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Chapter-5
Optimization of Laccase
Production By Solid State
Fermentation
Optimization of Laccase Production by Solid State Fermentation
94
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
Optimization of Laccase Production by Solid State Fermentation
95
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
Optimization of Laccase Production by Solid State Fermentation
96
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
Optimization of Laccase Production by Solid State Fermentation
97
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].
Optimization of Laccase Production by Solid State Fermentation
98
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.
Optimization of Laccase Production by Solid State Fermentation
99
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.
Optimization of Laccase Production by Solid State Fermentation
100
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)
Optimization of Laccase Production by Solid State Fermentation
101
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.
Optimization of Laccase Production by Solid State Fermentation
102
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
Optimization of Laccase Production by Solid State Fermentation
103
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
Optimization of Laccase Production by Solid State Fermentation
104
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
Optimization of Laccase Production by Solid State Fermentation
105
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..
Optimization of Laccase Production by Solid State Fermentation
106
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
Optimization of Laccase Production by Solid State Fermentation
107
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
Optimization of Laccase Production by Solid State Fermentation
108
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
Optimization of Laccase Production by Solid State Fermentation
109
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
Optimization of Laccase Production by Solid State Fermentation
110
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.
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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
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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
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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
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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).
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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.
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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 - -
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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.
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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
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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.
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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
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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.