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Chapter 1 Introduction

Chapter-1 Introduction

Ph.D. Thesis, UICT, North Maharastra University, Jalgaon 1

CHAPTER – 1

INTRODUCTION

The immense potential of fermentation lies in food processing and enzyme production

from agro-waste that has led to major research initiatives in this field. This acts as

motivation, leading the researchers to focus on its various aspects and contribute

scientifically through basic as well as applied research work. Chapter 1 summarizes

the fermentation process and various techniques involved in it.

1.1 Fermentation

Fermentation is a natural process which utilizes various micro-organisms to transmit

mixtures of solid – liquid substrate into various valuable products. In most of the

fermentation process, it requires a single species of micro-organisms to effect the

desired chemical change. In major of fermentation process are classified as either (i)

solid state or (ii) submerged culture. The basic difference between solid state

fermentation (SSF) and submerged fermentation (SmF) is that free flowing water is

present in SmF while it is absent or very minor in SSF. The role of the water content

in the substrate has been widely studied and reviewed by many researchers [1,2,3]. The

performance of SSF process depends on interface between the three key components

of the system (i) bioreactor (ii) substrate and (iii) micro-organism [5,6]. Relative studies

between SSF and SmF claim higher yields and few other advantages for products

made by SSF. Since SSF has found to be a good alternative in various applications[4],

this research is focused on SSF, specifically on PBSSF, since it is less energy

intensive with less capital investment. The pros and cons of the SSF process have

been deliberated further.



1.2 SSF: A unique fermentation process

SSF is defined as a fermentation process in which micro-organisms grow on solid

materials in the absence of free liquid [9]. SSF is mostly used for food processing and

production of enzymes using filamentous microorganism like fungi. In SSF technique,

microorganisms are grown on and inside the humidified solid substrate. Trinci

indirectly indicated that many of the filamentous fungi basically live and grow on

solid substrate[7]. Overall efficiency of the SSF basically depends on three ‘E’ factors

i.e. Energy, Economy and Environment [8]. In SSF, substrate itself is the source of

energy and requires no medium for growth of micro-organism. These solids are

polymeric in nature made up of carbohydrate and or proteinaceous in nature having

no tendency to break or stick with each other. SSF is heterogeneous in nature due to

the presence of three phase’s i.e. continuous air phase, which flows continuously

through solid phase substrate with biomass, and liquid phase as water or moisture

present in the substrate. SSF technology has been conventionally more applicable for

filamentous fungi, which grow on the surface of the solid particles. SSF is a well

adapted and cost effective process for the production of bio-products at a large

spectrum and has been used widely in Asian and African countries [10]. The SmF

method requires more cooling water and electricity than the SSF process, which

results in higher costs of utilities for the SmF method. In SSF downstream processing

is easy and cost effective than SmF. SSF technology did not get wide acceptance in

European countries, however owing to the benefits offered by SSF it is getting

acceptance [11]. Invariably use of SSF leads to minimization of the various problems

related to soil pollution, the potential application in bioremediation and coming up as

alternative for animal feeding lead to acceptance in European continent. SSF is a key

process and widely used for producing cellulase enzyme with a wide range of natural



substrate. SSF is very complex process where the fungal hyphae forms a mat on the

substrate surface and also penetrates through the substrate. SSF needs close process

parameters monitoring and controlling which is very difficult practically. In major

cases filamentous fungi is used in SSF while only in a few cases bacteria and yeasts

can be used [12]. Various bacteria, yeasts and fungi can secrete cellulases. Structurally

fungal cellulases are simpler as compared to bacterial cellulase systems, cellulosomes

[13,62-64]. Growth of filamentous fungi in SSF is an aerobic process. Previously, SSF

process was famous for “low volume - high cost” products due to its critical technical

problems associated with heat and mass transfer for large capacity. But due to

advancement in SSF technology, this process is inclining towards “high volume - high

cost” products. Further, classification of SSFr needs to be understood to narrow down

on a specific SSFr for a specific applications which is discussed in the below sub

section.

1.3 Types of solid state fermenter:

The SSF bioreactors are broadly classified into diverse types by various researchers

[10-11,61,73,76]. Majority of the researchers divided the SSFr broadly into following two

types, (i) small scale and (ii) large scale fermenter. According to the operations, the

SSFr can be divided into batch mode or continuous mode fermenter. Packed bed and

tray fermenter are used for batch SSF processes. The tunnel, paddles and rotating

drum fermenter are used for continuous or batch SSF processes. Based on design and

construction, the SSFr divided mainly into four types [10].

(i) Tray type fermenter

(ii) Fluidized bed type fermenter



(iii) Horizontal drum type fermenter and

(iv) Packed bed type fermenter

Generally, for large scale production, SSFr employs either tray type or drum type

fermenter. Even packed bed column can be used for large capacity; if operated in the

intermittently mixed mode. A leading enzyme manufacturer in India, ‘BIOCON’ uses

try type fermenter for large capacity production of immuno suppressants [61,77,78]. Each

fermenter having their own advantages and disadvantages hence the application and

ease of operations decide the selection of fermenter. Based on mixing and aeration

Mitchell[79] divided these SSFr into four group (Table 1.1).

Table 1.1 SSFr groups based on mixing and aeration[79]

Group-I Where bed is static or mixed only very infrequently and air is

circulated around the bed

Example: Tray column

Group-II Where bed is static or mixed only very infrequently and air is

passed forcefully through the bed.

Example: Packed bed fermenter

Group-III Where bed is continuously mixed and air is circulated around the

bed.

Example : Rotating drum fermenter

Group IV Where bed is agitated and air is passed forcefully through the

bed.

Example: Gas- solid fluidized bed

These groups are discussed in detail below



1.3.1 Group - I

A variety of SSFr have been designed and constructed in many small, pilot and large

scale applications. Koji fermenter is widely practiced in Japan. Koji is the simplest

and without forced aeration reactor which is a typical example of SSFr. Traditionally,

Koji fermentation is performed in wooden and bamboo trays these materials which

are now replaced by stainless steel chamber and trays. This type of SSFr is also

known as tray fermenter where trays are arranged one above the other with

appropriate gap between them. Thickness of the substrate bed within trays is between

5 to 15 cm deep. The bottom and sides of the trays are perforated for aeration.

Humidified air is circulated for heat transfer within the chamber to maintain uniform

temperature. However, loading and unloading is labor intensive and require large

operating area. Koji comprises soybean or other grains on which mould is grown,

which is traditionally used in oriental food preparation over the thousands of years

[10,61,80,81].

1.3.2 Group-II

Packed bed type fermenter comprises substrate kept in a natural way without any kind

of artificial mass transfer and facilities like agitation or mixing where the

microorganisms do not tolerate mixing. Packed bed is usually composed of a

cylindrical or rectangular column, oriented vertically, with the solid substrate retained

on a perforated plate. This column needs to be cooled by aeration in majority to avoid

overheating. Air is blown up through the perforated plate. Detail literature of this

column has been discussed in Chapter 2.



1.3.3 Group-III

Rotating drum fermenter mixes the substrate continuously or intermittently. If mixing

is done by the rotation of drum hence called as rotating drum fermenter. In this mode,

cylindrical drum is moving along the central axis. Typically with low speed of 1-15

rpm has been recommended. If a rotational rate is less than 10% of the critical speed

then it may be essential to include baffles within the drum[79]. Intermittent rotation is

potentially less destructive to fungal mycelium than the continuous rotation.

Efficiency of rotating and stirred drum fermentor depends strongly on the water

evaporation rate and the heat transfer between the solid bed and head space. In stirred

drum fermenter, straight agitator and curved agitator performance on comparison

shows that curved agitator gives better mixing in both radial and axial direction. [11,82-

84].

1.3.4 Group-IV

Gas solid fluidized bed fermenter is having perforated plate through which gas is

passed forcefully in upward direction so that substrate gets fluidized. Substrate used

in this bed essentially needs to be non sticky, having uniform size without

agglomeration. In such type of fermenter, controlling the bed temperature is

comparatively easy since, the gas flow rate is maintained high in fluidized the

substrate which makes heat transfer effective through convective cooling. This type of

fermentor needs to have enough height to allow bed expansion. The upper part of the

fermenter is needed to be wider than the bottom part and such shape of reactor helps

to separate substrate particles from each other [79,85]. It can be concluded from the

above discussion that choice of SSFr and quantum of process is dependent upon the

substrate, microorganism characteristics etc., therefore lot of changes were warranted

and researchers tried some innovations in SSF which is discussed below.

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suitable for closed static fermentation to produce value added product. Rivela and

Couto [90] worked on a new fermenter called as Immersion Fermenter. This fermenter

consisted of a jacketed cylindrical glass vessel with round bottom. Inside the vessel

many wired mesh baskets are placed which are colonized by the fungus for

fermentation.

Tunnel fermenter is an alteration of the static – bed fermenter. In tunnel fermenter the

bed of the solid is comparatively very long, but height is not more than 0.5 m. Perez-

Correa and Agosin[91] developed, a 50 kg capacity Rotating Basket Fermenter where

stationary blades were provided for mixing and nozzles were provided for water

sprinkling. Air can be passed from bed of substrate through perforated plate, provided

at the bottom of reactor. In this manner, various researchers applied several other

types of design in SSFr to fulfill their specific task. In the last few years, researchers

focused on packed bed column, as this type of fermenter can provide improved

process economics and easier material handling [92]. The importance of packed bed

column in SSF is again due to their simple design and construction. These innovations

in fermenter are widely accepted and hence applied for the production of enzymes on

commercial scale. The enzyme production by SSF is discussed in the below

subsection.

1.5 Enzyme production by SSF

Enzymes are among the vital products produced mostly by SSF. Generally, hydrolytic

enzymes (e.g. cellulases, xylanases, pectinases etc.)[93] are produced by fungal

cultures which are filamentous in nature and hence SSF is the method of choice for its

production. In current studies, SSF has been implied for the production of cellulases

by a locally isolated micro-organism. Cellulose is one of the most abundant and



renewable carbohydrate biopolymer on the earth. Cellulose is used as an energy

source by various bacteria, yeasts and fungi. Proper biotechnological utilization of

these materials in the environment has potential to eliminate pollution and convert

cellulosic biomass into useful by-product. Cellulases produced by microbes are a

group of hydrolytic enzymes capable of degrading cellulose to the smaller

monosaccharide glucose units [14]. It has been reported that SSF is an attractive

process to produce cellulase economically due to its lower capital investment and

operating cost [15]. For the maximum yield of enzyme, it is essential to provide

optimum parameters initially for the growth of organism followed by optimum

parameters for products formation. The optimum parameters include physico-

chemical parameters like substrate, inoculums concentration, humidity, pH,

temperature etc. In case of fungi, temperature is a very important parameter. Hence,

fungal growth in SSF is significantly affected by the temperature of the reactor. The

substrates used for SSF have low thermal conductivity which provides resistance to

heat transfer. Due to this metabolic heat accumulate in the bed which increases

temperature of the bed which may not be favorable for fungi. Therefore the key issue

in SSF is heat removal, and has become a focal point in research on SSF. Even

reducing the substrate bed height can solve the heat transfer problems, but it is

possible only in small scale SSF [16] while it remains unanswered in case of large scale

SSF.

1.5.1 Cellulase

Cellulase is an enzyme having potential applications in various industries.

Lignocellulosic agro-waste is the substrate choice for cellulase production which

decreases the cost of cellulase production. Lignocellulosic materials are cheaper and

abundantly available but needs pretreatment for its effective utilization. These



enzymes produced by a large number of microorganisms including both fungi and

bacteria. These microorganisms can be aerobic, anaerobic and thermophilic. SSF is

mostly preferred for cellulase production due to its lower capital investment and

operating cost. Cellulases have been commercially available from long time, however,

the challenge of increasing its yield has been a target for both academic as well as

industrial research [17,18]. Cellulase is a complex of enzymes having mainly endo and

exoexo-1,4 glucanase and β – glucosidase activities. Cellulases are used for the

production of glucose, ethanol, extraction of fruit and vegetable juices, deinking of

the recycled paper pulp and for improving cattle feed quality [10]. Tenerdy (1996)[19]

compared cellulase production in SmF and SSF system and interestingly found that

SSF was economical technology for the production of cellulase. Conversely, total

enzyme production was 12 times higher in SSF than in SmF under similar

experimental conditions [4]. The production of cellulase by SSF technology has been

studied by many researchers using different microorganisms, substrates and media.

Agricultural residues available abundant in quantity such as sorghum , wheat , rice

straw, bagasse, banana wastes, coconut coir pitch, coffee pulp, soybean hulls etc. can

be used for cellulase production. High content cellulosic composition is good for

fungal growth and cellulase production. Generally, these cellulosic materials need to

be pretreated for the efficient synthesis and production of enzyme cellulase. Fungal

strains that produce cellulases mainly are members of genera Aspergillus,

Trichoderma, Penicillium, Fusarium genera, Clostridium, Cellulomanas and

Thermomonospora [20,46-49]. Fungal strains of Aspergillus (one of the oldest named

genera of fungi, Aspergillus received its name from Micheli in 1729) are high in β –

glucosidase activities where as the funal strains of Trichoderma are poor in β –

glucosidase activities. Trichoderma shows high activity of endo and exo-glucanase



activity. In a few cases a combination of two fungi in SSF was found to enhance the

enzyme production. Brijwanit et. al. (2010)[20] used combination of T. reesi and

A.Oryzae in 1:1 ratio and found that the production of β – glucosidase level in SSF

increased when soybean hulls when used with wheat bran. Combination of

Aspergillus ellipticus and Aspergillus fumigates resulted in improved hydrolytic and β

– glucosidase activity [59,60]. Cellulase production is affected by various factors like

nature of solid substrate employed, particle size, water activity, temperature and pH,

etc. Table 1.2 indicates the bibliographic details of cellulases production by various

micro-organisms.

Table 1.2 Cellulases produced by various micro-organisms.

Sr.No.

Substrate Micro-organism Enzyme Reference(s)

1 Alfalfa Gliocladium spp.

Cellulase [22]

2 Cassava bagasse

Tricoderma reesei

Cellulase [36]

3

Chaff and Wheat bran mixture (Ratio of 7:3 (W/W))

Trichoderma viride

Cellulase [33]

4 Forage silage Streptomyces achromogenes

Cellulase Hemicellulase

[23]

5 Jowar straw (JS)

Aspergillus oryzae

Cellulase [32]

6 Rice straw (RS) Trichoderma

ressei

Cellulase [21]

7 Sugarcane bagasses

Trichoderma reesei

Phanerochaete Chrysoporium

Coriolus versicolor Streptomyces

viridosporus

Cellulase Ligninase

[29,30]



Sr.No.

Substrate Micro-organism Enzyme Reference(s)

8 Soy husks Coriolus

versicolor

Cellulase [31]

9

Soybean hulls and wheat bran (Ratio of 4:1 (W/W)

Mixtures of T.reesei and A.oryzae

Cellulase Β-

Glucosidase [20]

10

Sugar cane bagasse and wheat bran (Ratio of 80:20(W/W)

Trichoderma harzianum

Cellulase [34]

11 Vinegar waste Trichoderma

koniingii

Cellulase [17,35]

12 Wheat straw(WS)

Neurospora crassa

Thermoascus aurantiacus

Aspergillus niger several other

fungi

Cellulase Hemicellulase

[24,25,26]

13 Wheat bran

Trichoderma reesei

Aspergillus niger

Cellulase

[27,28]

In various agricultural residues mixed cultivation results in a better cellulase

production with efficient lignocelluloses degradation [39,57]. Various combinations of

cellulases, hemicellulases and pectinases were developed for better growth of crops

and controlling their diseases [17]. Microbial cellulases have become a crucial

biocatalyst due to their complex nature and extensive industrial applications. Different

combination of cellulases, hemicellulases and pectinases have potential applications

in agriculture to enhance growth of crops and control of crop diseases [37,38]. The

economy of cellulase production could be enhanced by the use of cheaper cellulosic

natural substrate [36,46, 53-56]. To produce these enzymes, the substrates are the key

ingredients, and are discussed in the below section.



1.6 Substrates for SSF

India is a large agricultural country. Varity of crops is produced in large quantities

across India e.g. wheat, rice, cotton, soyabean, jowar and tur etc. Crop waste referred

as agrowaste or straw is having economical value due to its nutritional potential and

can be used as substrate in SSF. Presently in most of the cases, these straw materials

are used either as animal feed or most of the material for burning as fuel or an easy

way of disposal resulting environmental pollution. After processing these materials in

SSFr, its nutritional value can be increased for use as animal feed while enzyme can

be separated a main product. However, it depends on the type of fungus used for

fermentation. This technique has a potential to increase the income of rural people. In

SSF processes, substrate is a source of energy for microorganisms. In nature,

lignocellulose is synthesized in tremendous amount in the form of grass, wood,

agricultural residues and forestry wastes etc. Solid substrate employed in SSF

processes are insoluble in water and act as a source of carbon, nitrogen and minerals

required for growth. Filamentous fungi penetrate deeply into solid substrate particles

to avail the required nutrients whereas, bacteria and yeast grow only on the surface of

the solid substrate particles [40].

Due to inadequate mixing characteristics of heterogeneous media of SSF, it is very

difficult to control parameters like pH and temperature of the SSF system [94].

Therefore, to address these challenges research work is essential to understand this

process critically. The ratio of C:N of the substrate carries high importance in view of

selection of substrate which generally needs to be 16.0. However, it is difficult to get

so, hence it needs to select substrate having better C:N ratio. Hence, various substrate

locally available having comparatively better C:N ratio is very important in view of

making the SSF process economically viable. Among various agrowaste available in



India especially in Maharashtra state, wheat, jowar and rice straw are abundantly

available. Jowar crop is very much habitual to unfavorable environmental conditions,

like drought and salinity hence; in the drought prone area it is always available.

Generally, a single type of substrate or mixture of two or more substrates can be used

in SSF. Mostly, all these solid wastes show the presence of nutrients and moisture

hence suitable for micro-organisms growth. Many of cost effective substrates are

generally insoluble in water and water is absorbed by it. This absorbed water supports

the biochemical reactions of life and substrate is used by microorganisms for growth.

The selection of a substrate for SSF process depends upon several factors, however;

their screening mainly depends on the C:N ratio as discussed, cost and its availability.

Substrate can be divided into three groups, substrate with soluble sugar, starchy

substrates and cellulosic substrate. Table 1.3 gives bibliographic revision of various

substrates used in SSF process.

Table 1.3 Various substrates used in SSF [9,31,60,72-75,95].

Sr. No. Substrate Materials

(i) Substrate

With

soluble

sugar

Banana waste, apple pomace, Sweet lime waste, grape

pomace , sweet sorghum waste, sugar beet waste, Kiwi

fruit peel ,pineapple waste, mixed fruit waste, Orange

peels and hemp with soluble sugars etc.

(ii) Starchy

substrate

Rice, cassava, buckwheat seeds, corn meal, sweet potato

residue, banana meal, jack fruit seed waste etc.

(iii) Cellulosic

substrate

WS, JS, corn rice stover , rice bran, wheat bran, sugar

beet pulp, TS, Soyabean straw, wood etc.



The mixtures of many different, two substrates have been reported such as mixtures of

soybean hulls with wheat at the ratio of (4:1 W/W) [20], groundnut oil cake with wheat

bran (1:1 W/W)[50], olive oil cake with sugarcane bagasse (1:1 W/W)[51], coconut oil

cake with sesame oil cake (1:1 W/W)[52] etc. The water activity plays a very crucial

role in SSF. Water activity (aw) of substrate is very crucial parameter in SSF. The aw

gives the amount of unbounded water available in the surroundings of the micro-

organism. The water activity (aw) is defined as the ratio of vapor pressure of an

aqueous solution to that of pure water at the same temperature [58]. It is closely related

,but not equal to, the water content of the bed. Generally in SSF low water activity

(aw) is preferred. Since, fungi can grow in SSF culture at low aw and low aw helps

avoiding bacterial growth because bacteria needs high aw for their growth[65-67].

Fermenter size reduces and a more concentrated product is produced due to low aw

requirement in SSF[68]. But, the substrate should have adequate moisture to support

growth and metabolic activities of the organisms. Derived water activity value

depends on the types of microorganism employed and substrate engaged in the

process. Fungi are selected as micro-organism for SSF because their hyphae can grow

on particle surfaces and penetrate into the inter-particle spaces efficiently and thereby

colonize solid substrates [41]. Lignocellulose material mainly consists of three type of

polymers (i) cellulose, (ii) hemicelluloses, and (iii) lignin. Studies with inert

substrates like polyurethane foam wetted with dissolved nutrients have been also

reported in literature [42]. In most of the SSF process, substrate needs preparation and

pre-treatment before loading it into fermenter, involving size reduction by grinding,

screening substrate particle sizes, cooking or vapor treatment, supplementation with

nutrients and pH settling. After treatment substrate should have high surface area to

volume ratio and absorb water amounting one or several times to its dry weight. The



C:N ratio plays very crucial role for selecting proper substrate for SSF and selection

of substrate also depends on the enzyme of interest for example, for the production of

cellulase, cellulosic rich substrates can be selected like RS, JS and WS. In the same

way, for the synthesis of pectinases substrate like apple, avocado pulp (generally fruit

pulp) can be selected [43]. Poor thermal conductivity of substrate presents a great

challenge to design SSFr. The particle shapes of substrate affect the flow pattern in

the bed, which also affects O2 distribution inside the bed. Fig. 1.3 shows the irregular

shapes and size of substrate after screening through a mesh size of 85 and Fig.1.4

shows the arrangement of wetted solid particles with a continuous gas phase in SSF

systems. Irregular shape of the substrate particles are bound to affect the packing

pattern inside the packed column. However, it is very difficult practically to find the

effect of these irregular shapes on various parameters of the SSF process, but it can

certainly be said that it has inherent ability to affect the fungal growth and

productivity of SSF.

Fig. 1.3:Size and shape of substrate after screening through a mesh size of 85.

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Advantages:

(i) Less water requirement

(ii) No foam formation inside the reactor

(iii) Low energy requirement

(iv) Downstream processing cost is very low and process is environment friendly

(v) Simpler technique

(vi) Better-quality and productivity of product

(vii) Low Substrate cost with abundant availability

(viii) Reduces the problems associated with solid wastes disposal.

(ix) Less chances of bacterial contamination

(x) Liquid waste is very less.

Thus, SSF gives numerous advantages for the production of bulk chemicals and

enzymes [69-71].SSF technology is still in research stage and waiting for advancements

in heat and mass transfer, biomass separation and process control etc. This technology

is also showing certain technical disadvantages [8-10,44] as listed.

Disadvantages:

(i) Uneven temperature gradients

(ii) Difficulties in handling large volume of solids

(iii) Difficulties in scale up

(iv) Difficulties in proper distribution of O2 to the biomass

(v) Difficulties in biomass determination

(vi) Longer cultivation duration

(vii) Channeling difficulties



(viii) Tedious and expensive substrate pretreatment methods

(ix) Labor intensive process and hard to control process parameters.

In spite of the fact that SSF poses enormous difficulties and critical process control,

low cost is the driving force behind its acceptance over the SmF and hence its

popularity is increasing day by day. Eventhough, there are many difficulties associated

with SSF. It has proved to be a cost effective technology for cellulase production.

Therefore, an important research task using local micro-organism on JS substrate in

SSF was undertaken where specific experiments related with process along with

modeling and parameter comparison of modeling were carried out. Keeping SSF as a

base further literature review on packed column was carried out in Chapter 2. All of

five objectives set for this research were as follows.

1.8 Objectives of the research:

The following are objectives set for carrying out the research on SSF

a. To find suitable local substrate for SSF

b. To find potent cellulase producer from local area

c. To find optimum parameters for lab scale SSF

d. To study axial temperature gradients formed inside the packed bed

e. The rigorous mathematical modeling and simulation to validate

experimental data to find out various parameters involved there in.



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