chapter-5 optimization of laccase production by...

Chapter-5

Optimization of Laccase

Production By Solid State

Fermentation

Optimization of Laccase Production by Solid State Fermentation

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CHAPTER V

OPTIMIZATION OF LACCASE PRODUCTION BY

SOLID STATE FERMENTATION

5.1 INTRODUCTION

Solid State Fermentation (SsF) is an imperative approach of fermentation where microorganisms

grow on the substrates in the absence or near-absence of free water and ooze aimed product competently

[Pandey et al., 2000 ; Veronique et al., 2003]. In scrutiny of the lower energy supplies, simplicity of

cultivation and media equipment, high product titres and lower waste water output, SsF method ignites

the interest of researchers again in the production of enzymes, fine chemicals, antibiotics etc [Kumar et

al., 2003 and Adinarayana et al., 2003]. SsF is ecological friendly as it resolves the problem of solid

wastes disposal. It has been generally claimed that product yields are mostly higher in SsF when

compared to submerged fermentation (SmF). Production of biocatalysts using agro-biotech substrates

under solid-state fermentation conditions provide several advantages in productivity, cost-effectiveness in

labour, time and medium components in addition to environmental advantages like less effluent

production, waste minimization, etc. [Pandey et al., 2000].

SsF involves two very different modes [Murado et al., 1998]. In the first one, a humidified solid

(organic material) acts as both support and nutrient source and the process essentially occurs in the

absence of free water [Kumar and Lonsane, 1987]. In the second mode, a nutritionally inert solid

(synthetic material), which exclusively acts as a support, is soaked in a nutrient solution. In both cases,

the success of the process is directly related to the physical characteristics of the support (particle size,

shape, porosity, consistency), which favour both gas and nutrient diffusion and the attachment of the

microorganisms [Mitchell, 1992]. Generally, smaller substrate particles provide a larger surface area for

microbial colonisation but if they are too small may result in substrate agglomeration as well as poor

growth. In contrast, larger particles provide better aeration but a limited surface for microbial

colonisation. Therefore, a compromised particle size must be selected for each particular process [Pandey

et al., 1999].

There are several reports describing use of agro-industrial residues for the production of laccase

e.g. banana skin by Trametes pubescens [Osma et al., 2007]. However, these production characteristics

would have to offer a competitive advantage over existing products. In general, each microbial strain is

unique in their molecular, biochemical, metabolic and enzyme production properties. This warrants

thorough characterization of isolated individual microbial species to evaluate its potential at commercial

level. Furthermore, most of these wastes contain lignin or/and cellulose and hemicellulose, which act as


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inducers of the ligninolytic activities. In addition, most of them are rich in sugars, which make the whole

process much more inexpensive.

There are large numbers of reports on the optimization of carbon and nitrogen source by the

classical method of medium optimization that changes one independent variable, while fixing other

variables at definite levels. Optimizing all the moving parameters by statistical experimental design,

Plackett-Burman and Response Surface Methodology can eliminate the limitations of single-factor

optimization process collectively [Gohel et al., 2006; Felse and Panda, 1999 and Joshi et al., 2007].

The present work attempts to formulate an appropriate production medium using statistical

optimization that can increase the laccase production from Streptomyces chartreusis strain NBRC 12753

(NCBI Accession no JQ086575) through SsF technology using Plackett-Burman [Plackett and Burman,

1946] and Central Composite Design.

5.2 MATERIALS AND METHODS

5.2.1 Chemicals

Wheat bran, rice bran, wheat straw, rice straw, sugarcane baggase and banana waste were

collected locally and used as lignocellulosic substrates. Casein enzyme hydrolysate, Yeast extract powder,

Sodium chloride and Dextrose were procured from Hi-Media (Mumbai, India). The o-anisidine and p-

anisidine were procured from CDH (Mumbai, India). All other chemicals were of analytical grade

procured from Qualigens (Mumbai, India). 2, 2-Azino-bis (3-ethylbenzthiozoline-6-sulphonic acid)

(ABTS) was purchased from Sigma (St. Louis M.O., U.S.A.).

5.2.2 Organism

Screening for laccase-producing microbes on Bennet’s agar plates (Appendix-I) containing

coloured indicators resulted in isolation of 20 Streptomyces strains. Bacterial isolates showing positive

Bavendamm’s reaction were maintained on Bennet’s agar at 30°C and stored at 4°C.

The best laccase producing isolate was identified by comparing the partial 16S ribosomal RNA

gene sequence which was deposited in GenBank data base and identified as Streptomyces chartreusis

strain NBRC 12753 (NCBI -Accession number JQ086575)[Chhaya and Modi, 2013a].

5.2.3 Media Preparation and Inoculation

Five grams of rice bran in 20 mL basal medium were added to a 250 mL Erlenmeyer flask and

was moistened with a salt solution containing (Appendix II) [Niladevi and Prema, 2008]. The requisite

volume of media constituents were pipetted out from their stock solution of higher concentration and


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were mixed together before sterilization. Rice bran was separately sterilized at 15 psi for 30 minutes and

mixed aseptically with the medium before inoculation. The pH of the medium was adjusted to 8.0 and

sterilized by autoclaving at 15 psi for 15 minutes. Each flask was inoculated with four mycelial agar plugs

of 8 mm in diameter (cut from the edge of an actively growing colony on Bennet’s agar plates), and

incubated under static condition at 30°C for 3 days.

5.2.4 Enzyme Assay

Laccase activity (E.C.1.10.3.2) [Bourbonnais and Paice, 1990] was measured by monitoring the

oxidation of 500μM ABTS. Increase in absorbance for 2 minutes was measured spectrophotometrically

(Make:-Wensor, Model:-WSP-UV800A) at 420 nm (å =36000cm-1 M-1). The reaction mixture contained

300μL of 50mM ABTS and 2400μL of 20mM Sodium Phosphate butter (pH-7.5) and 300μL of

appropriately diluted enzyme extract. One unit of enzyme was defined as amount of enzyme that oxidized

1μM of substrate per minute [Chhaya and Gupte, 2010].

5.2.5 Substrate Screening for SsF

Substrates used in SsF are generally insoluble in water. In practice, water is absorbed onto the

substrate particles, which can then be used by microorganisms for growth and metabolic activities

[Manpreet et al., 2005]. The collected agro-waste materials were used as substrates for solid-state

fermentation. 5 gm of each substrate was separately transferred into 250 mL Erlenmeyer flasks and was

moistened with a salt solution (Appendix I). Thirteen ml of the moistening solution was added to the

substrate and the initial moisture level in the substrate was adjusted to 50 % by adding an adequate

quantity of distilled water. The next day, water was drained off, and care was taken to retain sufficient

moisture.

The flasks were then sterilized in an autoclave. After cooling, all the flasks were inoculated with

2 days old, 9 % inoculum and incubated at 30 °C for 96 hrs [Gomez et al., 2005]. The substrates used

were wheat bran, rice bran, wheat straw, rice straw, banana waste and sugarcane bagasse [Winquist et

al., 2008; Singhania et al., 2009; Couto and Sanroman, 2005]. They were further mechanically

dehydrated until the moisture content of dried materials reached 4±1%, which generally took 3 to 4 hrs.

Dried materials were packed in polythene pouches for further studies [Balaraju et al., 2010].

5.2.6 Optimization of Substrate Weight for SsF

Each substrate in three separate sets of flasks containing 3, 4 and 5 gm [Irshad et al., 2013] were

incubated in order to find out the optimum substrate concentration for maximum laccase yield. After

carrying out the fermentation for optimum time, the contents of the flasks were filtered and extracted for


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laccase activity measurment. The study was carried out at 30 °C keeping all other conditions at their

optimum levels. The optimum amount of solid substrate achieved by this step was fixed for subsequent

experiments.

5.2.7 Optimization of Particle Size

The desired particle size of studied substrates was obtained by sieving (using Indian standard

sieves) the air dried substrates. Unsieved substrate that enclosed particles of different sizes was also

included in the study. The particle size was varied from 150 to 355 μm. The effect of penetration between

agro wastes and inoculated culture were studied using scanning electron microscopy.

Scanning electron microscopy was carried out at sophisticated instruments centre (SICART,

Anand) using Scanning electronmicroscope (Make:-Philips, Netherlands, Model:-ESEM EDAXXL- 30).

Different magnified angles are used to evaluate the level of penetration of both the substrates (Fig. 2.11)

towards inoculums.

5.2.8 Optimization of Initial Moisture Content

To investigate the influence of the initial total moisture content (before autoclaving) of the

substrate was carried out under various initial moisture content adjusted with salt solution (Appendix II).

Samples containing 5 moisture levels (30 %, 45 %, 50 %, 65 % and 70 %) were prepared by moistening 5

gm of studied substrates with salt solution (Appendix II) [Niladevi et al., 2007]. The optimum initial

moisture content of solid substrate achieved by this step was fixed in subsequent experiment. The other

condition was 9 % inoculum level and the fermentation was carried out for 96 hrs at 30°C [Divakar et

al., 2006]. After soaking, the sample was again dried as described above and percent moisture content

was calculated as follows, Percent of moisture content (initial) of solid medium = (wt. of the rice bran -

dry wt.) x 100 / dry wt [Ellaiah et al., 2002].

5.2.9 Optimization of Inoculum Size for SsF

For the development of inoculums, 30 mL of Bennet’s broth (Appendix I) was placed in a 100

mL Erlenmeyer flask, pH adjusted to 7.5 and the medium was sterilized. It was inoculated with a well-

sporulated slant culture of Streptomyces chartreusis (2 days old) and kept on a rotary shaker (7 rcf) for 24

hrs at 30°C. A 5 mL portion of this broth was used to inoculate 30 mL of the Inoculum medium contained

in a 100 mL Erlenmeyer flask. Usually, triplicate flasks were used for each test. The final concentration of

an inoculum contained 4 x 106 CFU/mL. Hence, for study the effect of isolate, Inoculum size ranges from

5 % to 20 % were applied for optimizing higher laccase production [Divakar et al., 2006; Chhaya and

Modi, 2013b].


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5.2.10 Optimization of Carbon and Nitrogen Supplements for SsF

Type, nature and concentration of carbon source are important factors for any fermentation

process. Supplementation with additional carbon sources [dextrose at 1% w/v while sucrose, and soluble

starch at 0.5 % w/v] were analyzed for laccase production.

Supplementation with a variety of organic nitrogen sources [yeast extract, peptone, and tryptone

at a concentration of 0.2 % w/v], inorganic nitrogen sources [ammonium sulphate, ammonium chloride,

and potassium nitrate at a concentration of 0.1 % w/v] were studied [Ramachandran et al., 2004] for

higher laccase production.

5.2.11 Optimization of pH and Temperature for SsF

Exact control of pH is very difficult in SsF process, but one can maintain pH during the process

by using pH-correcting solutions [Mitchell et al., 1991]. The pH was determined using 1.0 gm of

fermented material in 10 mL of distilled water, and then the mixture was agitated. After 10 minutes, the

pH was measured in the supernatant using a pH meter [Vastrad and Neelagund, 2011]. In the present

analysis initial pH of the moistening solution was varied from 5 to 9 with 1 N HCl or 1 N NaOH

[Niladevi et al., 2007; Irshad et al., 2013]. Here the fermentation was carried out at 30 ° C temperate to

study their effect on enzyme production.

In the present study the incubation temperature was studied in between 20-40 °C. All other

conditions were kept in their optimum level.

5.2.12 Inducers

In filamentous bacteria, extracellular laccases production can be considerably stimulated by the

presence of inducers mainly aromatic or phenolic compounds related to lignin or lignin derivatives.To

study the effect of inducers on laccase production, various aromatic compounds such as p-anisidine, o-

anisidine, ferulic acid, guaiacol, pyrogallol, cupric sulphate and vanillic acid were incorporated in the

fermentation medium. All the inducers were added at a concentration of 1 mM [El Aty and Mostafa,

2013]. Cupric sulphate and vanillic acid were dissolved in sterile water while all the other aromatic

inducers were dissolved in 50 % alcohol. The inducers were added to the flasks just before inoculation

[Laxmi and Khan, 2010]. Control flasks were supplied with basal medium without any inducers. The o-

anisidine and p-anisidine were both filter sterilized and added separately to the medium before

inoculation.


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5.2.13 Time Course Study

In order to evaluate the extracellular lignolytic activity, the content of each flask was soaked with

100 mL of aqueous solution of sodium phosphate buffer 20 mM, pH 7.5 and shaken on a rotary shaker

(200 rpm) for 1hr at 30°C. Finally, the suspension was squeezed through a double-layer muslin cloth and

solution was centrifuged at 4000×g for 20 minutes at 4°C, and the supernatant was filtered through a

membrane filter (pore size of 0.22μm). The clear filtrate obtained was assayed for laccase activity. Time

course up to 120 hrs were studied for SsF fermentation process.

Biomass was determined by weighing the dry mycelia after growth. The attached mycelia were

pressed to remove the medium, washed with the distilled water, and desiccated completely at 60° C.

Biomass was calculated by substrating the initial weight of the 5 gm solid substrate measured for the

uninoculated control from the final weight [Mazumder et al., 2009].

5.2.14 Statistical Optimization for Laccase Production by SsF

5.2.14.1 Identification of Nutrient Components by Placket and Burman Design

The Plackett-Burman design was used to find the nutrient components significantly influencing

laccase production by Streptomyces chartreusis NBRC 12753 (NCBI accession no. JQ086575). The

optimization of medium components for laccase production was fulfilled in two stages. In the first stage

Plackett-Burman design was used to find the nutrient components considerably influencing laccase

production by Streptomyces chartreusis strain NBRC 12753. Total 12 different components (variable

k=12) were selected for the study with each variable being represented at two levels, high (+) and low (-)

(Table 5.1). This model describes no interaction among factors and it is used to screen and evaluate the

important factors that influence enzyme production.

The factors that have confidence level above 95% are considered the most significant factors that

affect the enzyme production. The main effect of the medium components, regression coefficient, F

values and P values of the factors investigated in the present study. Table 5.1 shows selected experimental

variables for conducting twelve experimental trials. These 12 variables were selected based on the

previous experiments; three dummy variables were included and evaluated in 16 experiments. The

numbers of positive and negative signs per trial are (k+1)/2 and (k-1)/2 respectively. The effect of each

variable was determined by the equation as;

E (xi) =2(ΣMi+-Mi-)/N, ……………….(i)

where E (xi) is the concentration effect of the tested variable, Mi+ and Mi- are the laccase

production from the trial examination where the variable (xi) calculated was estimated by the variance

among the dummy variables as Veff = Σ (Ed2)/n, ……………………………………………………(ii)

Where Veff is the variance of the concentration effect, Ed is the concentration effect for the dummy

variables and n is the number of dummy variables.


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All experiments were performed in duplicate and the average of laccase activity was taken as

response. The variables showing highest positive effect on each category were considered to have greater

impact on laccase production and hence selected for further optimization using Central Composite Design

of Response Surface Methodology [Palvannan and Sathishkumar, 2010; Poojary and Mugeraya,

2012].

Table 5.1: Variables screening medium components used in Plackett-Burman design

Variables Components + Values - Values

K1 Cupric sulphatea 0.2 0.02

K2 Moisture contentb 65 50

K3 Time Coursec 72 24

K4 o-anisidinea 0.1 0.01

K5 Particle sized 350 usp 250 usp

K6 Sucrosea 2 0.2

K7 Temperaturee 40 30

K8 Pyrogallola* 0.5 0.05

K9 Yeast extracta 2.0 0.02

K10 Dextrosea 5.0 0.5

K11 p-anisidinea 0.1 0.01

K12 pHf 8.0 7.0

(a gm/L; a* mL/L, b % ; c time in hrs ; d size in µm & usp - unseived particles; e temp. in ° C ; f pH

value)


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5.2.14.2 Optimization of Screened Components using Central Composite Design

From the optimized nutrient composition for laccase producing Streptomyces chartreusis, the

effects of dextrose, yeast extract and pyrogallol were studied using Central Composite Design (CCD).

Response Surface Methodology was used to optimize the screened components for enhanced laccase

production using Central Composite Design (CCD).

The performance of the method was explained by the quadratic equation as,

Y=β0+Σβixi+Σβijxixj+Σβiixi,……………………………………..(iii),

Where Y is predicted response, β0 is offset term, βi is linear offset, βii is squared offset and βij is

interaction effect. Xi is dimensionless coded value of Xi. The above equation was solved by using the

software Design-Expert® (Version 7.0.2, Stat ease inc., USA) [Cheng et al., 2012]. A factorial design

with a total number of 20 trials was employed. The coded and actual values of the variables at various

levels are studied [Chhaya and Gupte, 2010].

5.3 RESULTS AND DISCUSSION

5.3.1 Organism

Extracellular laccase activity was found in twenty isolates. The isolates showed a brown colored

zone surrounding the growth on Actinomycete isolation agar plate containing o-anisidine, which is a

characteristic of phenol oxidase production on the solid medium [Chhaya and Modi, 2013a]. The

identification of Strain R1 was further corroborated by studies on its partial 16S rRNA gene sequencing

carried out at Department of Animal Biotechnology, College of Veterinary Science and Animal

Husbandry, Anand Agricultural University, Anand, Gujarat, India. The isolate was identified as a

Streptomyces chartreusis strain NBRC 12753 (Genbank Accession no JQ086575).

5.3.2 Substrate Screening

Among the different substrates screened, rice bran (39.6 U/gm) was the most suitable substrate

for laccase production, followed by wheat bran (25U/gm, Fig. 5.1) in SsF. This phenomenon might be

attributed to the presence of relatively higher availability of lignin related compounds in rice bran, which

favours ligninolytic enzyme production. SsF was usually performed with filamentous fungi due to their

ability to penetrate and colonize the solid substrate particles. In the present study, the filamentous nature

of Streptomyces chartreusis strain NBRC 12753 (Accession no JQ086575) was observed as an added

advantage which facilitated the penetration of rice bran particles and utilization of the nutrients; a

characteristic usually appreciated in solid-state culture conditions.


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Fig 5.1 Screening of agrowatse residues for laccase production in solid state fermentation

Selection of a suitable substrate is a key factor in solid substrate fermentation which concludes

the success of the process. Rice bran is a broken husks of the seeds of rice grains which are separated

from the flour by sifting and holds total carbohydrate 82% (w/w) around, oil is normally 14–24%; protein

12–18%; carbohydrate 45 % [Amissah et al., 2003]; water 7–14%; ash 8–12%. Besides, it is also rich in

vitamins E and B, protein with other nutrients [Randall et al., 1985] and the key composition (31%) was

hemicellulose [Claye et al., 1996]. Laccase synthesis was encouraged by phenolic compounds containing

in rice bran, leading to increasing of laccase production. This induction mechanism may help to humiliate

lignin or aromatic compounds in rice bran to supply further nutrients especially carbon and nitrogen

[Chawachart et al., 2004]. Rice bran was also used as a sole carbon source for xylanase production by

Streptomycete actuosus A-151 [Wang et al., 2003].

5.3.3 Effect of Substrate Weight

Surface-to-mass ratio [Prasad et al., 2011] of solid substrate was one of the important factors in

SsF, as it was directly related to the surface area available for the growth of cells. In this direction six

variations in solid substrate loading (3 gm, 4 gm, 5 gm, 10 gm, 15 gm and 20 gm of solid substrate) were

studied to enumerate its role on the laccase yield. It was found from the results that 5 gm of rice bran was

yielded effective highest yield of laccase production (Fig. 5.2). This condition indicated that too much

amount of substrate in a fixed container produced a thicker substrate bed which finally reduced the

substrate pore size and reduced the transferring of oxygen in between the substrate particles [Perez-

Guerra et al., 2003]. It also interferes with the oxygen diffusion in substrate, especially at the basement


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part of the flask where the substrate was not fully fermented or utilized. A thinner bed height allows for

better heat removal than a thicker bed height [Rashid et al., 2012].

Fig. 5.2 Laccase production in U/gm during Solid state fermentation

5.3.4 Effect of Particle Size

The adherence and penetration of microorganisms toward the solid substrate as well as enzyme

action on the substrate clearly depend upon the physical properties of the substrate such as the accessible

area, surface area, porosity and particle size, of which particle size plays a major role because all other

physical properties of the substrate depends on it [Niladevi et al., 2007]. The highest amount of laccase

production is achieved as 41 U/gm with mixed particle size of 355 µm and unseived particles (Fig. 5.3).

Fig. 5.3 Effect of particle size on laccase production by Streptomyces chartreusis


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As per shown in the Fig. 5.4 A (Mix particle size) and 5.4 B (Unseived particles), early growth

occurs with mixed particles rather then unseived particles. In the present study particle size in the range of

355 μm mixed with unsieved particles (Usp) was the optimum for laccase production.

A B

Fig. 5.4 Solid state fermentation using rice bran as substrate on day 5th (A = Mixed particles sizes,

B= Unseived particles)

The enzyme yield was low in the case of substrates with lower and higher particle size, which

was in correspondence with the general concept that lower particle size results in substrate agglomeration,

improved channeling problems and decreased heat transfer while larger particles reduce the production

due to limited surface area for microbial attack [Pandey et al., 2000]. It has been reported that in higher

bed thickness, the oxygen availability is getting decrease at the middle and bottom area of substrate.

Moreover, it was also reported that the oxygen availability has correlation with the substrate thickness

[Raghavarao et al., 2003]. This condition is due to the fast colonization of the filamentous bacteria on

the surface area which promotes the high density of the substrate.

This condition has caused the exhausting of oxygen which affects the bacterial growth as well as

the enzyme activity. Therefore, thicker bed height than the optimum leads to undesirable situations like

cell lysis and generating anaerobic conditions. On the other hand, a thinner bed height is usually chosen in

a tray system since it easily to be fermented [Suryanaran, 2003; Annuar et al., 2010] and permits better

oxygen supply and heat removal [Gervais and Molin, 2003]. Productivities were higher with the

substrate that contains particles of mixed sizes [Pandey, 1992].

To have a complete penetration of bacterial hyphae into solid substrates always become a major

problem in SsF. Therefore, the right size of substrate is important. The effects of penetration were studied


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in two solid substrates Rice bran and Wheat bran as per Fig. 5.5. The dense growth of Streptomyces

chartreusis was observed in Rice bran, while Wheat bran gave less absorbance of bacterial hyphae. In

which accessibility of surface area depends on substrate size which is crucial for mass transfer,

microorganism attachment, growth and also final product production [Prakasham et al., 2006].

Wheat Bran

Wheat Bran

Fig. 5.5 Scanning Electron Microscopy of Streptomyces chartreusis with wheat bran & rice bran

Cont..


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Rice Bran

Rice Bran

Fig. 5.5 Scanning Electron Microscopy of Streptomyces chartreusis with wheat bran & rice bran

5.3.5 Effect of Initial Moisture Content

In the present study an initial moisture content of 65 % (47 U/gm) was found optimum for laccase

production (Fig. 5.6). Moisture [Esfahani et al., 2004] is another key parameter to control the growth of

microorganisms and metabolite production in solid-state fermentation. Higher initial moisture in SsF

leads to suboptimal product formation due to reduced mass transfer while decrease in initial moisture

level results in reduced solubility and low availability of nutrients to the culture. Moisture content is one

of the key factors which could affect the metabolite production in SsF [Archana and Satyanarayana,

1997]. Water affects the physical properties of the solid substrate mainly by causing swelling of the


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substrate and facilitates effective absorption of the nutrients from the substrates for growth and metabolic

activities [How and Ibrahim, 2004].

The optimum initial moisture content of solid substrate achieved by this step was fixed in

subsequent experiment. However, too low moisture content might reduce the nutrient solubility in the

substrate and affect the initial growth of spores, which could disrupt the growth and enzyme activity

[Murthy et al., 1993; Battan et al., 2006].

Fig. 5.6 Effect of initial moisture content on laccase production by Streptomyces chartreusis

Low water content is usually related to insufficient substrate swelling which prevented the

nutrient absorption from the substrates. Low moisture content might reduce the nutrient solubility in the

substrate and disrupt the growth of organism [Murthy et al., 1993]. On the other hand, higher moisture

contents resulted reduction in substrate porosity and caused oxygen limitation within the substrates which

consequently affected the oxygen transfer within the substrate and thus resulting poor growth [Ramesh

and Lonsane, 1990; Sandhya et al., 2005].

5.3.6 Effect of Inoculum Size

Inoculum concentration with 9 % gives highest yield (47.6 U/gm) of laccase production (Fig. 5.7)

in SsF. Increase in inoculum size compact the level of the formed enzyme due to fast depletion of the

nutrients, resulting in a decrease in metabolic activity [Patel et al., 2009]. In concurrence with this

declaration, Sharma et al. (1995) reported that inoculum size controls and shortens the initial lag phase, as

smaller inoculum size increased the lag phase [Sharma et al., 1995]. When the inoculum size is low,

superfluous nutrients resulted and delayed enzyme production. However, insufficient nutrients and


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increasing temperature in the later period resulting from the rapid growth of mycelia inhibited enzyme

production through a high inoculum size.

Fig: 5.7 Effect of inoculum size on laccase production by Streptomyces chartreusis

In SsF, inoculum size must be distributed homogenously and must be in sufficient numbers for

the microorganism to grow well. The mycelia or fragments which initially attached on the outer surface of

the substrate particle is slowly growing, multiplying and penetrating in to the substrate. Lower inoculum

size was able to retard the proliferation of biomass [Ibrahim et al., 2012]. Thus, the degradation of the

substrates by the microbes is slower and affects the metabolite production [Ramachandran et al., 2004].

However, high inoculum sizes are inhibitory in nature where the overall trends shown that an increased in

spore concentration adversely affect the enzyme production.

This condition may be correlated to the amount of available oxygen and nutrients in the early

stage of inoculation where rapid growth resulted higher degradation of the substrates and increased

availability of the nutrients [Kashyap et al. 2002]. Higher inoculum than the optimum may produce too

much biomass and may reduce the nutrient that necessary for microbial metabolite production [Kumar et

al., 2010].

5.3.7 Effect of Supplemental Carbon and Nitrogen Source for SsF

Among the different supplemented carbon sources used, dextrose was comparatively less

repressive for laccase production, which yielded 48.1 U/gm (Fig. 5.8), while all the other carbon sources

reduced the enzyme yield considerably. As dextrose earlier noted was an excellent carbon source for


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antibiotic production by Streptomyces kanamyceticus, [Pandey et al., 2005]. Glucose and cellobiose were

efficiently and rapidly utilized by Trametes pubescens with high laccase activity [Galhaup et al., 2002].

The results were in accordance with those made by Sivakami et al., (2012) who reported that the dextrose

was the best carbon source for laccase production by Pleurotus ostreatus LIG 19 [Sivakami et al., 2012].

Fig 5.8 Effect of supplementary carbon source on laccase production in SsF by S. chartreusis

This was probably due to the reason that dextrose was a readily utilizable substrate which would

promote the biomass production. Growth conditions constitute an important factor influencing the

efficiency of bacteria to produce laccase. It has been frequently described that in a defined medium

absence of proper carbohydrate (carbon source) results in a dramatic decrease inenzyme production

[Gajju et al., 1996], so a carbon source is always an essential component of a fermentation medium.

Researches have reported the cause of different carbon and nitrogen sources on laccase production by

various white rot fungi and filamentous bacteria, such as Coriolopsis rigida [Gomez et al., 2005],

Pycnoporus sanguineus, Pleurotus species [Vikineswary et al., 2006 ; Stajic et al., 2004] and

Streptomyces psammoticus [Niladevi et al., 2007].

As per the results indicated the organic nitrogen source (yeast extract, peptone, tryptone) gave

maximum laccase activity then inorganic nitrogen source (ammonium sulphate, potassium nitrate and

ammonium chloride). Replacement of yeast extract with peptone failed to elicit laccase production. This

confirmed the suitability of yeast extract as the nitrogen source (49.8 U/gm) for laccase production (Fig.

5.9) by Streptomyces chartreusis and similar result has also been reported from Streptomyces

psammmoticus [Niladevi et al., 2007]. The lignolytic enzymes have been seen to be regulated by the


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usable concentration of the nitrogen in the media. The low nitrogen level can stimulate the lignolytic

enzyme production, whereas the high nitrogen level represses it [Patel et al., 2009]. Similar results were

obtained at different concentration of nitrogen in the medium.

Fig 5.9 Effect of supplementary nitrogen source on laccase production in SsF by S. chartreusis

5.3.8 Effect of pH and Temperature

The optimum pH for maximal laccase production during SsF was pH 8.0 (50.9 U/gm, Fig. 5.10).

The fermentation media shows increasing pH upto alkaline side during fermentation. The current results

confirmed that the enzyme production was favored by neutral to alkaline pH range, where as the acidic

pH decreased the enzyme yield considerably. The effect of increasing buffer concentration, effect of pH

on the maximum specific growth rate and the optimization of different buffers for the growth and enzyme

parameter were investigated [Nagel et al., 1999].

The optimum alkaline pH for laccase production observed earlier were of pH 7.5 in Streptomyces

psammoticus [Niladevi et al., 2007] and pH 7.0 in Streptomyces lydicus [Mahmoud et al., 2013]. The

optimunm pH for laccase production was substrate dependent and for phenolic substrates the highest

laccase activities were detected at alkaline conditions (pH 9.0 for 2, 6-dimethoxy phenol and guaiacol and

pH 8.0 for syringaldazine) by Streptomyces sviceus [Gunne and Urlacher, 2012], The culture was able

to grow and produce laccases in media of high pH values shows its importance and suitability for

industrial application.


111

Fig: 5.10 Effect of pH content on laccase production by Streptomyces chartreusis

Optimum cultivation temperature depends on the growth kinetics of the microorganism employed

rather than on the enzyme produced [Lonsane et al., 1985; Krishna, 1999]. As per below Fig. 5.11 a

gradual decrease in enzyme formation occurred at 30ºC. This can be interpreted by the alteration of cell

membrane composition and stimulation of protein catabolism. Similar to our findings, an incubation

temperature of 30 °C was optimum for laccase production by Streptomyces chartreusis and considerable

high activity was also observed at 30°C (52.3 U/gm). The same results for temperature optimization were

also noted earlier by Streptomyces psammoticus [Niladevi et al., 2007] in laccase and Streptomyces

antibioticus [Rao et al., 2012] for laccase and peroxidase free tyrosinase production.

Fig: 5.11 Effect of Temperature on laccase production by Streptomyces chartreusis


112

Microbial growth in SsF generates significant amount of metabolic heat. It has been reported that

100-300 kJ of heat per kg of cell mass is generated in SsF process [Prior et al., 1992]. Establishment of

temperature gradients and localized overheating of the substrate occurs because of inefficient removal of

heat from the substrate. Heat transfer problem in SsF includes temperature gradients that may cause

belated microbial activity, dehydration of the medium and undesirable metabolic deviations [Saucedo-

Castaneda et al., 1990]. Temperature can rise rapidly, because there is little water to absorb the heat or in

other words mean specific heat capacity of the fermenting mass is much lower than that of water.

Therefore, heat generated must be dissipated immediately as most of the microorganisms used in SsF are

mesophilic, having optimal temperature for growth between 20 and 40 °C and maximum growth below

50 ºC [Manpreet et al., 2005].

5.3.9 Effect of Inducers for SsF

A result of our study directs that inducers play a significant role in enhancing the production of

laccase. In the result pyrogallol enhanced the laccase production by two fold giving a yield of 72 U/gm

against the control 32 U/gm (Fig. 5.12). When an inducer is included several factors are important. The

chemical nature, the amount, and the time of addition of the inducer can influence laccase production.

Many compounds have been tested and shown to improve laccase production. Phenolic compounds that

are related to the natural substrate lignin or lignin derivatives are good inducers.

Fig 5.12 Effect of Inducers on Laccase production in SsF by Streptomyces chartreusis


113

The production of laccase can estimulated by the presence of a wide variety of an inducing

substrate, mainly aromatic and phenolic compounds related to lignin or lignin derivatives, such as ferulic

acid, and anilines such as o- and p-anisidine [Barbosa et al., 1996], phenols such as guaiacol, several

derivatives of benzoic acid such as vanillic acid and some lignin precusros such as ferulic acid and cupric

sulphate [Ikehata et al., 2004]. O-Anisidine caused a 62-fold increase in laccase production by T.

versicolor [Fahraeus and Tullander, 1956].

Earlier pyrogallol increased laccase production by Streptomyces psammoticus [Niladevi and

Prema, 2008], by Cerrena unicolor [Elisashvili et al., 2010] and in G. lucidum by 20 to 30 %

[Elisashvili et al., 2010]. Other inducers like ferulic acid, cupric sulphate, guaiacol, venilic acid and p-

anisidine were also found to be enhancing the laccase yield. Aromatic inducers and phenolic compounds

have been extensively used to extract improved laccase production by different organisms [De Souza et

al., 2004] and the personality of the compound that induces laccase production differs significantly with

the species.

5.3.10 Effect of Time Course Study

The result of time course of laccase production showed highest laccase production at 72 hrs with

biomass production of 39.8 mg/gm as shown in Fig. 5.13. This may be because laccase activity mounts up

during vegetative growth in accurate corresponding with mycelial mass, but go through rapid inactivation

shortly after the beginning of fruiting body formation [Thurston, 1994].

Fig. 5.13 Effect of time course study in laccase production by Streptomyces chartreusis


114

However, enzyme production was observed only after 24 hrs of incubation. Rapid and enhanced

enzyme production was recorded during 24 to 48 hrs and between 60 to 72 hrs. Results also indicated that

enzyme production was associated with actively growing log phase culture and not during stationary

phase, as the level of enzyme production remained stagnant during stationary phase of the culture. The

enzyme production was observed to be in linear relation with the biomass production as it is presented in

Fig. 5.13. The production of enzyme at a premature incubation time and the linear relation of enzyme

production with biomass were in fulfillment with the earlier report that ligninolytic enzyme production by

Actinomycetes is firmly a growth associated primary metabolic activity, while that of the fungi is a

secondary metabolic activity [McCarthy, 1987].

5.3.11 Screening of Variables for Laccase Production by SsF as per Plackett and Burman Design

Streptomyces chartreusis produces 72 U/gm of laccase [Chhaya and Modi, 2013b] during

primary screening at shake flask level under SsF. Variations ranging from 11.10 U/gm to 700 U/gm in the

production of laccase in 16 runs were observed in plackett- Burman experiments (Table 5.2). To enhance

the production of laccase, statistical method of medium optimization suggested by Plackett and Burman

was applied. A total of twelve variables with three dummy variables were analyzed with regard to their

effects on laccase production using a Plackett–Burman design (Table 5.2).

Statistical procedures have advantages basically due to utilization of fundamental principles of

statistics, randomization, replication and duplication [Rao et al., 2004]. The classical method follows

simultaneous optimization of each component by varying the concentration of only one of the component

and keeping all other at a constant value. This results in a large number of experiments which is both

costly and time consuming. Therefore, Plackett-Burman screening method was used for the purpose of

screening the medium components that indeed effected the production. The effectiveness of the medium

constituents was determined according to the test of significance (student’s t test, P value and confidence

level).


115

Table-5.2 Plackett-Burman design matrix of twelve variables (K1-K12) and three dummy variables

(D1-D3) along with observed response (Laccase production)

RunNo.

K1 K2 K3 K4 K5 K6 K7 K8 K9 K10 K11 K12 D1 D2 D3 ResultsU/gm

R1 + - + + - + - + - + - - + - + 456

R2 + + - + + - + - + - + - - + - 34

R3 - + + - + + - + - + - + - - + 121.56

R4 + - + + - + + - + - + - + - - 33.45

R5 - + - + + - + + - + - + - + - 700

R6 - - + - + + - + + - + - + - + 290.77

R7 + - - + - + + - + + - + - + - 31.23

R8 - + - - + - + + - + + - + - + 56.14

R9 + - + - - + - + + - + + - + - 46.78

R10 - + - + - - + - + + - + + - + 96.30

R11 + - + - + - - + - + + - + + - 90.14

R12 - + - + - + - - + - + + - + + 28.15

R13 + - + - + - + - - + - + + - + 60.56

R14 + + - + - + - + - - + - + + - 98.14

R15 - + + - + - + - + - - + - + + 61.27

R16 - - - - - - - - - - - - - - - 11.10

The design matrix selected for the screening of significant variables for laccase production and

the corresponding responses are shown in Table 5.3. The standard error (S.E.) of the concentration effect

was the square root of the variance of an effect and the significance level (p value) of each concentration

effect was measured using student’s t test, t(xi) =Exi/S.E where, Exi is the effect of variable xi. The

selected variables for the present study were carbon sources (dextrose, sucrose); nitrogen sources (yeast

extract); inducers (cupric sulfate, o- anisidine, p- anisidine and pyrogallol) and process parameters like

pH, time course, moisture content, particle size and temperature.


116

Table 5.3: Statistical analysis of variables in relation to Laccase production as per Plackett-

Burman design

Factors Variables Effect S.E. t(xi) p-value Confidence

level (%)

K1 Cupric sulphate -64.37 37.95 1.69 0.18 81.2

K2 Moisture content 21.94 37.95 0.57 0.60 39.6

K3 Time Course 13.18 37.95 0.34 0.75 24.9

K4 o-anisidine 92.36 37.95 2.43 0.09 90.7

K5 Particle size 76.66 37.95 2.01 0.13 86.3

K6 Sucrose 0.42 37.95 0.01 0.99 0.8

K7 Temperature 8.71 37.95 0.22 0.83 16.7

K8 Pyrogallol 187.93 37.95 4.95 0.01 98.4

K9 Yeast extract 121.46 37.95 3.2 0.04 95.1

K10 Dextrose 126.03 37.95 3.32 0.04 95.5

K11 p-anisidine 107.55 37.95 2.83 0.06 93.4

K12 pH 9.51 37.95 0.25 0.81 18.2

These twelve variables were chosen based on the earlier screening discussed before and were

evaluated in successive experiments. The confidence level of moisture content, time course, pH,

temperature, o-anisidine, p-anisidine, sucrose and cupric sulphate were below 95 % and hence were

considered insignificant, while the remaining variables dextrose, yeast extract and pyrogallol found above

95 % confidence level and selected for further optimization.

These results show the effectiveness of the Plackett-Burman design in recognizing the factors

with a significant influence on the laccase production. Subsequently the exact optimal values for the

individual factors were determined using central composite design experiments.


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5.3.12 Optimization of Screened Medium Components for Laccase Production by SsF using

Central Composite Design

Based on the results of Plackett-Burman Design the component with a significant confidence

level (dextrose, pyrogallol and yeast extract ) were set at their higher level, while the components with a

confidence level below 95% were set at their middle level. Table 5.4 represents the experimental design

for Central Composite Design and the results obtained for laccase production. The variables showing

positive effect with a confidence level of 98.4% (pyrogallol), 95.5% (dextrose) and 95.1% (yeast extract)

in the Plackett-Burman design were selected.

The variables used for factorial analysis were dextrose, pyrogallol and yeast extract for laccase

production. The actual and coded factor levels of laccase production are presented in Table 5.4. The data

were analyzed by a quadratic multiple regression using a Design- Expert® software (version 7.0.2, Stat

ease Inc., USA) and the following equation was obtained.

Y =234.30-65.24A-19.21B+33.27C+91.13AB-68.38AC-23.37BC+77.49A2-35.12B2-26.99C2…(iv)

Here Y is the predicted response and A, B, C, are the coded variables for pyrogallol, yeast extract and

dextrose respectively. To validate the regression coefficient, an analysis of variance (ANNOVA) of the

laccase production was performed (Table 5.4).


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Table-5.4 Central Composite Design matrix with coded values and actual values for Laccase

production.

Run

No.

Pyrogallol Yeast extract Dextrose Laccase in U/gm

Codedvalue

Actualvalue

Codedvalue

Actualvalue

Codedvalue

Actualvalue

Actualvalue

Predictedvalue

1 + 2 1.17 0 2.25 0 4.50 321.00 300.24

2 0 0.75 0 2.25 -2 1.98 123.00 124.26

3 - 2 0.33 0 2.25 0 4.50 109.00 126.31

4 0 0.75 +2 3.51 0 4.50 320.00 314.83

5 - 1 0.50 -1 1.50 +1 6.00 550.00 550.27

6 0 0.75 0 2.25 +2 7.02 123.00 100.79

7 +1 1.00 +1 3.00 -1 3.00 289.00 282.84

8 0 0.75 -2 0.99 0 4.50 182.00 197.86

9 0 0.75 0 2.25 0 4.50 560.00 563.19

10 -1 0.50 +1 3.00 +1 6.00 340.00 343.74

11 +1 1.00 -1 1.50 -1 3.00 145.00 167.28

12 0 0.75 0 2.25 0 4.50 118.00 102.65

13 +1 1.00 -1 1.50 +1 6.00 100.00 102.02

14 0 0.75 0 2.25 0 4.50 209.00 213.91

15 -1 0.50 -1 1.50 -1 3.00 302.00 234.30

16 +1 1.00 +1 3.00 +1 6.00 267.00 234.30

17 0 0.75 0 2.25 0 4.50 250.00 234.30

18 0 0.75 0 2.25 0 4.50 154.00 234.30

19 0 0.75 0 2.25 0 4.50 234.00 234.30

20 -1 0.50 +1 3.00 -1 3.00 200.00 234.30


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Table 5.5: Analysis of variance (ANNOVA) for the quadratic model

The goodness of fit of the model was checked by the determination coefficient (R2) as shown in

Table 5.6. The values of the adjusted determination coefficient (Adj R2=0.9519) was also very high

reconfirming the significance of the model. The lack of fit (0.9620) is found to be not significant. This

indicates an excellent correlation between the experimental and predicted values of laccase production.

At the same time relatively low coefficient variation (CV=16.26%) (Table 5.6) confirms the

precision and reliability of the experiment performed. Fig. 5.14 represents the relationship between the

actual laccase production and predicted values determined by the model equation (1) for Streptomyces

Sum of Source Squares Mean df F Square p-value Prob > F

Model 3.136E+005 9 34847.71 21.99 < 0.0001

A-Pyrogallol 58129.86 1 58129.86 36.68 0.0001

B-Yeast Extract 5042.02 1 5042.02 3.18 0.1048

C-Dextrose 15113.47 1 15113.47 9.54 0.0115

AB 66430.13 1 66430.13 41.91 < 0.0001

AC 37401.12 1 37401.12 23.60 0.0007

BC 4371.12 1 4371.12 2.76 0.1278

A2 86524.95 1 86524.95 54.59 < 0.0001

B2 17776.58 1 17776.58 11.22 0.0074

C2 10497.85 1 10497.85 6.62 0.0277

Residual 15849.83 10 1584.98 - -

Lack of Fit 2326.33 5 465.27 0.17 0.9620

Pure Error 13523.50 5 2704.70 - -

Cor Total 3.295E+005 19 - -


120

chartreusis. Clearly most of the points were near by the line adjustment which meant that the

experimentally determined values were similar to those determined by the model.

Table 5.6 Predicted R2 verses adjusted R2 value

Sum of Source Value Sum of Source Value

Std. Dev. 39.81 R-Squared 0.9519

Mean 244.80 Adj R-Squared 0.9086

C.V. % 16.26 Pred R-Squared 0.8826

PRESS 38682.80 Adeq Precision 16.425

Fig. 5.14. Predicted v/s Actual Laccase productions from Streptomyces chartreusis strain NBRC

12753.

Contour graph were obtained and analyzed based on feeding data on the laccase production in to

the design expert software. The software allows the laccase production to be predicted within the studied

range for the all three components of medium.


121

Figure: 5.15 Three dimensional Contour graph effect of Pyrogallol and Yeast extract at 6.0 (g/L) of

Dextrose.

Figure 5.16 Three dimensional Contour graph effect of Pyrogallol and Dextrose at 1.50 (g/L) of

Yeast extract


122

Figure 5.17 Three dimensional Contour graph effects of Yeast extract and Dextrose at 0.50 (mL/L)

of Pyrogallol.

The three dimensional response surface contour graph of laccase production based on the final

model are shown in figure 5.15 to 5.17 which were generated in pair wise combination of the three factors

while keeping the other one at its optimum level. Here each contour surface plot represents the effect of

two medium components at their studied concentration range, when the other components have a fixed

concentration. The values of the other components were then varied for that situation using the software

to determine the optimum values.

Based on the results of Plackett-Burman design the component with a significant confidence level

(pyrogallol, yeast extract and dextrose) were set at their higher level, while the components with a

confidence level below 95% were set at their middle level. Additional optimization was achieved using a

3-D surface plots (Fig. 5.18, 5.19, 5.20) were obtained and analyzed based on feeding data on the laccase

production in to the Design- Expert® software.


123

Figure: 5.18 3D Surface Plot effect of Pyrogallol and Yeast extract at 6.0 (g/L) of Dextrose.

Figure 5.19 3D Surface Plot effect of Pyrogallol and Dextrose at 1.50 (g/L) of Yeast extract


124

Fig. 5.20 3D Surface Plot effect of Yeast extract and Dextrose at 0.50 (mL/L) of Pyrogallol.

5.4 CONCLUSION

The outcome of medium components on the production of laccase by Streptomyces chartreusis

NBRC 12753 through SsF was studied using Plackett-Burman design. Medium components such as

pyrogallol, yeast extract and dextrose were found to influence the laccase production significantly. These

variables were selected for further optimization studies using RSM.

Response Surface Methodology was performed to optimize the medium components for laccase

production. A highly significant quadratic polynomial obtained by the central composite design was very

useful for determining the optimal concentrations of constituents that have significant effects on laccase

production. The production of laccase was 72 U/gm under the influence of pyrogallol as an inducer. After

the optimization of medium components by above mentioned approaches the increment of laccase

production was very appreciable, it was 700 U/gm greater production of laccase than the production of

optimal medium. Different statistical methods for medium optimization have been employed to improve

laccase production by Streptomyces chartreusis. The medium for laccase production was optimized by

Response Surface Methodology using Central Composite Design, and a 10 times increase in the laccase

activity was achieved. This methodology could therefore be successfully employed to any process, where

an analysis of the effects and interactions of many experimental factors are required. The maximum

information can be obtained by Central composite experimental design, while required very little amount

of the individual experiments.

chapter-5 optimization of laccase production by...

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