks by the
procedure of Kirsten Brinkmannet al., [382].
4.6.3 Bio-degradation of Cypermethrin
4.6.3.1 Screening of cypermethrin degrading organism
Commercially available cypermethrin [10% E.C.] was procured from
market and it was used for the study. 0.01% of cypermethrin was incorporated
in the SDA media and was poured into petri plates. After solidification the
plates were inoculated with well grown fungal discs of the following
fungi:Phanerochaete chrysosporium, Trichoderma harzianum, Trametes
hirsuta, Corioles versicolor and Aspergillus fumigatus. The plates were
incubated at 30°C for 2 weeks and the growth of the fungi was observed.
4.6.3.2 Biodegradation of cypermethrin by plate assay
The cypermethrin concentrations used in this study were: 0.01, 0.05,
0.1, 0.5, 1.0 and 5.0.SDA were prepared as per standard procedure and was
inoculated with 0.01, 0.05, 0.1, 0.5, 1.0 and 5.0 concentrations of
cypermethrin separately. The media along with the cypermethrin were added
to sterile petri plates and were inoculated with Aspergillus fumigatus and the
plates were incubated for 3-4 weeks at 30°C.
4.6.3.3 Bio-degradation in liquid media
SD broths were inoculated with 0.01, 0.05, 0.1, 0.5, 1.0 and 5.0
concentration of cypermethrin separately and were inoculated with Aspergillus
120
fumigatus. The flasks were incubated for 3-4 weeks at 30°C. The contents of
the flaks were filtered and were assayed for cypermethrin degradation by
Dhundhel & Rai, procedure [384].
4.6.3.4 Assay procedure for the biodegradation of cypermethrin
The assay was done as per the procedure of Dhundhel & Rai [384].An
aliquot of test solution containing 0.01 to 10 g of cypermethrin was taken in a
25 mL graduated tube and to it; 2 ml of 20% sodium hydroxide was added.
The solution was kept for 10 min at room temperature for complete
hydrolysis. Then 1 mL of 0.1% potassium iodide was added in acidic medium,
to liberate iodine and then 1 mL p-amino acetophenone reagent was added and
shaken thoroughly and kept for 15 min for full colour development and
absorbance was measured at 400 nm against a reagent blank and a standard
graph was constructed. The test samples were filtered and O.D. was taken at
400nm and the absorbance was compared with the standard graph and from
that the concentration of residual cypermethrin was calculated. The percentage
of degradation was also calculated.
4.6.3.5 Test for the ability of Aspergillus fumigatus to use cypermethrin as
sole carbon and nitrogen source
The ability of Aspergillus fumigatus to use cypermethrin as sole carbon
and nitrogen source was detected by growing P.chrysosporium on Mineral
Salt Medium (MS Medium) which had the following composition:K2HPO4
1.0g.,(NH4)SO4 5.0g., NaCl1.0g., MgSO4.7H2O 0.5g., FeSO4.7H2O
0.1g.,ZnSO4.7H2O0.1g.,CuSO4.5H2O0.1g., MnSO4.7H2O 0.1g., in 1000
ml distilled water. The pH of the medium was 7.2. MS media was inoculated
with 0.01, 0.05, 0.1, 0.5, 1.0 and 5.0 concentrations of cypermethrin
separately. The flasks were inoculated with Aspergillus fumigatus and the
121
flasks were incubated for 3-4 weeks at 30°C. After the incubation the flasks
were assayed for cypermethrin degradation by Dhundhel & Rai, (2011)
procedure [384].
4.6.3.6 Biodegradation of cypermethrin by laccase enzyme
Conical flasks with 0.01, 0.05, 0.1, 0.5, 1.0 and 5.0 concentration of
cypermethrin in Acetate buffer (pH 5.5) were taken and were inoculated with
one unit of laccase enzyme and were incubated at 30°C for about 1 hour.
Samples were withdrawn at 5 minutes interval and were assayed for
cypermethrin degradation by Dhundhel & Rai, (2011) procedure [384].
4.6.3.7 Time scale for cypermethrin degradation with laccase enzyme
Conical flasks with 0.01% concentration of cypermethrin in Acetate
buffer (pH 5.5) was taken and were inoculated with one unit of laccase
enzyme and was incubated at 30°C for 1 hour. Samples were withdrawn at an
interval of 5 minutes and assayed for cypermethrin degradation by Dhundhel
& Rai, (2011) procedure [384].
4.6.3.8 Effectof temperature on cypermethrin biodegradation
Conical flasks with 0.01concentration of cypermethrin in Acetate
buffer (pH 5.5) were taken and were inoculated with one unit of laccase
enzyme and were incubated at 25°C, 30°C,35°C,40°C and 45°C for 1 hour.
Samples were withdrawn at an interval of 5 minutes and assayed for
cypermethrin degradation by Dhundhel & Rai, (2011) procedure [384].
122
4.6.3.9 Effect of pH on cypermethrin biodegradation
Conical flasks with 0.01% concentration of cypermethrin in Acetate
buffer (pH 5.0, 5.5, 6.0, 6.5 and 7.0) were taken and were inoculated with one
unit of laccase enzyme and were incubated at 30°C for 1 hour. Samples were
withdrawn at an interval of 5 minutes and assayed for cypermethrin
degradation by Dhundhel & Rai, (2011) procedure [384].
4.6.3.10 Optimization laccase enzyme concentration for the
biodegradation of cypermethrin
The optimum concentration of laccase enzyme needed for the
biodegradation of cypermethrin was calculated by carrying out a degradation
study by taking varying concentrations of laccase enzyme (like 0.5,1.0,1.5,2.0
and 2.5 U/ml) and fixed concentration of cypermethrin (0.01%). The Conical
flasks were incubated at 30°C for 1 hour. Samples were withdrawn and
assayed for cypermethrin degradation by Dhundhel & Rai, (2011) procedure
[384].
4.6.3.11 Comparison of biodegradation of cypermethrin under optimized
and un-optimized conditions
The comparison of cypermethrin biodegradation under un-optimized
and optimized conditions (i.e. at optimum temperature, pH, time and Laccase
enzyme concentration) was carried out by taking conical flasks with 0.01%
concentration of cypermethrin in Acetate buffer (pH 6.0) were taken and were
inoculated with 1.5 unit of laccase enzyme and were incubated at 30°C for 1
hour. Samples were withdrawn and assayed for cypermethrin degradation by
Dhundhel & Rai, (2011) procedure [384].
123
4.7 Statistical analysis
The statistical analyses of the above experiments were carried out using
SPSS software (version 16.0).SPSS (Statistical Package for the Social
Sciences) is a statistical analysis and data management software package.
SPSS can take data from almost any type of file and use them to generate
tabulated reports, charts, and plots of distributions and trends, descriptive
statistics, and conduct complex statistical analyses. All the above experiments
were carried out in duplicates and their significance was analysed using the
SPSS software.
