chapter 5 results and discussion 5.1 screening and...

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CHAPTER 5

RESULTS AND DISCUSSION

5.1 Screening and isolation of laccase producing fungi

A total of 16 isolates were tested for their ability to produce laccase

enzyme. The isolates were assigned the labels from SMWF 1-16. SMWF is

the abbreviation for saw mill waste fungi. The results of the screening tests are

given in the Table 5.1. The isolates SMWF-6, 10, 11, 14 and 16 gave positive

result for the entire 5 indicator compound, namely ABTS, syringaldazine,

Guaiacol, tannic acid and Gallic acid. SMWF-3, 9, 12 and 15 did not give

positive result with any of the indicator compounds thereby indicating they are

laccase negative strains. The isolate SMWF-7, 8 & 13 was negative for ABTS

and positive for the rest of the indicators. Similarly SMWF-4 was negative for

ABTS and Syringaldazine but not for the others.SMWF-2 gave positive result

with all except Syringaldazine and SMWF-5 gave positive result with all

indicators but ABTS and Guaiacol. While SMWF-1 showed positive result

only with Guaiacol and not with others.

Microbes that produce laccases have been screened for either on solid

media containing coloured indicator compounds that enable the visual

detection of laccase production [385, 386,387] or with liquid cultivations

monitored with enzyme activity measurements [388, 389, 390]. The use of

coloured indicators is generally simpler as no sample handling and

measurement is required. As laccases oxidize various types of substrates,

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several different compounds have been used as indicators for laccase

production.

The variability of fungal strains response to the indicator compounds

was discussed by Baldrin [62] in his review article. Bourbonnais et al., [391],

Kwang-Soo Shin et al.,[392] and several other authors have used ABTS as the

substrate for laccase detection and quantification. 2, 2’-azino-bis (3-

ethylbenzothiazoline-6-sulphonic acid) or ABTS is chemical compound used

to observe the reaction kinetics of specific enzymes. A common use for it is in

the enzyme-linked immunosorbent assay (ELISA) to detect for binding of

molecules to each other.

Table5.1: Response of fungal isolates to the indicators*

Strain ABTS Syringaldazine GuaiacolTannic

acidGallicacid

SMWF-1 - - + - -SMWF-2 + - + + +SMWF-3 - - - - -SMWF-4 - - + + +SMWF-5 - + - + +SMWF-6 + + + + +SMWF-7 - + + + +SMWF-8 - + + + +SMWF-9 - - - - -

SMWF-10 + + + + +SMWF-11 + + + + +SMWF-12 - - - - -SMWF-13 - + + + +SMWF-14 + + + + +SMWF-15 - - - - -SMWF-16 + + + + +

*Positive result is shown by (+) symbol and negative result is shown by (-)

symbol. Positive result means that the fungal isolate is able to produce colour

halo with the indicator compound around its colony and negative result means

the absence of colour halo.

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The formal reduction potentials for ABTS are high enough for it to act

as an electron donor for the reduction of oxo species such as molecular oxygen

and hydrogen peroxide, particularly at the less-extreme pH values encountered

in biological catalysis.

Harkin and Obst [393] were the first to demonstrate the use of

Syringaldazine as a substrate for laccase assay and later several other workers

have also used syringaldazine as an assay substrate for laccase. Laccase

catalyses the oxidation of syringaldazine to tetramethoxy-azo-bis (methylene

quinone) that is measured spectrophotometrically at 530 nm.

The traditional screening reagents tannic and gallic acid [393] have

nowadays mostly been replaced with synthetic phenolic reagents, such as

guaiacol and syringaldazine [394, 395] or with the polymeric dyes Remazol

Brilliant Blue R (RBBR) and Poly R-478 [396, 397and 398]. RBBR and Poly

R-478 are decolourized by lignin-degrading fungi [399, 396], and the

production of ligninolytic enzymes is observed as a colourless halo around

microbial growth. With guaiacol a positive reaction is indicated by the

formation of a reddish-brown halo [394], while with tannic and gallic acid the

positive reaction is a dark-brown coloured zone [393].

Airong Li et al., [400], Xiao et al., [401] etc., have shown the ability of

Guaiacol to detect laccase enzyme. While Alina Manole et al.,[402] Pointing

,[22] Marièlle Bar.,[403] etc., have used Tannic acid as an substrate for

laccase and so as Gallic acid was used by Wang et al.,[404] Pointing,[22]

Baldrin [62]etc.,

Based on the above results SMWF-6, SMWF-10, SMWF-11, SMWF-

14 and SMWF-16 were selected for further studies.

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5.2 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. The isolates were

identified as in Table 5.2.

Trametes versicolor — formerly known as Coriolus versicolor and

Polyporus versicolor— is an extremely common polypore mushroom which

can be found throughout the world. T. versicolor is commonly called Turkey

Tail because of its resemblance to the tail of the wild turkey

(Wikipedia).Trametes (Coriolus=Polyporus) versicolor is the most-studied

laccase producing fungus [211]. Trametes (Coriolus) versicolor is a white-rot

fungus that produces extracellular enzymes including laccase,manganese

peroxidase (MnP), lignin peroxidase (LiP), cellulase and avicelase. This

fungus is a common habitant of dead woods, causing their decay due to the

ability to degrade lignin (non-hydrolysable part of wood) using extracellular

enzymes .

Table 5.2: Fungal cultures identified by morphological, cultural and

biochemical characteristics

Sl.No. Code No. Fungi identified as

1 SMWF-16 Trametes versicolor

2 SMWF-11 Trametes hirsuta

3 SMWF-6 Phanerochaete chrysosporium

4 SMWF-14 Aspergillus fumigates

5 SMWF-10 Trichoderma harzanium

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Han et al., [204] reported Trametesversicolor 951022 which had first

been isolated in Korea, washighly active in degrading polycyclic aromatic

hydrocarbons and capable of producing laccase.Previous studies [380, 405, 6]

have shown that Trametes versicolor CCT 4521 was the best laccase producer

in aliquid medium containing 2, 5-xylidine as inducer in the presence of

copper sulphate and produced different isoenzymes. T. versicolor was also

able to produce laccase isoenzymes in submerged culture medium depending

on the lignocellulose material employed with ratio of activity Lac II/Lac I

from 0.9 (barley straw) to 4.4 (grapes stalks) [233].The cultivation of

Trametes versicolor for laccase production and cell growth were strongly

dependent on experimental conditions namely physical and chemical

parameters as well as nutrient availability and inducer stimulation [245].

Trametes hirsuta has been described as a very promising candidate for

the production of laccase [406]. Moreover, laccase from T. hirsuta can

efficiently degrade a wide variety of synthetic dyes [337, 407]. Moldes et al.,

[233] have also shown the laccase producing capabilities of T.hirsuta.

Phanerocheate chrysosporium, a model organism for lignin and

xenobiotics biodegradation studies produces a family of Lac, Lip and MnP

isoenzymes [408]. Viswanath et al., [409] has shown the laccase producing

ability of P.chrysosporium and its ability to decolourize textile dyes along

with some other fungi. Srinivasan et al., [410] has shown that P.

chrysosporium produces low but consistent levels of laccase presented

evidence for the presence of laccase in P. chrysosporium grown in low-

nitrogen (2.4 mM) or high-nitrogen (24 mM) defined media containing

cellulose as the carbon source. Buddolla Viswanath et al., [409] found

P.chrysosporium as the best source for laccase production along with Stereum

ostrea. Paulraj Kanmani et al., [411] have studied the production of laccase

enzyme on coir waste substrate. Jhadav et al., [178] have optimized, produced

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and partially purified laccase from P.chrysosporium using submerged

fermentation.

Safari Sinegani et al., [412] have demonstrated that Laccase activity of

Phanerochaete chrysosporium under liquid conditions was strikingly low.

However, under solid conditions it increased more than that of the other fungi.

Dittmer et al., [413] produced multiple isoforms of laccase from

P.chrysosporium.

Aspergillus fumigatus is the most ubiquitous of the airborne

saprophytic fungi. A. fumigatus plays an essential role in recycling

environmental carbon and nitrogen. Laccase enzyme plays a major role in the

biosynthesis of melanin in A.fumigatus [414]. Jie-Jie Hao et al., [415] have

shown the ability of A.fumigatus, a soil borne fungi, to produce laccase

enzyme. Banana Peel was used as a potential substrate for laccase production

by Aspergillus fumigatus VkJ2.4.5 in Solid-State Fermentation by Vivekanand

et al., [416].

Sadhasivam et al., [417] used a newly isolated Trichoderma harzianum

WL1 for the production, purification and characterization of mid-redox

potential laccase from.

5.3 Screening of semi-synthetic culture media for laccase enzyme

production

The laccase enzyme production in the YPD-Cu and GPB media showed

marked variations as depicted in the Table 5.3 and Figure 5.1. Trametes

versicolor produced highest quantity of laccase enzyme in both the media viz.,

1.31 U/ml and 0.187 U/ml respectively. Trichoderma harzanium was the

lowest producer of laccase enzyme; the quantity produced being 0.22 and

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Table 5.3: Comparison of laccase enzyme production by the 5 isolated fungal

species in two semi synthetic media namely, YPD-Cu and GPB

Sl.No. Isolate(s)Laccase enzyme in U/ml

YPD-Cu GPB1 Trametes versicolor 1.317±0.007 0.187± 0.0032 Trameteshirsuta 0.792±0.007 0.11± 0.013 Phanerochaete

chrysosporium0.0497±0.007 0.042± 0.006

4 Aspergillus fumigatus 0.366±0.008 0.028± 0.0035 Trichoderma harzanium 0.221 ± 0.002 0.023± 0.002

The result shown above is the mean value of duplicate experiments along with the standard deviation. The P is < 0.05 at 95% confidence interval.

Figure 5.1: Comparison of laccase enzyme production by the 5 isolated fungal

species in two semi synthetic media namely, YPD-Cu and GPB

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0.023 U/ml respectively. P.chrysosporium produced almost same quantity of

laccase enzyme (0.05 and 0.042 U/ml) in both the culture media used for the

study. T.hirsuta was the second highest producer of laccase enzyme (0.78 &

0.11 U/ml), while A.fumigatus yielded 0.36 and 0.028 U/ml quantity of

enzymes in YPD-Cu and GPB media.

YPD-Cu media yielded more laccase enzyme than GPB and the isolate

Trametes versicolor produced maximum enzyme (1.315 U/ml in YPD-Cu and

0.187 U/ml in GPB). The presence of copper as an inducing agent for laccase

production in the YPD-Cu media could be the reason why all strains yielded

maximum laccase when compared to GPB. Copper has been reported to be a

strong laccase inducer in several species, among them, T. versicolor [225], P.

chrysosporium [407] and Pleurotus eryngii [266]. It is known that Cu induces

both laccase transcription and activity [266], and the increase in activity is

proportional to the amount of copper added. But in the case of T. trogii,

induction of MnP and GLOX activity was observed as well. In T. trogii

maximal laccase, MnP, and GLOX production were attained with1 mM Cu. At

the highest concentrations Cu appeared to be toxic, since growth was reduced.

Even though ligninolytic enzyme production rose while increasing Cu (up to 1

mM), other enzyme systems may be affected, considering that no increase in

extracellular proteins was detected. Heavy metals are known as inhibitors of

many enzymes belonging to both primary and secondary metabolic pathways

[418].

The maximum laccase production by T.versicolor is well documented

by several other authors also and it is attributed as the best laccase producer

when compared to the rest of the fungi under study. Similar result for laccase

enzyme was obtained by Minussi et al., [300] by growing T.versicolor in a

liquid medium containing (g/L): peptone, 10; malt extract, 5; CuSO4.5H2O,

0.005 and glucose, 20; at pH 5.4. 0.57 U/ml laccase enzyme was obtained with

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culture media (pH 6.0) containing glucose, 1%; peptone, 0.3%,; KH2PO4,

0.06%; ZnSO4,0.0001%; K2HPO4, 0.04%; FeSO4, 0.0005%; MnSO4, 0.05%

and MgSO4, 0.05% by Vaidyanathan Vinoth Kumar et al., [419].

0.126U/ml quantity of laccase enzyme was produced by T. hirsuta in

media having 2% glucose in YPD media [420].

P. chrysosporium BKM-F1767 produces extracellular laccase in a

defined culture medium containing cellulose (10 g/liter) and either 2.4 or 24

mM ammonium tartrate [410]. Maximum laccase activity occurred in the

filtrates of P. chrysosporium grown in Olga et al., medium with 0.983n kat/ml

and 0.933 n kat/ml by Buddolla Viswanath et al., [409].

5.4 Time scale for laccase enzyme production

The time taken by each of the fungal strain to start synthesizing the

laccase enzyme and also the time taken for them to produce maximum amount

of laccase enzyme is highly variable with respect to the fungal strain. SMWF-

16 started producing laccase enzyme as early as 4th day onwards while strains

SMWF-6 and SMWF-11 did not produce laccase enzyme in 14 days time.

While strain SMWF-14 started producing laccase enzyme in the 5th day

onwards and SMWF-16 from 7th day onwards. SMWF-16 yielded a maximum

of 1.31 U/ml of laccase by the 14th day, while the rest of them yielded

significantly less quantity of laccase by the end of 8 weeks of incubation.

The time taken by the 5 selected strains of fungi, which showed

positive results in the plate assay, are given in Table 5.4 and Figure 5.2.

The time taken by the 5 isolated fungi showed that the isolate SMWF-

10 and SMWF-14 produced Laccase enzyme in the first week itself, SMWF-

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16 started producing in the second week, SMWF-6 in the fourth week and

SMWF-11 in the eight week of incubation. The results are shown in the Table

5.5.

Table 5.4: Time taken by the 5 isolated fungal species for the production of

laccase enzyme in two weeks time.

Day

(s)

SMW

F-6SMWF-10

SMW

F-11SMWF-14 SMWF-16

0 0.0 0.0 0.0 0.0 0.01 0.0 0.0 0.0 0.0 0.02 0.0 0.0 0.0 0.0 0.03 0.0 0.0 0.0 0.0 0.04 0.0 0.006 ± 0.001 0.0 0.0 0.05 0.0 0.017± 0.003 0.0 0.01±0.001 0.06 0.0 0.038± 0.001 0.0 0.04±0.001 0.07 0.0 0.065± 0.001 0.0 0.09± 0.001 0.0001± 0.08 0.0 0.089± 0.002 0.0 0.14± 0.001 0.02±0.00019 0.0 0.10± 0.001 0.0 0.17± 0.001 0.03±0.001

10 0.0 0.122± 0.002 0.0 0.19± 0.001 0.05±0.00111 0.0 0.141± 0.006 0.0 0.21± 0.001 0.08±0.00112 0.0 0.158± 0.032 0.0 0.22± 0.002 0.13±0.00113 0.0 0.176± 0.027 0.0 0.25± 0.001 0.18± 0.00214 0.0 1.31± 0.013 0.0 0.36± 0.001 0.22±0.001


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Figure 5.2: Time taken by the 5 isolated fungal species for the production of

laccase enzyme in two weeks time.

Table 5.5: Time taken by the 5 isolated fungal species for the production of

laccase enzyme in 8 weeks of time.

Fungal isolateLaccase production in

1 week 2weeks 4weeks 8weeks

SMWF-6 +

SMWF-10 +

SMWF-11 +

SMWF-14 +SMWF-16 +

+ production of laccase enzyme

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The secretion of laccase enzyme by T.versicolor from 4th day onwards

is also reported by Han et al., [296]. They reported that T. versicolor 951022

strains secrete laccase during 3-4 days of incubation in liquid culture.

Bourbonnais et al., [211] and Xavier Font et al.,[421] have also reported the

same result for T.versicolor.

T.hirsuta produced laccase enzyme from 6th day onwards and the

maximum yield was achieved is less than that of T.versicolor, hence

T.versicolor was chosen for further studies based on the quantity and time

taken for laccase enzyme production (Table 5.4).

5.4 Screening of Agro waste based media for laccase enzyme production

All the 7 agro wastes (rice bran, wheat bran, sugarcane bagasse, cotton

seed, green gram husk, ground nut shell and sapota seeds) used for the study

supported the growth and multiplication of laccase producing fungi. But these

substrates showed marked variation with respect to the laccase enzyme

production. Sugar cane bagasse, cotton seed and sapota seed supported much

higher production of laccase enzyme while the rice bran, wheat bran, ground

nut shell and green gram husk resulted less enzyme production than the above

three substrates. The results were shown in Table 5.6 and Figure 5.3.

Sapota seed supported maximal laccase enzyme (2.34 U/ml) for

Trametes versicolor, 2.11 U/ml for Trametes hirsuta, and 1.78 U/ml for

Phanerochaete chrysosporium, 1.67 U/ml for Trichoderma harzianum and

1.56U/ml for Aspergillus fumigatus. From this it is concluded that sapota seed

extract supported the maximal laccase enzyme production and Trametes

versicolor produced maximal laccase enzyme.

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Various agroindustrial by-products have been used for submerged

fermentation of Cerrenaunicolor (Bull.: Fr.) Murrill and C. maxima (Fr.)

Ryvarden for ligninolytic enzyme production. Six agroindustrial byproducts

were tested in shake flask trails. Wheat bran appeared to be the best growth

substrate for fermentation of C. unicolor, enabling a very high accumulation

of laccase (87.450 IU/Lon day 7) in culture liquid. Kiwi, as a growth substrate,

supported remarkable secretion of manganese peroxidase (2016 IU/L on day

7). Ethanol production wastes also provided a high yield of laccase, whereas

other substrates (banana peels, peanut shells, and cotton stalks) appeared to be

rather poor growth substrates for laccase and manganese peroxidase

production. Testing of C. maxima as a ligninolytic enzyme producer showed

that this fungus is a weaker producer of laccase and peroxidase than C.

unicolor. However, the supplementation of wheat bran, ethanol production

wastes, and kiwi highly stimulated laccase production, which reached 6247

IU/L, 6141 IU/L, and 5569 IU/L, respectively. In contrast to C. unicolor, the

second tested fungus, C. maxima, produced very low titers of manganese

peroxidase (9.2–43.7 IU/L) in fermentation of five lignocellulosic substrates,

whereas no manganese peroxidase was detected in submerged fermentation of

kiwi.

The data prove that the composition of lignocelluloses substrates

appear to determine the type and amount of enzyme produced by the wood-

rotting Basidiomycetes [422].

T. versicolor was the most potent laccase producer organism among the

ones examined among the Trametes versicolor ATCC 200801, Phanerochaete

chrysosporium ME 496 and Pleurotus sajor-caju, Pleurotus ostreatus,

Pleurotus florida, Pleurotus sapidus and Pleurotus eryngii. T. versicolor had

maximum laccase production (6.1 U/ml) capacity when compared to the other

examined white-rot fungus.

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Table-5.6: Comparison of Laccase enzyme production by the five isolated fungal species in AWMS media with various

agro wastes.


FungiLaccase Enzyme produced in AWMS media (U/ml)

Rice bran wheat bran

sugarcane bagasse

cottonseed

green gram husk

ground nut shell

sapotaseeds

Phanerochaete chrysosporium 0.94± 0.01 0.89±0.003 1.16±0.011 1.43±0.013 0.98±0.001 1.24±0.001 1.78±0.003

Trichoderma harzianum 0.86±0.003 0.91±0.001 1.45±0.013 1.56±0.001 1.02±0.001 1.00±0.001 1.67±0.001

Trametes hirsuta1.12±0.001 1.39±0.012 1.52±0.001 1.73±0.003 1.34±0.015 1.45±0.003 2.11±0.001

Trametes versicolor1.35±0.021 1.37±0.022 1.71±0.003 1.80±0.001 1.28±0.023 1.33±0.012 2.34±0.002

Aspergillus fumigatus0.86±0.022 0.89±0.001 1.03±0.002 1.07±0.01 0.64±0.001 0.72±0.001 1.56±0.001

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Figure 5.3: Comparison of Laccase enzyme production by the five isolated fungal species in AWMS media with various agro

wastes.

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According to some researchers, T. versicolor has been known as a good

laccase source [423, 424, 425 and 355]. The maximum yield was obtained at

pH 5.0 and 150 µM initial substrate concentration, 1.78 U/ml laccase activities

and at 25 °C of reaction temperature. It is known that, T. versicolor is a good

laccase source [423, 424, 425 and 355].

Based on the above studies, the isolate Trametes versicolor, was

selected for further studies and sapota seed powder was selected as the agro

based material for laccase production by the above selected fungi.

5.5 Optimization of laccase enzyme production

5.5.1. Optimization by classical method

5.5.1.1 Optimization of semi synthetic culture media

The yeast extract peptone dextrose–copper sulphate (YPD-Cu) medium

was used as the basal medium for optimization of laccase enzyme production

study and the fungal strain used was SMWF-16 (Trametes versicolor)

5.5.1.1.1 Selection of carbon sources

The results of RAPID HICARBOHYDRATE TEST showed that

Trametes versicolor could use 28 out of 35 carbohydrates present in the test

kit. Out of this 28 carbohydrates Glucose, Sucrose, Lactose, Mannitol and

Maltose were selected as carbon source as these were used very quickly by the

Trametes versicolor when compared to the rest of the carbohydrates. The

results are shown in Table 5.7 and Figures 5.4-5.6.

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Table5.7. Screening of Carbohydrates for Trametes versicolor by Hi

carbohydrate Test Kit test*

Sl.No. Test

Originalcolour of

themedium

Positivecolour

Negativecolour

Resultobtained

1. Lactose PinkishRed/ Red Yellow Red/Pink Yellow

2. Xylose PinkishRed/ Red Yellow Red/Pink Yellow

3. Maltose PinkishRed/ Red Yellow Red/Pink Yellow

4. Fructose PinkishRed/ Red Yellow Red/Pink Yellow

5. Dextrose PinkishRed/ Red Yellow Red/Pink Yellow

6. Galactose PinkishRed/ Red Yellow Red/Pink Red/Pink

7. Raffinose PinkishRed/ Red Yellow Red/Pink Yellow

8. Trehalose PinkishRed/ Red Yellow Red/Pink Yellow

9. Melibiose PinkishRed/ Red Yellow Red/Pink Yellow

10. Sucrose PinkishRed/ Red Yellow Red/Pink Yellow

11. L-Arabinose PinkishRed/ Red Yellow Red/Pink Yellow

12. Mannose PinkishRed/ Red Yellow Red/Pink Yellow

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Table 5.7 continued…..

13. Inulin PinkishRed/ Red Yellow Red/Pink Yellow

14. Sodium gluconate

PinkishRed/ Red Yellow Red/Pink Red/Pink

15. Glycerol PinkishRed/ Red Yellow Red/Pink Yellow

16. Salicin PinkishRed/ Red Yellow Red/Pink Yellow

17. Glucosamine

PinkishRed/ Red Yellow Red/Pink Yellow

18. Dulcitol PinkishRed/ Red Yellow Red/Pink Yellow

19. Inositol PinkishRed/ Red Yellow Red/Pink Yellow

20. Sorbitol PinkishRed/ Red Yellow Red/Pink Yellow

21. Mannitol PinkishRed/ Red Yellow Red/Pink Yellow

22. Adonitol PinkishRed/ Red Yellow Red/Pink Yellow

23.α-Methyl-

D-Glucoside


24. Ribose PinkishRed/ Red Yellow Red/Pink Red/Pink

25. Rhamnose PinkishRed/ Red Yellow Red/Pink Yellow

26. Cellobiose PinkishRed/ Red Yellow Red/Pink Yellow

27. Melezitose PinkishRed/ Red Yellow Red/Pink Yellow

28.α-Methyl-

D-mannoside


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Table 5.17 continued…..

29. Xylitol PinkishRed/ Red Yellow Red/Pink Yellow

30. ONPG Colourless Yellow Colourless Yellow

31. Esculinhydrolysis Cream Black Cream Black

32. D-Arabinose

PinkishRed/ Red Yellow Red/Pink Red/Pink

33. Citrateutilization Green Blue Green Green

34. Malonateutilization

LightGreen Blue Light

GreenLightGreen

35. Sorbase PinkishRed/ Red Yellow Red/Pink Yellow

*The positive result is indicated by the colour change.

Figure 5.4. Screening of Carbohydrates for Trametes versicolor by

carbohydrate utilization test (Part-A). The top one is the control and the

bottom one is the test result.

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Figure 5.5: Screening of Carbohydrates for Trametes versicolor by carbohydrate utilization test (Part-B). The top one is the control and the bottom one is the test result.

Figure 5.6: Screening of Carbohydrates for Trametes versicolor by

carbohydrate utilization test (Part-C). The top one is the control and the

bottom one is the test result.

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5.5.1.1.2 Effect of different carbon sources on laccase enzyme production

in YPD-Cu media

From the five carbon sources [Glucose, Sucrose, Maltose, Lactose &

Mannitol] tested, Glucose was found to be the best carbon source, as it

supported 1.31 U/ml of laccase enzyme, when compared to the rest of the

carbon sources. Hence Glucose, as the carbon source, is used in the rest of the

media optimization studies for semi synthetic medium. The results are given in

the Table 5.8 and Figure 5.7.

Glucose, mannose, maltose, fructose and lactose were tested as carbon

sources for fungus mycelial biomass yield and laccase production. Figure 3

showed that the carbon sources induced different fungus mycelial biomass

yield and activity of laccase. The best fungus mycelial biomass yield was

achieved with glucose (88.32±1.50 mg/cm3), while the best laccase activity of

(47.5±1.85 U/min) was induced by mannose [Oluseyi Damilola Adejoye and

Isola. O. Fasidi, 2010].

Among the different carbon sources tested namely Glucose, Sucrose,

Mannitol, Maltose, Glycerol and Fructose at 1% concentration, the level of

laccase production was maximum at 161.1 U/ml, in mannitol amended

medium. The enzyme production of 149 U/ml and 90.9 U/ml was recorded

with Fructose and Glycerol on 15th day of incubation. The carbon sources

like glucose and maltose showed decreased effect on laccase production from

Pleurotus sp., [248].

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Table 5.8: Effect of different carbon sources on laccase enzyme production in

YPD-Cu media by Trametes versicolor

Sl.NO. CARBON SOURCE LACCASE (U/ml)

1 Glucose 1.31± 0.001

2 Sucrose 1.20± 0.001

3 Maltose 0.96± 0.022

4 Lactose 0.99± 0.003

5 Mannitol 0.85± 0.012


Figure 5.8: Effect of different carbon sources on laccase enzyme production

in YPD-Cu media by Trametes versicolor.

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Churapa Teerapatsakul et al., [426] showed that Ganoderma sp.

produced maximum laccase activity in the presence of glycerol as a carbon

source in liquid medium. In addition, Rodriguez Couto et al.,[427] reported

glycerol as a sole carbon source in Trametes hirsuta, which showed a

maximum laccase activity of 19,400 U/L, which was reported the highest from

the fungus [428].

5.5.1.1.2.1Effect of varying concentrations of Glucose on laccase enzyme

production in YPD-Cu media

The study on the effect of varying concentrations of Glucose on laccase

enzyme production shows that a concentration of 20g/l is the optimum

concentration needed for the maximal growth and enzyme productions by

Trametes versicolor, any increase in concentration did not increase the yield

were as a decrease in the glucose concentration decreased the yield

considerable. The results are given in Table5.9 and Figures 5.8.

5.5.1.1.3 Effect of various nitrogen sources on laccase enzyme production

The study on the selection of Nitrogen sources showed that Peptone as

the best Nitrogen source when compared to the rest [Peptone,

Urea,Ammonium Sulphate , Tryptone And Ammonium Chloride ] as it

supported the maximal laccase yield (1.31 U/ml) by Trametes versicolor.

147

Table 5.9: Effect of varying conc. of Glucose on laccase enzyme production

by Trametes versicolor in YPD-Cu media

Sl.No. Concentration of Glucose(g/L)

Laccase U/ml

1 5 1.12±0.0112 10 1.19± 0.0013 15 1.26± 0.0214 20 1.31± 0.0105 25 1.31± 0.010


Figure 5.8: Effect of varying conc. of Glucose on laccase enzyme production


148

The reason for this is peptone apart from serving as nitrogen source it

can also act as a source of B-complex vitamins. Among the inorganic sources

ammonium sulphate and ammonium chloride gave almost close result (1.0 and

0.99 U/ml respectively). The results are given in the Table 5.10 and Figure

5.9.

In the literature, contradictory evidence exists for the effects of the

nature and concentration of the nitrogen source on ligninolytic enzyme

production. While high nitrogen media gave the highest laccase activity in

L.edodes, Rigidoporus lignonus, and Trametes pubescens, nitrogen-limited

conditions enhanced the production of the enzyme in Pycnoporus

cinnabarinus, P. sanguineus, and Phlebia radiata [429, 205 and 142].

The role of these compounds in the regulation of enzyme synthesis

depends not only on the physiology of the tested fungi but also on the medium

composition, especially on presence of lignocellulosic substrate [379, 430].

Tekere et al., [431] showed that some Trametes species, T. cingulata, T.

elegans and T. pocas produced the highest MnP activities in a medium

containing high carbon and low nitrogen conditions. At the same time, high

MnP activity was notable for T. versicolor when both carbon and nitrogen in

the medium were present at high levels. Laccase production by these species

was highest under conditions of high nitrogen. Sun et al., [432] demonstrated

that stationary cultivation conditions and low nitrogen concentration favoured

MnP production by T. gallica, while during solid-state fermentation of wheat

straw; lignocellulolytic enzyme production needed high nitrogen content

[433].

149

Table 5.10: Effect of various nitrogen sources on laccase enzyme production



Figure 5.9: Effect of various nitrogen sources on laccase enzyme production


Sl.No. Nitrogen Source Laccase (U/ml )

1 Peptone 1.31± 0.001

2 Urea 1.06± 0.013

3 Ammonium Sulphate 1.00± 0.011

4 Tryptone 1.10±0.001

5 Ammonium Chloride 0.99±0.001

150

Pleurotus sp. produced maximum laccase activity when beef extract

was sole nitrogen source (114 U/ml). However, most of the fungal laccases

were stimulated by organic nitrogen than inorganic nitrogen source. Kirk and

Farrell [434], Higuchi [435], Cullen [436] reported laccase and other

ligninolytic enzyme activity were increased due to carbon and organic

nitrogen.

Saravanakumar and Kaviyarasan [437] reported maximum laccase

production (84 U/ml) by using vanillin. Monteiro and De Carvalho [438]

reported high laccase activity with semi-continuous production in shake-flasks

using a low carbon to nitrogen ratio (7: 8 g). Buswell et al., [242] found that

laccases were produced at high nitrogen concentrations, although it is

generally accepted that a high carbon to nitrogen ratio is required for laccase

production. Laccase was also produced earlier when the fungus was cultivated

in a substrate with a high nitrogen concentration and these changes did not

reflect differences in biomass. Heinzkill et al., [439] also reported a higher

yield of laccase using nitrogen rich media rather than nitrogen limited media

usually employed for production of oxido-reductase [437].

Similar results were observed with different nitrogen sources (Yeast

extract, peptone, urea, (NH4)2SO4 and NaN03) in submerged medium. All the

nitrogen sources used in this study significantly promoted biomass yield and

laccase production by the fungus. The best stimulatory nitrogen source for

fungus mycelial biomass yield (125.25±3.45 mg/30cm3) was achieved using

urea as nitrogen source, while the highest laccase activity (65.5±2.52

U/min)was induced with yeast extract [440].

151

5.5.1.1.3.1 Effect of varying concentrations of peptone on laccase enzyme

production

The study on the effect of varying concentrations of peptone on laccase

production shows that 5g/l as the optimum concentration needed by Trametes

versicolor for producing maximum laccase enzyme (1.31U/ml). The increase

in peptone concentration beyond 5g/l did not affect the yield while lower

concentration resulted in lower yields. The results are given in the Table 5.11

and Figures 5.10.

5.5.1.1.4 Effect of different incubation temperatures on laccase enzyme

production

The effect of various incubation temperatures on laccase production in

YPD-Cu media shows that 30°C was the best temperature for the production

of laccase enzyme. The growth and enzyme production was from seen 25°C

between 35°C. Temperature above 35°C resulted in decreased enzyme

production by Trametes versicolor and beyond 50°C there was no fungal

growth and consequently no enzyme production. The results are in league with

growth temperature of the fungi. The results are shown in Table 12 and Figure

5.11.

Temperature is of much significance in the liquid state, even though the

impact of temperature is more prominent in the scale up processes, it remains

an inevitable factor in all systems due to its impact an microbial growth and

enzyme production. Result showed that the temperature of 25oC was optimum

for laccase production (53.4U/ml and no considerable activity was observed at

any of the other temperatures considered (20, 30, 35, 40ºC). The culture of

Cyathus bulleri showed maximum laccase production at 30ºC.The optimal

152

temperature of laccase production is been reported to differ greatly from one

strain to another.

Table 5.11: Effect of varying concentrations of peptone on laccase enzyme

production by Trametes versicolor

Sl.No.Concentration of

Peptone (g/L)Laccase U/ml

1 1 0.98± 0.002

2 2 1.11± 0.001

3 3 1.18± 0.015

4 4 1.22± 0.001

5 5 1.31± 0.001

6 6 1.31± 0.002


Figure 5.10: Effect of varying concentrations of peptone on laccase enzyme

production by Trametes versicolor

153

Table 5.12: Effect of incubation temperatures on laccase enzyme production

in YPD-Cu media by Trametes versicolor

Sl.No. Temperature Laccase( U/ml )

1 25°C 1.24± 0.022

2 30°C 1.31± 0.013

3 35°C 1.30± 0.001

4 40°C 0.52± 0.005

5 45°C 0.18± 0.001


Figure 5.11: Effect of incubation temperatures on laccase enzyme production

in YPD-Cu media by Trametes versicolor.

154

In general fungi are cultivated at temperatures between 25°C and 30°C

for optimal laccase production. When cultivated at temperatures higher than

30°C the activity of the ligninolytic enzyme was reduced. Biomass production

and laccase activity was observed to be high at temperature 25-30°C, with

temperature 25°C as the optimum. Biomass production and laccase activity of

this fungus was not favoured by low nor high temperatures. Similar

observations were made by Gbolagade et al., [441] on biomass production of

Pleurotus florida [442].

Effect of different carbon and nitrogen sources reported that

temperatures higher than 30°C reduce the activity of ligninolytic

enzymes[443, 444, 255, 445,442 and 441]. When fungi cultivated at

temperatures higher than 30°C the activity of ligninolytic enzymes was

reduced [442].

Pleurous sp. was grown in CDB at different temperatures ranging from

20 and 40˚C at 5°C interval. Maximum production of 44 U/ml was recorded at

25˚C. The temperature above and below 25˚C did not enhance the Laccase

production. Saravanakumar and Kaviyarasan [437] studied the optimal

parameter of maximum laccase enzyme production was found to be pH 8.0,

temperature 25°C, glycerol, vanillin and CuSo4, but the laccase production

was decreased at 30°C [2]. In general, the fungi were cultivated at

temperatures between 25°C and 30°C for optimal laccase production [445,

446, 447 and 61]. When the temperatures higher than 30°C the activity of

ligninolytic enzymes was reduced [442,428].

5.5.1.1.5 Effect of different pH on laccase enzyme production

The effect of various pH on laccase production in YPD-Cu media

shows that maximum laccase enzyme was produced at the pH of 5.5

155

corresponding to the fungal growth range of pH. The enzyme production was

seen between the pH ranges from 5.0 to 7.0. There was no enzyme production

at pH of 9.5 and above and 3.0 and below. The result is given in the Table

5.13 and Figure 5.12.

The pH is one of the important parameter in fungal cultivation. Various

pH (4.0-8.0) of the laccase production medium were adjusted with pH meter.

Optimal pH for maximum laccase production (56.7U/ml) was observed at 5.5

in 17th day of incubation. Exponential increase in laccase activity was

observed from pH 4.0-5.5 but there after laccase production was decreased

with increase in pH. This may be attributed to the fact that change in pH may

alter the three dimensional structure of the enzymes.

The pH of 4.5-5.0 has been shown to be optimal for laccase activity

with marked suppression above 5.5 and below 3.5 [444].The optimal pH for

laccase production was found to be at pH 6.5 in submerged culture of

Chaetomium globosum [448]. The laccase production was 25-fold higher in

Botryosphaeria rhodina showed a significant effect with increased pH of 3.5 -

7.5 for both induced and non-induced cultures [449]. Whereas, the maximum

laccase activity of 42 U/ml, at pH 8.0 in Lentinus sp. than less acidic pH [437].

156

Table-5.13: Effect of pH on laccase enzyme production in YPD-Cu media by

Trametes versicolor

Sl.NO. pH Laccase (U/ml )

1 5.0 1.22± 0.011

2 5.5 1.31± 0.010

3 6.0 1.28± 0.001

4 6.5 1.30± 0.21

5 7.0 1.30± 0.012


Figure 5.12: Effect of pH on laccase enzyme production in YPD-Cu media by

Trametes versicolor

157

The pH showed a significant influence on the production of extra

cellular laccase in the Pleurotus Sp. It produced laccase between the pH of 5.0

to 9.0. Maximum laccase production of 94.3 and 94.0 U/ml was recorded at

pH 5.5 and 7.5 respectively, and the moderate activity was observed in pH 9.0

and then declined. The optimal pH for laccase production was found to be at

pH 6.5 in submerged culture of Chaetomium globosum [448]. The laccase

production was 25-fold higher in Botryosphaeria rhodina showed a significant

effect with increased pH of 3.5 - 7.5 for both induced and non-induced

cultures [450,437].

5.5.1.1.6 Effect of shaking speed on laccase enzyme production

Shaking of the culture medium facilitates better interaction between the

fungi and culture media components thereby leading to better growth and

enzyme production. The effect of shaking speed on laccase enzyme production

shows that maximum laccase enzyme production was noticed when the YPD-

Cu flasks were incubated at 150rpm (Revolutions-Per-Minute) shaking speed

in orbital shaker incubator maintained at the temperature of 30°C, 1.28U/ml of

laccase enzyme was produced by Trametes versicolor; an increase in shaking

speed up to 160rpm did not alter the enzyme yield, but a speed of 200 and

above resulted in poor fungal growth and very less enzyme production due to

media dispersion culture media and fungal mass. The results are shown in the

Table 5.14 and Figure 5.13.

158

Table 5.14: Effect of shaking speed on laccase enzyme production in YPD-Cu

media by Trametes versicolor

Sl.No Shaking Speed (RPM) Laccase enzyme (U/ml)

1 80 0.98 ± 0.031

2 100 1.02 ± 0.002

3 120 1.08 ± 0.014

4 140 1.14 ± 0.032

5 150 1.28 ± 0.011

6 160 1.28 ± 0.001


Figure 5.13: Effect of shaking speed on laccase enzyme production in YPD-

Cu media by Trametes versicolor

159

5.5.1.2 Optimization of Agro waste based media

5.5.1.2.1 Effect of Achras sapota (Sapota) seeds conc. on growth & 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 on Trametes versicolor

were studied by incorporating the various concentrations of dried sapota seed

powder in MS media (Sapota Seed Broth- SSB). The results show that 2% of

Sapota seed powder supported the maximal laccase enzyme production (2.34

U/ml) while further increase in sapota seed concentration doesn’t increased the

enzyme yield further. Table 5.15 and Figure 5.14shows the result.

5.5.1.2.2 Effect of temperature on laccase enzyme production in SSB

The effect of various temperature ranges on laccase enzyme production

in SSB shows a pattern similar to that of semi-synthetic media as 30°C was the

optimum temperature for maximal quantity of laccase enzyme (2.34 U/ml)

production by Trametes versicolor. Table 5.16 and Figure 5.15 shows the

results.

160

Table 5.15: Effect of varying concentration of Achras zapota (Sapota) seeds

on growth and laccase production by Trametes versicolor

Sl.No.Concentration of Sapota seed

powder Laccase (U/ml)

1 0.5% 1.34± 0.0032 1.0% 1.63± 0.0013 1.5% 1.92± 0.0154 2.0% 2.34 ± 0.0025 2.5% 2.34± 0.0136 3.0% 2.34± 0.008


Figure 5.14: Effect of varying concentration of Achras sapota (Sapota) seeds

on growth and laccase production by Trametes versicolor

161

Table-5.16: Effect of temperature on laccase enzyme production by Trametes

versicolor

Sl.No. TemperatureLaccase (U/ml)

SSB

1. 25°C 1.92± 0.054

2. 30°C 2.34 ± 0.003

3. 35°C 1.87± 0.001

4. 40°C 0.78± 0.013

5. 45°C 0.01± 0.001


Figure 5.15: Effect of temperature on laccase enzyme production by Trametes

versicolor

162

5.5.1.2.3 Effect of pH on laccase enzyme production in SSB

The effect of various pH ranges on laccase enzyme production by

Trametes versicolor in SSB shows that 6.0 was the optimum pH which is

slightly (0.5) higher than that of semi-synthetic media. The pH range 5.0 to 7.0

supported more or less same quantity of enzyme. However highly acidic or

basic ranges supported very low yield as with semi-synthetic media. The

enzyme yield at pH 6.0 was 2.34 U/ml. Table 5.17 and Figure 5.16 shows the

results.

5.5.1.2.4 Effect of shaking speed on laccase enzyme production in SSB

Like semi-synthetic media, here also 150 rpm was found to be the best

for maximal laccase enzyme production by Trametes versicolor. The quantity

of laccase enzyme produced at 150rpm is 1.918 U/ml. the results are given in

Table 5.18and Figure 5.17.

5.5.1.3 Effect of Vermiwash on laccase enzyme production

The effect of varying concentrations of vermiwash on laccase enzyme

production by Trametes versicolor showed that 50% concentration of vermiwash

resulted in maximal laccase enzyme production while the higher concentrations

doesn’t increased the yield any further. There was an increase of 0.35 U/ml of

laccase enzyme production in YPD-Cu + 50%media when compared to YPD-Cu

media. Similar comparison was done in SSB, there also the results showed that 50%

concentration of vermiwash supports maximal enzyme production (2.61U/ml), which

is 0.27 U/ml higher than the media without vermi wash. The results are shown in the

Table 5.19 and 5.20 and Figure 5.18&5.19.

163

Table 5.17: Effect of pH on Laccase enzyme production by Trametes

versicolor

SL.NO. pH

Laccaseenzyme

(U/ml)

SSB

1. 5.0 1.88± 0.004

2. 5.5 1.90± 0.031

3. 6.0 2.34± 0.001

4. 6.5 2.12± 0.021

5. 7.0 1.92± 0.034


Figure 5.16: Effect of pH on laccase enzyme production by Trametes

versicolor

164

Table 5.18: Effect of shaking speed on laccase enzyme production

SL.NO.

Shaking

speed

(rpm)

Laccase (U/ml)

SSB

1. 80 1.38± 0.003

2. 100 1.467± 0.021

3. 120 1.80± 0.004

4. 140 1.910± 0.031

5. 150 1.918± 0.001

6. 160 1.916± 0.013


Figure 5.17: Effect of shaking speed on laccase enzyme production

165

Table 5.19: Effect of vermiwash on laccase enzyme production in YPD-Cu


Sl.No. Media Media Composition Laccase activity (U/ml)

1 YPD yeast extract, peptone and Dextrose 1.31± 0.002

2 YPD + VW 1yeast extract, peptone,

Dextrose and 25% vermi wash

1.49± 0.012



1.66± 0.032

4 YPD + VW 3yeast extract, peptone ,


1.66± 0.004



1.66± 0.015


Figure 5.18: Effect of vermiwash on laccase enzyme production in YPD-Cu


166

Table5.20: Effect of vermiwash on laccase enzyme production in SSB media

by Trametes versicolor

Sl.No. Media Media Composition Laccase activity (U/ml)

1 SSB 2% of sapota seed powder 2.34± 0.002

2 SSB + VW 12% of sapota seed powder

and 25% vermi wash2.47± 0.031








Figure 5.19: Effect of vermiwash on laccase enzyme production in SSB


167

5.5.1.3.1 Comparison of laccase enzyme production in vermiwash (50%)

supplemented YPD-Cu and SSB

The comparison of laccase enzyme production in vermiwash (50%)

supplemented YPD-Cu and SSB shows that (Table 5.21 & Figure 5.20) SSB

amended with 50% vermiwash supports maximum (2.61 U/ml) laccase

enzyme.

Based on the results, the optimization of laccase enzyme production

was carried out in SSB by submerged and solid state fermentation using

T.versicolor.

5.5.1.4 Comparison of laccase enzyme production by the submerged and

solid state fermentation using SSB

The production of laccase enzyme was carried out in SSB using

submerged and solid state fermentation under the optimized conditions as

determined by the above methods. It was found the solid state fermentation

yielded better result than that of submerged fermentation. The results are given

in Table 5.22 and Figure 5.21.

Since there was 4.98% increase in the laccase enzyme production under

solid state fermentation when compared to submerged fermentation, SSF

(solid state fermentation) method was followed for further optimization by

response-to- surface methodology under statistical optimization.

Trametes versicolor ATCC200801 was grown on submerged cultures

in potato dextrose broth (PDB) using wheat bran as inducer. After 12 days of

culture, supernatant was filtered and used as crude laccase source for 2,4-d

degradation [451].

168

Table 5.21: comparison of laccase enzyme production in vermiwash (50%)

supplemented YPD-Cu and SSB along with YPD-Cu and SSB by Trametes

versicolor

Media Laccase (U/ml)YPD-Cu 1.31± 0.002YPD-Cu+ Vermiwash (50%) 1.66± 0.032SSB 2.34± 0.002SSB+ vermiwash (50%) 2.61± 0.001


Figure 5.20: Comparison of laccase enzyme production in vermiwash (50%)

supplemented YPD-Cu and SSB along with YPD-Cu and SSB by Trametes

versicolor

169

Table 5.22: Comparative production of laccase enzyme under various

fermentation conditions

Fermentation Method Laccase enzyme Produced (U/ml)

Submerged Fermentation 2.61± 0.013

Solid State Fermentation 2.74± 0.010


Figure 5.21: Comparative production of laccase enzyme under various

fermentation conditions

170

The highest laccase activity (200 U mL-1) was obtained from

Coriolopsis byrsina in the 3rd week of fermentation in medium containing

wheat bran, although a good activity was detected in crude enzyme from P.

sanguineus in the same medium (93U mL-1 after 3rd week) . When rice straw

was used as substrate, laccase production was lower than in wheat bran for all

strains (Lentinus strigellus SXS355 and Lentinus sp SXS48 (Lentinaceae),

Picnoporus sanguineus SXS 4 3 (Polyporaceae) and Phellinus rimosus

SXS47).

The highest laccase activity (200 UmL-1) was obtained from C. byrsina

in the 3rd week of fermentation in medium containing wheat bran, although a

good activity was detected in crude enzyme from P. sanguineus in the same

medium (93 UmL-1after 3rd week). When rice straw was used as substrate,

laccase production was lower than in wheat bran for all strains [452].

An attempt was made to use cyanobacterial biomass of water bloom,

groundnut shell (GNS) and dye effluent as culture medium for laccase enzyme

production by Coriolus versicolor. Laccase production was found to be 10.15

± 2.21 U/ml in the medium containing groundnut shell and cyanobacterial

bloom in a ratio of 9:1 (dry weight basis) in submerged fermentation at initial

pH 5.0 and 28 ± 2 °C temperature [453].

5.5.2Statistical optimization of laccase enzyme production

5.5.2.1 Plackett- Burman design

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

171

in this experiment are shown in the following Table 5.23. The values were

chosen based on the previous study.

Plackett-Burman design evaluated 15 factors in 20 runs with 1 block

and 1 replicate. The design is given in Table 5.24. This is used to evaluate the

selected 15 factors for selecting appropriate factors for media optimization.

Table 5.23: Variables used in the study and their high and low levels

SYMBOL VARIABLES HIGH(+) LOW(-)

ASugarcaneBagasse

2 1

B Cotton seed 2 1

CSapota 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 0.5

I 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

The results for the above run are given in the Table 5.25 which gives

the quantity of laccase enzyme produced for each of the run. From results,

the 5 factors which have maximum T value where selected for the next level

of optimization using Box-Benhken method.Estimated effects and

coefficients for the 20 run PB design are given in the Table 5.26. The

results for analysis of variance (ANOVA) are given in the Table 5.27.

172

Table 5.24: The coded values for the PB design for the 15 factors at two levels [High (+) and Low(-)].

Run A B C D E F G H I J K L M N O1 1 1 1 0.5 0.5 0.5 0.5 0.5 0.002 0 0.001 0.001 0.001 0.001 0.0012 2 2 1 1 1 0.5 0.5 0.5 0.002 0.001 0.001 0.005 0.001 0.002 0.0023 2 2 2 0.5 0.5 1 1 0.5 0.001 0.001 0.001 0.001 0.001 0.001 0.0024 2 1 2 1 0.5 0.5 0.5 0.5 0.001 0.002 0.005 0.001 0.002 0.002 0.0025 2 2 1 0.5 1 1 0.5 1 0.001 0.002 0.001 0.001 0.001 0.002 0.0016 2 1 1 0.5 0.5 1 0.5 1 0.002 0.001 0.005 0.005 0.002 0.001 0.0017 2 1 1 1 1 0.5 1 1 0.002 0.002 0.001 0.001 0.002 0.001 0.0028 2 1 2 1 1 1 0.5 0.5 0.001 0.001 0.001 0.005 0.002 0.001 0.0019 1 2 2 0.5 1 1 0.5 0.5 0.002 0.002 0.005 0.001 0.002 0.001 0.002

10 1 1 2 1 0.5 1 1 0.5 0.002 0.002 0.001 0.005 0.001 0.002 0.00111 1 2 1 1 1 1 1 0.5 0.002 0.001 0.005 0.001 0.002 0.002 0.00112 1 2 1 1 0.5 1 1 1 0.001 0.002 0.001 0.005 0.002 0.001 0.00213 2 1 2 0.5 1 1 1 1 0.002 0.002 0.005 0.005 0.001 0.002 0.00214 1 1 2 0.5 1 0.5 1 1 0.001 0.001 0.001 0.001 0.002 0.002 0.00115 1 1 1 1 0.5 1 0.5 1 0.001 0.001 0.005 0.001 0.001 0.002 0.00216 1 2 2 1 1 0.5 0.5 1 0.001 0.002 0.005 0.005 0.001 0.001 0.00117 2 2 1 0.5 0.5 0.5 1 0.5 0.001 0.002 0.005 0.005 0.002 0.002 0.00118 1 2 2 0.5 0.5 0.5 0.5 1 0.002 0.001 0.001 0.005 0.002 0.002 0.00219 1 1 1 0.5 1 0.5 1 0.5 0.001 0.001 0.005 0.005 0.001 0.001 0.00220 2 2 2 1 0.5 0.5 1 1 0.002 0.001 0.005 0.001 0.001 0.001 0.001

173

Table 5.25: Results of the 20 run of the PB design

Run Result

1 1.21 ± 0.003

2 1.98 ± 0.021

3 2.1 ± 0.033

4 2.2 ± 0.004

5 3.6 ± 0.002

6 1.76 ± 0.003

7 1.23 ± 0.001

8 2.18 ± 0.002

9 1.92 ± 0.012

10 2.33 ± 0.023

11 1.37 ± 0.019

12 0.98 ± 0.002

13 1.56 ± 0.001

14 3.24 ± 0.027

15 2.91 ± 0.022

16 1.9 ± 0.001

17 1.28 ± 0.017

18 2.88 ± 0.047

19 1.89 ± 0.026

20 1.74 ± 0.007


174

Table5.26: Estimated effects and coefficients for the 20 run PB design

175

Table 5.27: ANOVA test for the 20 parameters of PB design

176

5.5.2.2 Box-Benhken design

The five factors which were selected includes the following with new

codes (Table 5.28)

Table5.28: Selected variables and their codes

A 46 run Box-Benhken Design (with 6 center points and 1 block and 1

replicate) was used to evaluate the above 5 factors (at three levels, -1, 0, +1)

to find out their optimum values which supports the highest quantity of

laccase enzyme production. The results for the 46 run are given in the

Table-5.29. 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 (Table 5.30) 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 (Table 5.31 and Figures 5.22.A-I).

Variable Code

Sapota Seed Powder A

Yeast Extract B

Glucose C

CuSO4 D

ZnSO4 E

177

Table 5.29: 3 levels of the variables taken for optimization by BB

design

Variables3 levels of the 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

Table 5.30: Hold values for the 5 variables used in Box-Benhken

design

Symbol Component Quantity

(g/100ml)

A Sapota Seed Powder 1.97

B Yeast Extract 0.9

C Glucose 1.1

D CuSO4 0.025

E ZnSO4 0.008

178

Table 5.31: Result of the 46 run BB design for the 5 variables

RunOrder RESULT

1 0.91 ± 0.0012 0.94 ± 0.0213 0.65 ± 0.0164 0.57 ± 0.0245 0.59 ± 0.0316 1.31 ± 0.1327 0.87 ± 0.0088 0.76 ± 0.0139 0.66 ± 0.120

10 0.98 ± 0.02311 0.59 ± 0.11412 0.54 ± 02313 0.60 ± 0.11514 0.45 ± 0.00615 0.51 ± 0.12416 0.62 ± 0.00917 0.56 ± 0.05318 1.43 ± 0.01219 0.89 ± 0.00520 0.86 ± 0.05721 0.78 ± 0.01122 0.66 ± 0.01823 0.71 ± 0.011

RunOrder RESULT

24 0.44 ± 0.02125 0.98 ± 0.00426 0.66 ± 0.03127 0.76 ± 0.03428 0.71 ± 0.00629 0.38 ± 0.01530 0.56 ± 0.01131 1.44 ± 0.05132 0.62 ± 0.00833 0.64 ± 0.03334 0.66 ± 0.00335 0.28 ± 0.01936 0.72 ± 0.01137 1.37 ± 0.02838 0.68 ± 0.06139 0.75 ± 0.04240 0.60 ± 0.06141 0.48 ± 0.00542 0.56 ± 0.01043 0.55 ± 0.00344 0.61 ± 0.02145 1.22 ± 0.00646 0.56 ± 0.016


179

sapota seed extract

Glu

cose

1.00.50.0-0.5-1.0

1.0

0.5

0.0

-0.5

-1.0

B 0.5D 0.002E 0.002

Hold Values

> – – < 2.0

2.0 2.22.2 2.4

2.4

C10

contour plots for sapota seed extract vs glucose in BB desogn

Figure 5.22 A: Contour plot for the variables sapota seed extract Vs

glucose in the BB design

Sapota seed extract

Co

pp

er

sulp

ha

te

1.00.50.0-0.5-1.0

1.0

0.5

0.0

-0.5

-1.0

B 0.5C 1E 0.002

Hold Values

> – – < 1.8

1.8 2.02.0 2.2

2.2

C10

contour plots for sapota seed extract vs copper sulphate in BB desogn

Figure 5. 22 B: Contour plot for the variable sapota seed extract Vs CuSo4

in the BB design

180

sapota seed extract

ZnS

o4

1.00.50.0-0.5-1.0

1.0

0.5

0.0

-0.5

-1.0

B 0.5C 1D 0.002

Hold Values

> – – < 1.8

1.8 2.02.0 2.2

2.2

C10

contour plots for sapota seed extract Vs ZnSo4 in BB desogn

Figure 5. 22 C: Contour plot for the variable sapota seed extract Vs ZnSo4

in the BB design

Yeast extract

Glu

cose

1.00.50.0-0.5-1.0

1.0

0.5

0.0

-0.5

-1.0

A 1D 0.002E 0.002

Hold Values

> – – < 2.0

2.0 2.22.2 2.4

2.4

C10

contour plots for yeast extract vs Glucosein BB desogn

Figure 5. 22 D: Contour plot for the variables yeast extract Vs glucose for

BB design

181

yeast extract

Cu

So

4

1.00.50.0-0.5-1.0

1.0

0.5

0.0

-0.5

-1.0

A 1C 1E 0.002

Hold Values

> – – < 2.0

2.0 2.22.2 2.4

2.4

C10

contour plots for yeast extract vs CuSo4 in BB desogn

Figure 5.22 E: Contour plot for the variables yeast extract Vs CuSo4 for the

BB design

Figure5.22 F: Contour plot for the variables yeast extract Vs ZnSo4 for the

BB design

yeast extract

ZnS

o4

1.00.50.0-0.5-1.0

1.0

0.5

0.0

-0.5

-1.0

A 1C 1D 0.002

Hold Values

> – – < 2.0

2.0 2.22.2 2.4

2.4

C10

contour plots for yeast extract vs ZnSo4 in BB desogn

182

Glucose

Cu

So

4

1.00.50.0-0.5-1.0

1.0

0.5

0.0

-0.5

-1.0

A 1B 0.5E 0.002

Hold Values

> – – < 2.10

2.10 2.252.25 2.40

2.40

C10

contour plots for Glucose vs CuSo4 in BB desogn

Figure 5. 22 G: Contour plot for the variables glucose Vs CuSo4 in the BB

design

Glucose

ZnS

o4

1.00.50.0-0.5-1.0

1.0

0.5

0.0

-0.5

-1.0

A 1B 0.5D 0.002

Hold Values

> – – < 2.10

2.10 2.252.25 2.40

2.40

C10

contour plots for Glucose vs ZnSo4 in BB desogn

Figure 5. 22 H: Contour plot for the variables glucose Vs ZnSo4 in the BB

design

183

CuSo4

ZnS

o4

1.00.50.0-0.5-1.0

1.0

0.5

0.0

-0.5

-1.0

A 1B 0.5C 1

Hold Values

> – – < 2.10

2.10 2.252.25 2.40

2.40

C10

Contour plots for CuSo4 vs ZnSo4 in BB desogn

Figure 5.22 I: Contour plot for the variables CuSo4 Vs ZnSo4 in the BB

design

The estimated regression coefficients of responses are given in the

Table 5.32 and the results of ANOVa are given in Table 5.33.

The value of “R” (correlation coefficient) for the production of

laccase is 0.9730 which indicates a good aggrement between experimental

and predicted values. The corresponding analysis of variance (ANOVA) is

presented in the table. The F- value is measure of variation of the data about

the mean. Generally, the calculated F value should be several times greater

than the tabulated value, if the model is a good prediction of their

experimental results and the estimated factors effects are real [453]. Also

the high F-value and a very low probability (P>F = 0.0001) indicates that

the present model is in a good prediction of the experimental results [454].

The p-value serves as a tool for checking the significance of each of the

coefficients. The pattern of interaction between the variables is indicated by

these coefficients.

184

Table 5.32 : Response Surface Regression: Results versus Variables (A, B,

C, D & E) The analysis was done using coded units. Estimated Regression

Coefficients for Result.

Term Coef SE Coef T P

Constant 2.47000 0.06065 40.725 0.000

A 0.30188 0.03714 8.128 0.000

B -0.03500 0.03714 -0.942 0.355

C 0.12875 0.03714 3.467 0.002

D -0.02438 0.03714 -0.656 0.518

E 0.08375 0.03714 2.255 0.033

A*A -0.27354 0.05029 -5.439 0.000

B*B -0.10604 0.05029 -2.109 0.045

C*C -0.27271 0.05029 -5.423 0.000

D*D -0.15188 0.05029 -3.020 0.006

E*E -0.08771 0.05029 -1.744 0.093

A*B -0.03250 0.07428 -0.438 0.665

A*C 0.06250 0.07428 0.841 0.408

A*D -0.01750 0.07428 -0.236 0.816

A*E 0.01000 0.07428 0.135 0.894

B*C -0.32250 0.07428 -4.342 0.000

B*D -0.00000 0.07428 -0.000 1.000

B*E 0.16000 0.07428 2.154 0.041

C*D -0.15250 0.07428 -2.053 0.051

C*E -0.05250 0.07428 -0.707 0.486

D*E -0.15750 0.07428 -2.120 0.044

185

Table 5.33: Analysis of Variance for the Result of the BB design for

evaluating 5 variables

The variables with low probability levels contribute to the model,

whereas the others can be neglected and eliminated from the model. Values

of prob >F less than 0.0500 indicates model terms are significant. In the

present study terms are significant model terms [455].High F-values and

non significant lack of fit indicated that model was a good fit.

Source DF Seq SS Adj SS Adj MS F PRegression 20 3.65713 3.65713 0.18286 8.29 0.000

Linear 5 1.86461 1.86461 0.37292 16.90 0.000

A 1 1.45806 1.45806 1.45806 66.06 0.000

B 1 0.01960 0.01960 0.01960 0.89 0.355

C 1 0.26523 0.26523 0.26523 12.02 0.002

D 1 0.00951 0.00951 0.00951 0.43 0.518

E 1 0.11222 0.11222 0.11222 5.08 0.033

Square 5 1.04934 1.04934 0.20987 9.51 0.000

A*A 1 0.34107 0.65302 0.65302 29.59 0.000

B*B 1 0.00249 0.09814 0.09814 4.45 0.045

C*C 1 0.48428 0.64905 0.64905 29.41 0.000

D*D 1 0.15437 0.20130 0.20130 9.12 0.006

E*E 1 0.06714 0.06714 0.06714 3.04 0.093

Interaction 10 0.74318 0.74318 0.07432 3.37 0.007

A*B 1 0.00422 0.00422 0.00422 0.19 0.665

A*C 1 0.01562 0.01562 0.01562 0.71 0.408

A*D 1 0.00123 0.00123 0.00123 0.06 0.816

A*E 1 0.00040 0.00040 0.00040 0.02 0.894

B*C 1 0.41602 0.41602 0.41602 18.85 0.000

B*D 1 0.00000 0.00000 0.00000 0.00 1.000

B*E 1 0.10240 0.10240 0.10240 4.64 0.041

C*D 1 0.09303 0.09303 0.09303 4.21 0.051

C*E 1 0.01103 0.01103 0.01103 0.50 0.486

D*E 1 0.09923 0.09923 0.09923 4.50 0.044

Residual Error 25 0.55177 0.55177 0.02207

Lack-of-Fit 20 0.55177 0.55177 0.02759 * *

Pure Error 5 0.00000 0.00000 0.00000

Total 45 4.20890

186

From the optimum values of the five factors obtained from the BB

analysis, a media was formulated and was inoculated with T.versicolor and

incubated for 2 weeks at 30°C. The laccase enzyme was extracted and

separated and was assayed by Bourbanis and paice method [7]. The results

showed an increase of 102% in laccase enzyme production. Maximum yield

was 2.94U/ml and was obtained in 14 days of incubation.

A response surface methodology allowed calculation of maximum

production based on a few sets of experiments in which all the factors were

varied within chosen ranges. This method has been successfully applied in

the optimization of medium compositions [456] conditions of enzymatic

hydrolysis [457] and fermentation processes [458] and Sonia et al., [459].

In optimizing the effect of carbon and nitrogen sources on laccase

production from Pleurotus sp., 4 factorial designs were applied using

Mannitol, peptone, pH and metal ion. Box-Behnken design aims to select

most important variables in the system that influence over all enzyme

productivity. Each variable varies for a desired response represented at high

and low levels. Generally calculated F values should be several times more

than tabulated value, if the model was a good prediction of experimental

results and estimated factors effects are real. Also high F value and a very

low probability (P> F = 0.0001) indicate that present model is in a good

prediction of experimental results. The F value of model implies that model

was significant [376]. The goodness of fit was checked by determination

coefficient (R2), and value of the determination coefficient (R2 = 89.5%)

indicates that only 10.5% was not explained by the model. The adjusted

coefficient (Adj. R2 = 75 %) was also very high, which indicates a high

significance of the experiment [460, 461, 376, 277]. Box et al., [275]

reported a higher value of the correlation coefficient (R = 98.1%) signifies

an excellent correlation between the independent variables [460,461.376].

The application of Response Surface Methodology [460, 277] was yielded

higher enzyme production [437].

187

5.5.3. Comparison of laccase enzyme production under optimized and

un-optimized conditions in SSB media

The results of the comparative study among optimized and un-

optimized conditions for laccase enzyme production, it was found that there

was considerable increase in laccase enzyme production on all the

incubation days under optimized condition than un-optimized condition.

The results are given in Table 5.34 and Figure 5.23 (a & b).

Table 5.34: Quantity of laccase produced in Optimized conditions

Day(s)Laccase U/ml

Normal Optimized

3 0.32 ± 0.002 0.46± 0.012

5 0.71± 0.015 0.92± 0.022

7 1.06± 0.011 1.48± 0.001

9 1.92 ± 0.001 1.86± 0.032

11 2.41 ± 0.011 2.61± 0.021

14 2.74 ± 0.001 2.94± 0.001


188

Figure 5.23a: Comparison of laccase enzyme production in normal and

optimized conditions

Figure 5.23b: comparison of laccase enzyme production in normal

and optimized conditions

189

5. 6 Production of laccase enzyme

5. 6.1 Production of laccase enzyme by solid state fermentation

Laccase enzyme production was carried out in SSB with the media

optimized by RSM method. The result shows that the maximum laccase

enzyme produced is 2.94 U/ml. The enzyme was separated and was

subjected for purification. Figure 5.24 shows the conical flask, having fully

grown Trametes versicolor, after the incubation period on a SSB media

having vermi wash.

Figure 5.24: Laccase enzyme production in SSF using the media optimized

by RSM design by Trametes versicolor

Most studies on laccase production have been performed in liquid

cultures, which do not reflect the natural living conditions of ligninolytic

fungi (wood). Solid-state fermentation (SSF) is defined as any fermentation

process occurring in absence or near absence of free liquid, employing an

inert substrate or a natural substrate as a solid support [219]. The former

only functions as an attachment place for the microorganism, whereas the

190

latter also acts as a carbon source, reducing considerably the production

costs [462]. The selection of an adequate support for performing SSF is

essential, since the success of the process depends on it. The most important

factors to take into account are particle size, porosity and chemical

composition. In addition to this, availability and cost are also criteria of

great importance.

5.7 Purification and characterization of laccase enzyme

5.7.1 Ammonium sulphate precipitation

The crude extract was precipitated using Ammonium sulphate and

was subjected for dialysis using cellulose nitrate membrane and then was

separated by column chromatography and finally was purified by native

PAGE. The molecular mass was determined by SDS-PAGE and was found

to be 55 kDa. The results are depicted in the Table 5.35 and Figures 5.25.

Table 5.35: Purification steps for the laccase enzyme produced by Trametes

versicolor using SSB media under solid state fermentation

Purification step

TotalActivity(Units)

Totalyield (%)

Specificactivity(U/mg)

Purification fold

Crude Culture filtrate

2.4 100 0.01 1

AmmoniumSulphate

precipitation26.7 122 0.03 2.51

Dialysis 28.8 134 0.04 3.05Column

chromatography7.37 44 2.36 179

Native page 1.46 14 0.71 53.6

191

Figure 5.25: SDS-PAGE of the laccase enzyme from Trametes versicolor.

Lane-1 is the markers; Lane -2 & 3 is the laccase enzyme. The gel is stained

with guaiacol.

The laccase secreted by T. versicolor 951022 was purified to

homogeneity, and the purified laccase protein appeared to be a monomeric

protein similar to other fungal laccases [463, 464 and 401]. It did not have

any isozyme and the molecular mass of denatured laccase was estimated to

be 97 kDa by SDS-PAGE analysis. The molecular mass of this laccase is

larger than other laccases from the same or related species of Trametes or

other fungi, which range 61 ~ 81 kDa [463, 464, 401, 290, 168 and

465].The high molecular mass of the protein might lower the recovery rate

of this enzyme, especially at the final stage of purification. Many laccases

contain some carbohydrate [466, 160], and the high molecular weight of

laccase in this study may be partly due to the presence of carbohydrate.

Measurement of carbohydrate content should be carried out in a further

study. T. versicolor 951022 have a very high specific activity of 91,443

192

U/mg with ABTS as a substrate, although specific activity can be different

with the substrate used and the conditions of assay. Furthermore, units of

enzyme activity in each study may be defined differently. In spite of the

differences in the conditions of each enzyme assay, the specific activity in

this study is much higher than those in other studies using the same

substrate ABTS, which showed activities between 152 ~ 610 U/mg [160,

290, 467, 464, 468, 296].

Two peaks of laccase activity which were detected in culture media

of T. trogii [469] were probably owing to the specific induction of both

laccase isoforms secreted by the fungus. Patterns after PAGE of T. trogii

laccase isoenzymes were similar in media with different C/N ratio and Cu-

concentrations. Likewise, identical laccase isoforms were consistently seen

in cultures of Ganodermalucidum grown in low N synthetic medium, malt

extract or wood [397]. The molecular masses (38 and 60 kDa) reported in

this study for T.trogii laccases, are in the range observed for laccases

isolated from other white rot fungi [397, 2]. Two main laccases (67 and 70

kDa) were purified from T. versicolor [211]. Two to three laccase isoforms

with molecular masses 64to70 kDa were described in T. gibbosa, T. hirsuta,

a different strain of T. trogii [406] and T. hispida [327].

5.7.2 Temperature maxima for laccase activity

The optimum temperature for the laccase was determined by

measuring the enzyme activity at various temperatures ranging from 20oC

to 90oC in a 100 mM sodium acetate buffer (pH 5.0). After incubation, the

remaining activity was determined. The thermal stability of the enzyme was

also determined by incubating the enzyme at pH 5.0 for 1 h. The enzyme

remained stable up to 40oC, yet the stability decreased rapidly above 40oC,

which was similar to the results previously reported for the laccases from L.

edodes and A. blazei . The results are given in Table 5.36 and Figure 5.26.

193

Table 5.36: Effect of temperature on laccase enzyme stability

Sl.No.Laccase

(ml)

Substrate

ABTS

(ml)

Tempe

rature

(°C)

1%SDS

(ml)

Absorbance

(OD)

1 0.1 0.1 20 0.1 0.949 ±0.0012 0.1 0.1 25 0.1 0.964 ±0.0023 0.1 0.1 30 0.1 0.988 ±0.0034 0.1 0.1 35 0.1 0.986 ±0.0015 0.1 0.1 40 0.1 0.984 ±0.0126 0.1 0.1 45 0.1 0.770 ±0.0117 0.1 0.1 50 0.1 0.102 ±0.0138 0.1 0.1 55 0.1 0.008 ±0.0019 0.1 0.1 60 0.1 0.001 ±0.00110 0.1 0.1 65 0.1 0.000 ±0.0011 0.1 0.1 70 0.1 0.000 ±0.0012 0.1 0.1 75 0.1 0.000 ±0.0013 0.1 0.1 80 0.1 0.000 ±0.0014 0.1 0.1 85 0.1 0.000 ±0.0015 0.1 0.1 90 0.1 0.000 ±0.00


Figure 5.26: Effect of temperature on laccase enzyme stability

0

0.2

0.4

0.6

0.8

1

1.2

20 25 30 35 40 45 50 55 60 65 70 75 80 85 90

Abs

orba

nce

(OD

)

Temperature in° C

194

The optimum temperature of the laccase for ABTS oxidation was

70oC, which was much higher than the optimum temperatures previously

reported for other fungal laccases, ranging from 40oC to 50oC. The thermal

stability of the enzyme was also determined by incubating the enzyme at

pH 3.0 for 1 h. The enzyme remained stable up to 40oC, yet the stability

decreased rapidly above 40°C, which was similar to the results previously

reported for the laccases from L. edodes and A. blazei. However, the

enzyme was less stable than the laccases from P. ribis and Trametes sp.

strain AH28-2, which remained stable at 55oC and 70oC for more than 1 h,

respectively.

5.7.3 pH optima for laccase enzyme

The effect of pH on the enzyme activity was investigated at pH

values ranging from 3.5 to 10.0 with ABTS as the substrate. The optimum

pH for the enzyme was identified as 5.0, which was consistent with the

optimum pH for the laccase isozyme fromCorioles subvermispora and

enzyme from C. hirsutus. Other studies have also reported very low optimal

pH’s (between 3.0 and 5.7) for fungal laccases, except for the laccase from

Rhizoctonia praticola, which exhibited a neutral optimal pH with various

substrates. The results are shown in Table 5.37 and Figure 5.27.

The optimum pH for the laccase was estimated using ABTS as the

substrate in a 100 mM sodium citrate buffer (pH 3.5-6.0) and 100 mM

sodium phosphate buffer (pH 6.5-10.0). The effect of pH on the enzyme

stability was measured after 1 h of incubation at various pH’s at 25oC.

195

Table 5.37: Effect of pH on laccase enzyme stability

Sl.No. Laccase(ml)

SubstrateABTS (ml)

pH 1%SDS (ml)

Absorbance (OD)

1 0.1 0.1 3.5 0.1 0.101 ±0.0012 0.1 0.1 4.0 0.1 0.386 ±0.0123 0.1 0.1 4.5 0.1 0.768 ±0.0214 0.1 0.1 5.0 0.1 0.988 ±0.0035 0.1 0.1 5.5 0.1 0.986 ±0.0116 0.1 0.1 6.0 0.1 0.977 ±0.0127 0.1 0.1 6.5 0.1 0.946 ±0.0048 0.1 0.1 7.0 0.1 0.922 ±0.0329 0.1 0.1 7.5 0.1 0.901 ±0.02710 0.1 0.1 8.0 0.1 0.864 ±0.02011 0.1 0.1 8.5 0.1 0.823± 0.01712 0.1 0.1 9.0 0.1 0.654± 0.00213 0.1 0.1 9.5 0.1 0.154 ±0.01014 0.1 0.1 10.0 0.1 0.000 ±0.001


Figure 5.27: Effect of pH on laccase enzyme stability

0

0.2

0.4

0.6

0.8

1

1.2

3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10

Abs

orba

nce

(OD

)

pH

196

The effect of pH on the enzyme activity was investigated at pH

values ranging from 2.5 to 8.0 with ABTS as the substrate. The optimum

pH for the enzyme was identified as 3.0, which was consistent with the

optimum pH for the laccase isozyme from C. subvermispora and enzyme

from C. hirsutus. Other studies have also reported very low optimal pH’s

(between 3.0 and 5.7) for fungal laccases, except for the laccase from

Rhizoctonia praticola, which exhibited a neutral optimal pH with various

substrates. When the effect of pH on the enzyme stability was examined at

25oC for 1 h, the enzyme remained stable within an acidic pH range from

3.0 to 5.0.

5.8 Applications of laccase enzyme

5.8.1 Bio- decolourization of Azo dyes

5.8.1.1 Screening of Azo dye decolourizing fungi

Following incubation at room temperature for 2 weeks in Acid

orange-7 containing media, it was found that Phanerochaete chrysosporium

was the best among the five isolates. It was used for further studies in

decolourization experiment.

Decolourization activity of Phanerochaete chrysosporium for three

synthetic dyes viz., congo red, malachite green and crystal violet were

demonstrated by Deepak Pant et al., [470]. About 100 % decolorization

was achieved with P. chrysosporium RP78 after 24 h for two most widely

used groups of azo dyes in textile industry consisting reactive and acidic.

The physical adsorption of the azo dyes by mycelia was not significant

which indicated that the enzymatic degradation of the dyes was occurred

[471]. P. chrysosporium was able to transform the three azo dyes viz., Acid

197

orange-1,2 and 12. The decolorized Acid Orange-12 and Orange-II more

effectively than Orange-I M. B. Pasti-Grigsby et al., [104, 105].

5.8.1.2 Bio-decolourization in solid media

The SDA plates were observed for fungal growth and

decolourization periodically. The decolourization was seen up-to the

concentration of 1.0 % of azo dye in the SDA media.The results are shown

in Figure5.28. The maximum fungal growth and decolourization was best in

the initial concentration (0.01% of dye) and gradually decreased as the

concentration increases. The growth and decolourization at 1% dye

concentration indicates that the dye is not toxic to P.chrysosporium.

Figure 5.28: Biodecolourization of varying concentrations of acid orange-7

in SDA plates by P.chrysosporium. The fungal growth and decolourization

is evident in the photograph.

198

There was no fungal study available for diodegradation of Acid

orange-7 in solid media. However some bacterial species like

Enterobactersp., Pseudomonas sp. and Morganella sp.were shown to grow

on solid media containing acid orange-7 as the sole carbon and energy

source [472].

5.8.1.3 Bio-decolourization in liquid media

SDB flasks containing 0.01% to 1.0% concentration were

periodically observed for decolourization and the decolourization

percentage was calculated. The results showed that decolourization could

be seen up to 1% dye concentration. Here the growth and decolourizaion

was more prominent than solid media, the reason being the interaction of

dye and enzyme is more prominent in liquid media. Maximum

decolourization was seen with 0.01% dye concentration (94.4%) while the

minimum decolourization was with 1.0% dye concentration (68.4%). The

results are given in the Table5.38 and Figure 5.29, 5.29a and 5.29b.

5.8.1.4 Bio-decolourization in mineral salt media

To study on the ability P.chrysosporium to use acid orange-7 dyes as

sole source of carbon and nitrogen, Mineral Salt Medium (MS Medium)

was used and the results showed that the decolourization could take place

up to the concentration of 0.1%, indicating the ability of the fungi to use

acid orange-7 as sole carbon and energy source (Table 5.39). However the

decolourization percentage was very low when compared to SDA or SDB.

Blanca E. Barragan et al.,[472] discussed the ability of

microorganisms to use acid orange-7 as sole carbon and energy source.

199

Table 5. 38: Decolourization of Acid orange-7 in SDB by P.chrysosporium

S.No. ConcentrationDecolourization

(%)

1. 0.01 94.4± 0.010

2. 0.05 91.8± 0.011

3. 0.1 89± 0.001

4. 0.5 84.1± 0.012

5. 0.76 74.6± 0.023

6. 1.0 68.4± 0.002


Figure 5.29: Decolourization of Acid orange-7 in SDB by P.chrysosporium

200

Figure 5.29a: Control vials havingvarying concentrations of Acid orange-7

in SDB for Decolourization by P.chrysosporium

201

Figure 5.29b: Decolourization of Acid orange-7 in SDB by

P.chrysosporium

Table 5.39: Comparison of Acid orange-7 decolourization by

P.chrysosporium in SDA,SDB and MS media

S.No. Concentration

Decolourization (%) in different culture

media

SDA SDB MS

1. 0.01 +++ 94.4 16.6

2. 0.05 +++ 91.8 14.2

3. 0.1 ++ 68.4 8.3

4. 0.5 ++ 84.1 -

5. 0.76 ++ 74.6 -

6. 1.0 + 68.4 -

+++ High biomass and decolourization

++ Less biomass and decolourization

+ Least biomass and decolourization

- No growth and no decolourization

5.8.1.5 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. Of the various carbon

sources and nitrogen sources, glucose and peptone (76.2 % and 68%

respectively) showed the highest percentage of decolourization. The

maximum decolourization was seen at 30°C temperature (95.15%) and at

the pH of 5.5 (89.9%). Using the best carbon, nitrogen sources and

optimum temperature and pH the bio-decolourization was carried out in

202

batch culture and it gave 98.95% decolourization, which was the maximum

decolourization obtained in this study. All the above results depicted in the

figures and Tables 5.40 to 44 and Figures 5.30 to 5.34.

The effect of added carbon and nitrogen sources on the azo dye

decolourization by microbes were studied by Sofia Nosheen et al., [473].

Deepak Pant et al.,[470] proved that maximum decolourizing capacity of

P.chrysosporium was observed up to 15 ppm. The addition of urea as

nitrogen source and glucose as carbon source significantly enhanced

decolourizing capacity (up to 87%). In all the cases, both colour and COD

were reduced more in non-sterilized treatments as compared to sterilized

ones. Significant reductions in COD content of dye solutions (79- 84%)

were recorded by fungus supplied with additional carbon and nitrogen. A

highly significant correlation (r = 0.78, p<0.001) between colour and COD

of dye solutions was recorded. Thus, a readily available carbon and nitrogen

source is imperative to enhance the bioremediation activity of this fungus

which has been the most suitable for synthetic dyes and textile industry

wastewater treatment.

203

Table 5.40: Effect of different carbon sources on the decolourization of

Acid orange-7 byP.chrysosporium


Figure 5.30: Effect of different carbon sources on the decolourization of

Acid orange-7 by P.chrysosporium

Sl.No.Conc. Of

Dye(gm)

Sources of

Carbohydrate

(1%)

Degradation

(%)

1. 0.01 Glucose 76.2± 0.010

2. 0.01 Mannitol 62.5± 0.001

3. 0.01 Maltose 65.2± 0.230

4. 0.01 Mannose 54.0± 0.021

5. 0.01 Sucrose 52.8± 0.050

6. 0.01 Xylose 39.9± 0.071

204

Table 5.41: Effect of different nitrogen sources on the decolourization of

Acid orange -7 by P.chrysosporium

Sl.No.

Conc. of

Dye

(gm)

Sources of

Nitrogen (0.5%)

Degradation

(%)

1. 0.01 Peptone 68.0± 0.011

2. 0.01 Tryptone 64.3± 0.021

3. 0.01 Yeast extract 59.0± 0.003

4. 0.01 Beef extract 38.0± 0.034


Figure 5.31: Effect of different nitrogen sources on the decolourization of

acid orange-7 by P.chrysosporium

205

Table 5.42: Effect of various temperatures on the decolourization of acid

orange-7 in SDB by P.chrysosporium

Sl.No. Temperature Degradation (%)

1 25°C 86.8± 0.012

2 30°C 95.1± 0.002

3 35°C 91.3± 0.012

4 40°C 88.9± 0.034

5 45°C 89.7± 0.023


Figure 5.32: Effect of various temperatures on the decolourization of acid

orange-7 by P.chrysosporium

206

Table 5.43: Effect of pH on the decolourization of acid orange-7 in SDB by

P.chrysosporium


Figure 5.33: Effect of pH on decolourization of acid orange-7 in SDB by

P.chrysosporium

Sl.No. pH Degradation (%)

1 5.5 89.9± 0.021

2 6.0 86± 0.022

3 6.5 87.6± 0.003

4 7.0 88.2± 0.034

5 7.5 85.2± 0.021

207

Table 5.44: Decolourization of Acid orange -7 in SDB by P.chrysosporium

under optimized conditions

S.No.Concentration

[%]

Decolourization

(%)

1. 0.01 98.9± 0.023

2. 0.05 96.2± 0.003

3. 0.1 92.3± 0.001

4. 0.5 87.4± 0.025

5. 0.76 81.2± 0.034

6. 1.0 75.4± 0.004


Figure 5.34: Decolourization of Acid orange-7 in SDB by P.chrysosporium

under optimized conditions

208

5.8.1.6 Comparative decolourization of Acid orange-7 under optimized

and un-optimized conditions

The study on the decolourization of acid orange-7 under optimized

and un-optimized conditions shows marked increase in the decolourization

percentage. The maximum decolourization was seen with 0.01% dye

concentration (98.9%). The percentage of decolourization was better for all

concentrations of the dye when compared to decolourization carried out

under un-optimized condition. The results are given in Table 5.45 and

Figure 5.35.

5.8 .1.7 Tests for absence of Re-colourization

The flasks remained colourless even after 8 weeks of incubation at

room temperature indicating that there was no re-colourization of the

decolourized acid orange-7. The results are given in the Figure 5.36.

Table 5.45: Comparison of Acid orange-7 by P.chrysosporium under un-

optimized and optimized conditions

S.No.

Acid orange -7

concentrations

(%)

Decolourization (%)

Un optimized

condition

Optimized

condition

1. 0.01 94.4± 0.032 98.9± 0.005

2. 0.05 91.8± 0.021 96.2± 0.054

3. 0.1 89± 0.002 92.3± 0.043

4. 0.5 84.1± 0.034 87.4± 0.003

5. 0.76 74.6± 0.071 81.2± 0.001

6. 1.0 68.4± 0.0.008 75.4± 0.023


209

Figure 5.35:Comparative decolourization of acid orange-7 under normal

and optimized conditions by P.chrysosporium

Figure 36: Test for Re-colourization. The vial on left hand side is the

control and the vial on the right hand side is the test one. The test vial

shows the absences of recolourization.

210

5.8.1.8 Biodecolourization of Acid orange-7 by immobilized laccase

enzyme

The immobilized beads were used for the decolourization study. The

immobilized enzyme beads (Figure 5.37) were added to flaks having

varying concentrations of acid orange-7 (from0.01% to 1.0%) and were

incubated at 25°C for 4 hours and were observed for decolourization at the

interval of 20 minutes. The results show a pattern similar to that of

decolourization in the SDB media with free enzyme; however the

decolourization was slightly lower when compared to free enzyme, the

reason being the diffusion problems faced by the immobilized enzyme.

Ghasemi and Tabandeh [471] showed that the physical adsorption of

the azo dyes by mycelia was not significant which indicated that the

enzymatic degradation of the dyes was occurred. The laccase enzyme

treated azo dyes showed varying results. Acid Orange-52 and Direct Blue-

71 showed the highest decolorization rates (more than 50% decolorization

after 2 h) at the absorption maximum in the visual region. Reactive Orange-

16 and Reactive Orange-107 showed no decolorization. Formation of bands

at 320 and 350 nm was found in the cases of Acid Orange-5 and Acid

Orange-52, respectively. Acid Orange-52 was degraded twice as fast as

Acid Orange-5 by the laccase from T. modesta. Various textile dyes have

been decolorized by a Trametes hirsuta laccase to an extent of 80% [474].

5.8.1.9Comparison of Acid orange-7 decolourization by free and

immobilized laccase enzyme

The comparison of bio-decolourization of acid orange-7 by the free

and immobilized laccase enzyme shows that the free enzyme had a better

percentage of decolourization; this is due to difficulty in permeability of the

211

Figure 5.37: Immobilized laccase enzyme beads

dye across the immobilized layer and to reach the entrapped enzyme. The

results are given in Table 5.46 and Figure 5.38.

5.8.1.10 Effect of Incubation time of Acid orange-7 decolourization by

immobilized laccase enzyme

The effect of various incubation times (in Minutes) shows that the

maximum decolourization by the immobilized laccase enzyme is 60 min.

and there was no change in the decolourization percentage with increase in

incubation time. The results are given in Table 5.47 and Figure 5.39.

212

Table 5.46: Comparison of acid orange-7 decolourization by laccase

enzyme in free and immobilized state

S.No.

Acid orange-7

concentration

(%)

Decolourization (%)

Free enzymeImmobilized

enzyme

1. 0.01 98.9± 0.005 94.7± 0.001

2. 0.05 96.2± 0.01 90.4± 0.003

3. 0.1 92.3± 0.021 87.3± 0.022

4. 0.5 87.4± 0.022 80.1± 0.003

5. 0.76 81.2± 0.002 72.6± 0.004

6. 1.0 75.4± 0.30 66.9± 0.045


Figure 5.37: Comparison of acid orange-7 decolourization by laccase

enzyme in free and immobilized state

213

Table 5.47: Effect of incubation time on decolourization of acid orange-7

by immobilized laccase enzyme

Sl.No. Time(in minutes)Percentage

decolourization (%)

1 20 83.5± 0.004

2 40 91.2± 0.032

3 60 94.7± 0.017

4 80 94.7± 0.006

5 100 94.7± 0.044

6 120 94.7± 0.011


Table 5.38: Effect of incubation time on decolourization of acid orange-7

by immobilized laccase enzyme

214

5.8.1.11 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 and

it was found that the immobilized enzyme retained its activity up to 5 cycles

and thereafter there was a slow decrease in activity up to 23 cycles and then

it declined very rapidly (Table 5.48 and Figure 5.40). The study indicates

that the immobilized laccase enzyme could be used for 23 cycles of acid

orange-7 decolourization. Though the decolourization percentage with

immobilized enzyme was low, when compared to free enzyme, it serves the

advantage that it can be re-used for 23 cycles of decolourization in batch

mode.

5.8.2 Bio-delignification of Eucalyptus sp.

5.8.2.1 Bio-delignification of Eucalyptus sp.

5.8.2.2 Tests for delignification

The delignification of Eucalyptus sp. by different fungi is given in

the Table 5.49 and Figure 5.41. Phanerochaete chrysosporium resulted in

the highest percentage of delignification (85.8%) while Trichoderma

harzianum resulted in least delignification (48.5%) in the present study.

There was no change in the control flask which had Eucalyptus wood alone.

Trametes hirsuta gave the second best delignification (81.2%) followed by

Trametes versicolor (79.4%). The delignification done by Aspergillus

fumigatus is 59.4%. The results correlate to the quantity of lignolytic

enzymes produced by the respective fungal species.

215

Table5.48:Cycles of decolourization of acid orange-7 with immobilized

laccase enzyme

Sl.No. Cycle(s) Decolourization (%)

1 1 94.7± 0.006

2 5 94.7± 0.016

3 10 93.5± 0.033

4 20 91.6± 0.018

5 23 89.3± 0.008


Figure 5.40:Cycles of decolourization of acid orange-7 with immobilized

laccase enzyme

216

Table 5.49: Delignification of Eucalyptus sp. by various fungi.

Sl.No. OrganismInitiallignin

content

Finallignin

content

Delignification (%)

1 Control 3.84 g 3.84 g 0± 0

2 Phanerochaete chrysosporium 3.84 g 0.54g 85.8± 0.013

3 Trichoderma harzianum 3.84 g 1.98g 48.5± 0.022

4 Trameteshirsuta 3.84 g 0.53g 81.2± 0.005

5 Trametes versicolor 3.84 g 0.79g 79.4± 0.013

6 Aspergillus fumigatus 3.84 g 1.56g 59.4± 0.036


Figure 5.41: Delignification of Eucalyptus Sp. by various fungi along with

control flask

217

Fungi that cause white rot belong to the Basidiomycotina and have

the capacity to degrade all cell wall components, including lignin. The

extent of lignin degradation can vary considerably among species of white-

rot fungi [73]. Some species, such as Trametes versicolor, are nonselective

in how they degrade the wood, i.e., they simultaneously degrade lignin,

cellulose, and hemicelluloses. Other species, such as Phellinus pini,

Ceriporiopsis subvermispora, and Phlebia tremellosa, cause preferential

degradation of lignin [475].

White-rot fungi, most of which belong to the Basidiomycetes, are the

best lignin degraders among all known microorganisms and secrete

extracellular ligninolytic enzymes including lignin peroxidase (LiP),

manganese peroxidase (MnP) and laccase [476, 72, 477, 69]. These

enzymes have been found in culture filtrates of various white-rot

Basidiomycetes including Phanerochaete chrysosporium [478], Coriolus

versicolor [479], Phlebia radiata, Bjerkandera adusta, Pleurotus

ostreatus,Lentinus edodes [478, 479].

Several methods have been developed to select fungal species with

selective lignin-degrading ability. However, one of the most appropriate

methods appeared to be the assessment of decay (chemical analyses of

lignin and wood sugar content) using wood blocks in accelerated decay

chambers. P. chrysosporium, C. subvermispora, Phlebia brevispora,

Phlebia tremellosa, Dichomitus squalens, and Phellinus pini were the best

lignin degrading fungi. Different strains of these selected species varied in

their selectivity towards lignin. Two fungi P. chrysosporium and C.

subvermispora were effective with hardwood only, whereas others were

effective on both hardwood and softwood. These results clearly showed

large differences among the strains in capacity to degrade lignin and in

selectivity. Of these, more emphasis was given to P. chrysosporium because

218

it is by far the most studied white-rot fungus, grows rapidly, and competes

well with indigenous microorganisms of wood chips [480].

5.8.2.3 Tests for lignolytic enzymes

The lignolytic enzymes produced by Phanerochaete

chrysosporium is given in the Table 5.50.

The production of laccase, lignin peroxidase and manganese

peroxidase were seen in all the fungal species. The ability of Phanerochaete

chrysosporium to produce lignolytic enzymes were reported by Asit Datta

et al., [481], Fatma Gassaraet al., [482] and several others in the years to

follow. Laccase producing ability of Trichoderma harzanium was

demonstrated by Sadhasivam et al., [417]. Trametes versicolor’s ability to

produce laccase, LiP and MnP was demonstrated by Moturi & Singara

charya [483]. The presence of laccase, LiP and MnP enzymes in Trametes

hirsutawas demonstrated by several workers example: Alberto Domınguez

et al., [484] and Shanmugam et al.,[485]. Hao et al., [486] and several

others demonstrated the presence of lignolytic enzymes in Aspergillus

fumigatus. The results are shown in the Table 5.51 and Figure 5.42.

219

Table 5.50: Production of lignolytic enzymes (Lignin peroxidase,

Manganese peroxidase and Laccase) by the five fungal species

Sl.No. FungiEnzyme

Ligninperoxidase

Manganese peroxidase

Laccase

1.Phanerochaete chrysosporium + + +

2.Trichoderma harzianum + + +

3.Trametes hirsuta + + +

4.Trametes versicolor

+ + +

5.Aspergillus fumigatus.

+ + +


Phanerochaete chrysosporium was selected for further studies on

delignification. Though the quantity of laccase enzyme produced by P.

chrysosporium is less than that of Trametes versicolor and Trametes

hirsuta, but the LiP and MnP produced by it was considerable higher than

that of the other fungal species. Since it has been reported that Lac, LiP and

MnP are involved in the delignification, particularly LiP and MnP, P.

chrysosporium was used for further study as it produced all the enzymes

(except laccase) in high proportion when compared to rest of the fungi

under study.

220

Table 5.51:Comparison of lignolytic enzyme producing capability of the

five fungal species under study

Sl.No. FungiQuantity of Enzyme(s) U/ml

Ligninperoxidase

Manganese peroxidase Laccase

1.Phanerochaete chrysosporium 0.84± 0.010 0.91± 0.021 1.78± 0.009

2.Trichoderma harzianum 0.71± 0.001 0.90± 0.001 1.67± 0.045

3.Trameteshirsuta

0.83± 0.022 0.89± 0.023 2.11± 0.019

4.Trametes versicolor

0.84± 0.031 0.89± 0.011 2.34± 0.022

5.Aspergillus fumigatus.

0.47± 0.061 0.38± 0.019 1.56± 0.032


Figure 5.42: Comparison of lignolytic enzyme producing capability of the

various fungal strains under study.

221

Evidence to date indicates that three oxidizing enzymes, lignin

peroxidase (LiP), manganese peroxidase (MnP), and laccase are responsible

for the initial fragmentation of the lignin polymer and production of low

molecular mass breakdown products in white-rot fungi. Not all white-rot

fungi apparently produce all three enzymes, although some, including T.

versicolor, P.chrysosporium produces Lac,LiP and MnP [25,410].

5.8.2.4 Effect of physical and chemical parameters physical parameters

optimization of delignification process

The effect of temperature, shaking speed, glucose and peptone

concentration on the delignification process were studied and it was found

that a temperature 30°C was needed for the maximal delignification

(86.2%) and a shaking speed of 150 rpm was needed to achieve maximum

delignification (89.98%). The addition of 1% of glucose increased the

delignification percentage to 92.3% and the addition of 0.5% of peptone

also increased the delignification percentage to 89.8% from 86.2%. All the

results are shown in the Tables 5.52 to 5.55 and Figures 5.43 to 5.46.

222

Table 5.52: Effect of temperature on the delignification of Eucalyptus sp.

byP.chrysosporium

Sl.No. Temp. Percentage Delignification (%)

1 25°C 85.80± 0.065

2 30°C 86.20± 0.011

3 35°C 84.50 ± 0.012

4 40°C 84.95 ± 0.033

5 45°C 84.70 ± 0.028


Figure 5.43: Effect of temperature on the delignification of Eucalyptus sp.

by P.chrysosporium

223

Table 5.53: Effect of shaking speed on the delignification of Eucalyptus sp.

byP.chrysosporium

Theresult shown above is the mean value of duplicate experiments along with the standard deviation. The P is < 0.05 at 95% confidence interval.

Figure 5.44: Effect of shaking speed on the delignification of Eucalyptus

sp. by P.chrysosporium

Sl.No.Shaking speed

(rpm)

Percentage

delignification (%)

1 80 86.10± 0.076

2 100 87.30± 0.033

3 120 87.80± 0.002

4 140 89.95± 0.025

5 150 89.98± 0.001

6 160 89.98± 0.004

224

Table 5.54: Effect of glucose concentration on the delignification of

Eucalyptus sp. by P.chrysosporium


Figure 5.45: Effect of glucose concentration on the delignification of


Sl.No. Glucose conc. (%)Percentage delignification

(%)

1 0.5 90.20± 0.013

2 1 92.30± 0.006

3 1.5 92.10± 0.071

4 2 92.00± 0.004

5 2.5 91.70± 0.081

225

Table 5.55: Effect of peptone concentration on the delignification of



Figure-5.46: Effect of peptone concentration on the delignification of


Sl.No.Peptone

conc.(%)Percentage delignification (%)

1 0.1 84.20 ± 0.009

2 0.2 84.65 ± 0.131

3 0.3 85.40 ± 0.034

4 0.4 87.90 ± 0.011

5 0.5 89.80 ± 0.003

226

5.8.2.5 Comparative delignification of Eucalyptus sp. under un-

optimized and optimized conditions

The delignifications of Eucalyptus sp. under optimized conditions

were carried out. The optimized conditions include the addition of 1%

Glucose, 0.5% Peptone and an incubation temperature of 30°C and a

shaking speed of 150rpm. The percentage of delignification obtained with

P.chrysosporium is 94.6% which is 10.25% higher than the delignification

under un-optimized condition by the same organism. The results are given

in Table 5.56 and Figure 5.47.The control flask showed no change in the

lignin content while in test flask there was 10.25% increase in

delignification. The final delignification along with control flask is shown

in the Figure 5.48.

Table 5.56:Comparative delignification of Eucalyptus sp. under un-

optimized and optimized conditions by P.chrysosporium.

Delignification (%)Un-optimized

conditionOptimized condition

Control flask 0 0

Test flask 85.8 ± 0.034 94.6 ± 0.019


Eucalyptus chemithermo-mechanical pulp (CTMP) was treated with

three white rot fungi namely P.chrysosporium, T.hirsuta 19-6W and

T.hirsuta 19-6. The results showed that lignin reduction by both T.hirsuta

19-6W and T.hirsuta 19-6 was twice that of P.chrysosporium; however the

selectivity of the selectivity of T.hirsuta was less [487].

227

Figure 5.47:Comparative delignification of Eucalyptus sp. under un-

optimized and optimized conditions by P.chrysosporium.

Figure 5.48: Photograph showing the control (left hand side) and test (right

hand side) of the delignification process by P.chrysosporium

228

The ability of Curvularia lunata LW6 to produce two lignolytic

enzymes namely lignin peroxidase (3.31U/mL) and laccase (6.12 U/ml)

using sugar cane bagasse as the substrate and its subsequent delignification

was studied by Narkhede and Vidhale [488].P.chrysosporium produced

30.11% lignocelluloses degradation of Cymbopogon martini (palma rosa) in

28days at 40°C [489].

5.8.3 Bio-Degradation of Cypermethrin

5.8.3.1 Screening of Cypermethrin degrading organism

From the results on screening for cypermethrin biodegrading fungi it

was observed that Aspergillus fumigatus grows more profoundly when

compared to the rest of the fungi (Figure 5.49 A-E). Based on this

observation Aspergillus fumigatus was selected for the further study on

cypermethrin biodegradation.

P.chrysosporium

0.01%0.05%

0.1%

0.5%0.76%

1.0%

Figure 5.49A: Growth of P.chrysosporium on Cypermethrin containing

plates

229

T.harzianum

0.01% 0.05% 0.1%

0.5%0.76%

1.0%

Figure 5.49B: Growth of T.harzianum on Cypermethrin containing plates

T.versicolor

0.01% 0.05% 0.1%

0.5%0.76% 1.0%

Figure 5.49C: Growth of T.versicolor on Cypermethrin containing plates

230

T.hirsuta

0.01% 0.05%0.1%

0.5% 0.76%1.0%

Figure 5.49D: Growth of T.hirsuta Cypermethrin containing plates

A. fumigatus

0.01%0.05% 0.1%

0.5%

0.76%1.0%

Figure 5.49E: Growth of A.fumigatus on Cypermethrin containing plates

231

5.8.3.2 Biodegradation of cypermethrin by plate assay

The results of plate assay shows that Aspergillus fumigatus can grow

at all the concentrations of cypermethrin (Figure 5.48E). However there

was a gradual decrease in the growth of Aspergillus fumigatus as the

cypermethrin containing SDA plates as the concentration of cypermethrin

increases. It is observed that the sporulating potential of A.fumigatus was

lost beyond 0.5% of cypermethrin concentration. The growth was very

much less at the concentration of 1.0% of cypermethrin.There was only one

report available on fungal degradation of cypermethrin in solid media. Juan

De Jesús et al., showed that Aspergillus niger can grow on a Malt extract

agar (MEA) plate containing 250 ppm of cypermethrin.

5.8.3.3 Bio-Degradation in liquid media

SD broth containing flasks were added with 0.01, 0.05, 0.1, 0.5, 1.0

and 5.0 concentration of cypermethrin separately and were inoculated with

Aspergillus fumigatus. The flasks were incubated for 3-4 weeks at 30°C.

The contents of the flaks were filtered and were assayed for cypermethrin.

The highest degradation (97%) was seen with the 0.01% cypermethrin

concentration and least degradation (8%) was observed with 5.0%

cypermethrin concentration.The biodegradation results are given in Table

5.57 and Figures5.50A, 5.50B, 5.51A, 5.51B and 5.52.

The results on cypermethrin biodegradation in liquid media are

available only with that of bacteria and Actinomycetes. Micrococcus sp.

was grown on Seubert’s mineral salts medium [490] containing 0.1%

(wt/volume) cypermethrin as sole source of carbon, in 500 ml Erlenmeyer

flask on a rotary shaker (150 rpm) at room temperature and growth was

measured turbidometrically at 660 nm [491].

232

RESULTS

CONTROL

0.01% 0.05% 0.1%

Figure 5.50A:Control flasks with 0.01%, 0.05%, and 0.1% concentrations

of Cypermethrin

TEST

0.01% 0.05%0.1%

Figure 5.50B: Test flasks with 0.01%, 0.05% and 0.1% concentrations of

Cypermethrin inoculated with A.fumigatus

233

CONTROL

0.5% 0.76% 1.0%

Figure 5.51 A: Control flasks with 0.5%, 0.76% and 1.0% concentrations

of Cypermethrin

Figure 5.51 B: Test flasks with 0.5% and 1.0% concentrations of

Cypermethrin

234

Table-5.57: Percentage biodegradation of cypermethrin in liquid media.

Sl.No.Cypermethrin

Conc. (g/ 100 ml)Degradation (%)

1 0.01 97 ± 0.008

2 0.05 88 ± 0.011

3 0.1 72 ± 0.019

4 0.5 41 ± 0.023

5 0.76 28 ± 0.018

6 1.0 8 ± 0.092


Figure 5.52: Percentage biodegradation of cypermethrin in liquid media.

Gradual decrease in degradation percentage is seen with increasing

concentration of cypermethrin.

0

20

40

60

80

100

120

0.01 0.05 0.1 0.5 0.76 1

Perc

enta

ge D

egra

dati

on

Cypermethrin Concentration (g/100ml)

235

Jilani and Altaf Khan [123] demonstrated that Pseudomonas can

grow in Nutrient broth containing cypermethrin. They also showed that

Cypermethrin (20 mg/L) was almost completely degraded in just over 48 h

at ambient temperature. Deepak Malik et al.,[492] showed that a new

Pseudomonas species called strainCyp19 degrades cypermethrin up to

100% within two days.

5.8.3.4 Test for the ability of Aspergillus fumigatusto use cypermethrin

as sole Carbon and Nitrogen source

The growth of A. fumigatesin the MS media indicated that A.

fumigatescan use cypermethrin as sole carbon and nitrogen source. The

assay for laccase enzyme showed positive result. The 0.01% cypermethrin

supported the maximum degradation (12%) while 1% degradation was seen

with 0.1% of cypermethrin. The remaining concentration did not support

the fungal growth. Concentrations above 0.5% did not support the fungal

growth; the lower concentrations the fungal growth and subsequent

degradation of cypermethrin was seen. However the degradation percentage

was very low when compared to SDA.The results are given in Table 5.58

and Figure 5.53. Similar results were reported by Preeti N. Tallur et al.,

[491] with Micrococcus sp.

5.8.3 .5 Biodegradation of cypermethrin by Laccase enzyme

Conical flasks with 0.01, 0.05, 0.1, 0.5, 0.76 and 1.0 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 and were assayed for cypermethrin

degradation. The biodegradation of cypermethrin by pure laccase enzyme

showed almost same result as that of fungal degradation.

236

Table 5.58:Percentage biodegradation of Cypermethrin in MS media.

Sl.No.Cypermethrin

Conc.(g/100 ml)

Degradation(%)

1 0.01 12± 0.0472 0.05 6± 0.0343 0.1 1± 0.0664 0.5 0± 05 0.76 0± 06 1.0 0± 0


Figure 5.53 : Percentage biodegradation of cypermethrin in MS media. The

degradation indicates that A.fumigates could use cypermethrin as carbon

and nitrogen source

0

2

4

6

8

10

12

14

0.01 0.05 0.1 0.5 0.76 1

Perc

enta

ge D

egra

dati

on


237

The maximum degradation was 98% (with 0.01% cypermethrin

concentration) and minimal degradation was 14% (with 1.0% cypermethrin

concentration).The results are shown in the Table 5.59 and Figure 5.54.

The role of esterase enzyme in the biodegradation of cypermethrin

was demonstrated by Preeti Tallur et al., [491] in Micrococcus sp. The role

Laccase enzyme in the biodegradation of Polyaromatic hydrocarbons and

pesticides were discussed by Baldrin [493].

5.8.3 .6 Time scale for Cypermethrin degradation with Laccase enzyme

Conical flask with 0.01concentration of cypermethrin in Acetate

buffer (pH 5.5) was taken and was 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 were assayed for cypermethrin degradation.

There was 98.4% degradation at the end 60 minutes. There was in sharp

increase in degradation percentage from 5 minutes to 35 minutes then there

was very slight change in the cypermethrin degradation. Highest

degradation was seen with 60 min of time.The results are depicted in the

Table 5.60 and Figure 5.55.

5.8.3.7 Effect of Temperature and pH on cypermethrin Biodegradation

The studies on the effect of temperature and pH on the degradation

of cypermethrin shows that the optimum temperature needed is 35°C and

pH is 6.0. The degradation at these conditions being 98.4% and 96.2%

respectively. The degradation at 30°C and 35°C was almost same (98.4% &

98.3% respectively); however the degradation decreased sharply at 45°C

and above 50°C the degradation was very negligible. Similarly, the pH

profile also showed significant variation. The degradation was significant at

pH range 5.0 to 7.0. The degradation at pH above 7.5 and below 4.5 was

238

very low. The results are given in the Tables 5.61 & 5.62 and Figures 5.56

& 5.57.

Table 5.59:Percentage biodegradation of Cypermethrin by laccase enzyme

Sl.No.Cypermethrin

Conc.(g/ 100 ml)

Degradation(%)

1 0.01 98± 0.0222 0.05 86± 0.0033 0.1 68± 0.0164 0.5 40± 0.0235 0.76 30± 0.1136 1.0 14± 0.048


Figure 5.54: Percentage biodegradation of cypermethrin by laccase

enzyme. The results indicate the decrease in degradation percentage with

higher concentrations of cypermethrin.

Table 5.60 :Time profile of cypermethrin degradation with laccase enzyme.

0

20

40

60

80

100

120

0.01 0.05 0.1 0.5 0.76 1

Perc

enta

ge D

egra

dati

on


239

Sl.No. Time (in Minutes) Degradation (%)1 5 71.1± 0.0032 10 82.8± 0.0213 15 88.6± 0.0114 20 91.2± 0.0145 25 96.3± 0.0326 30 97.7± 0.0177 35 98.0± 0.0818 40 98.0± 0.0079 45 98.0± 0.034

10 50 98.0± 0.01211 55 98.0± 0.02312 60 98.0± 0.043


Figure 5.55: Time profile of cypermethrin degradation with laccase

enzyme. Highest degradation was seen with 60 min of time.

Table 5.61: Effect of temperature on cypermethrin biodegradationby

laccase enzyme

0

20

40

60

80

100

120

5 10 15 20 25 30 35 40 45 50 55 60

Perc

enta

ge D

egra

dati

on o

f Cy

perm

ethr

in

Time in Minutes

240

Sl.NoTemperatures

(°C)Degradation (%)

1 25 96.5± 0.004

2 30 98.4± 0.013

3 35 98.3± 0.023

4 40 96.0± 0.022

5 45 93.9± 0.031


Figure 5.56: Effect of temperature on cypermethrin biodegradationby

laccase enzyme

Table 5.62: Effect of pH on cypermethrin biodegradation by laccase

enzyme

241

Sl.No. pH Degradation (%)

1 5.0 88.4± 0.006

2 5.5 95.1± 0.012

3 6.0 96.2± 0.023

4 6.5 95.1± 0.002

5 7.0 92.0± 0.019


Figure 5.57: Effect of pH on cypermethrin biodegradation by laccase

enzyme

5.8.3.8 Optimization laccase enzyme concentration for the

biodegradation of cypermethrin

242

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 and

fixed concentration of cypermethrin (0.01%). The results showed that 1.5

unit of laccase enzyme resulted in the maximal degradation (99.2%) at

30°C and 6.0 pH. The results are depicted in the Table 5.63 and Figure

5.58.

Table 5.63: Effect of varying concentrations of laccase enzyme on the

cypermethrin biodegradation

Sl.No.

Laccase enzyme

concentration

(Units)

Cypermethrin

Concentration (%)

Biodegradation

(%)

1 0.5 0.01 96.7± 0.012

2 1.0 0.01 98.4± 0.005

3 1.5 0.01 99.2± 0.034

4 2.0 0.01 99.2± 0.005

5 2.5 0.01 99.2± 0.004


243

Figure 5.58 : Effect of varying concentrations of laccase enzyme on the

cypermethrin biodegradation

5.8.3.9 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) shows that there was slightly better

degradation under optimized condition. There was1.22% increase in the

cypermethrin biodegradation under optimized condition. The results are

given in the Table 5.64 and Figure 5.59 a & b.

Table 5.64: Comparison of degradation of cypermethrin by laccase enzyme

under un-optimized and optimized conditions.

95

95.5

96

96.5

97

97.5

98

98.5

99

99.5

0.5 1 1.5 2 2.5

Cype

rmet

hrin

bio

degr

adat

ion

perc

enta

ge (%

)

Concentration of Laccase enzymes (Units)

244

Sl.No Cypermethrin

Conc.

(g/ 100 ml)

Percentage of degradation under

optimized condition

Percentage of degradation under

un-optimized condition

1 0.01 98 ± 0.011 99.2 ± 0.080

2 0.05 86 ± 0.021 89.2 ± 0.0112

3 0.1 68 ± 0.019 76.0 ± 0.045

4 0.5 40 ± 0.20 41.4 ± 0.006

5 0.76 30 ± 0.10 38.1 ± 0.002

6 1.0 14 ± 0.23 18.11 ± 0.017


Figuree 5.59 a : Comparison of degradation of cypermethrin by laccase

enzyme under un-optimized and optimized conditions.

0

20

40

60

80

100

120

0.01 0.05 0.1 0.5 0.76 1

Perc

enta

ge b

iode

grad

atio

n of

cyp

erm

ethr

in

by la

ccas

e en

zym

e

Cypermethrin concentrations

Biodegradation under un-optimized condition

Biodegradation underoptimized condition

245

Figure 5.59 b: Comparison of degradation of cypermethrin by laccase

enzyme under un-optimized and optimized conditions.

chapter 5 results and discussion 5.1 screening and...

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