Fermentor systems

November 12

2012SUBMITTED TO: DR ZAFFAR MEHMOODSUBMITTED BY: TAHIRA KHAN HASSAN CHAUDHRY MADIHA HAMID KANWAL SHAHEEN AISHA NAEEM

Food Biotechnology

TABLE OF CONTENTS:

Introduction to fermentor systems…………………………………………... 1

Fermentor design……………………………………………………………... 3

Types of fermentor systems………………………………………………….. 7

Submerged culture fermentor……………………………………………….. 9

Solid state fermentor…………………………………………………………. 15

Applications and types of Fermentors used in food industry……………… 20

Murree brewery an industrial model fermentor……………………………. 26

References……………………………………………………………………. 37

FERMENTER SYSTEMS

Fermentation is carried out in vessels known as Fermenters.

A fermenter can be a simple vessel but if it is connected to complex integrated system of

automated control, then it is termed as fermenter system.

HISTORY OF FERMENTATION

Divided into four stages:

1. Pre 1900

2. 1900-1940

3. 1940-date

4. 1964-date

5. 1979 –date

1. First Stage:

Wooden vessels were used. These wooden vessels had the capacity of approximately 1500

barrels. In the later years the trend of carrying out fermentation in copper vessels was seen.

2. Second Stage:

Steel vessels were used which had the ample capacity of 200m3. Such vessels were mainly used

for acetone or butanol fermentation. Air spargers were also introduced in the second stage for the

aeration of yeast and mechanical stirring also came into practice for small vessels.

3. Third Stage:

Mechanical aerated vessels are among the salient features of the third stage. True fermenters i.e.

the ones which are operated aseptically also came into practice.

4. Fourth Stage:

Development of pressure cycle and pressure jet vessels occurred in fourth stage which led to the

exclusion of some major problems like gas exchange and heat exchange.

5. Fifth Stage:

Fermenters actually developed in the third and fourth stage. Along with fermenters animal cell

reactors were also developed.

FERMENTER VS BIOREACTOR

Fermenter system is used for the growth and maintenance of a population of bacterial or fungal

cells. While, a bioreactor is used for the growth and maintenance of a population of mammalian

or insect cells.

FERMENTER DESIGN

1. MATERIALS USED IN A FERMENTER:

Due to the strict aseptic environment, the materials used in fermenter should be able to withstand

repeated sterilizations usually done with steam. Secondly the use of appropriate material depends

mainly upon the scale. For a small scale, it is a common practice to use glass or stainless steel.

Glass because it has a smooth surface, it is non-toxic and corrosion proof and most importantly it

is easy to examine the interior of the vessel. Pilot-scale and industrial scale vessels are normally

constructed of stainless steel or at least have a stainless-steel cladding to limit corrosion.

2. CONDITIONS FOR A FERMENTER:

Following conditions should be met in order to make a proper fermenter and for it to work in an

efficient way. To achieve these, the fermenter should have:

• Heat and oxygen transfer configuration

• Impeccable sterilization procedures

• Foam control

• Fast and thorough cleaning system

• Proper monitoring and control system

• Productivity and yield

• Fermenter operability and reliability

• Product purification

• Water management

• Energy requirements

• Waste treatment

Other few significant things to be taken in account include:

• Design in features so that process control will be possible over reasonable ranges of

process variables.

• Operation should be reliable

• Operation should be contamination free

3. STERILIZATION:

A. Sterilization Of The Fermenter:

Fermenters are designed in such a way so that it may be steam sterilized with pressure when

necessary. Moreover the medium also needs to be sterilized either in the vessel or outside the

vessel before it is added into the vessel aseptically.

B. STERILIZATION OF AIR SUPPLY:

Aerobic fermentation processes an ample amount of air is needed and the air should be sterilized

before entering the fermenter. There are two conventional ways of sterilizing air i.e. is by heating

and by filtration. Heat is discouraged because of its being too costly to implement in full-scale

operation.

C. STERILIZATION OF THE EXHAUST GAS FROM THE

FERMENTER:

Exhaust gas can be sterilized by using filters of 0.2 µm placed on the exhaust pipe. Usually the

exhaust gas contains moisture and solid particles leading to aerosol formation. This can be

avoided by either using a cyclone operator before solids or the coalesce for liquids before the

exhaust pipe opening in series. To make sure that no viable cells are leaving the exhaust pipe.

The filter needs to be constantly checked.

4. SENSOR PROBES:

Glass electrodes are used as sensor probes but in order to seal these probes rings are used

commonly known as Double ‘O’ rings. These work as an aseptic seal and allow minimum

release of micro-organisms and if in any case a leakage is inevitable then there are simple

disinfection protocols to deal with it.

Pre-installed back up probes are also necessary because if in a case a probe fails then there is a

chance of leakage of broth if a retractable probe housing is used during the fermentation.

5. AGITATOR:

Also known as impeller. It is required for the purpose of mixing, e.g. broth mixing, transfer of

oxygen & heat, solid particles suspension and for maintaining a constant environment in the

fermenting vessel.

6. SPARGER:

Sparger is also called aeration system. It is a device for introducing air into the fermenter broth.

There are three standard types of spargers namely:

Porous sparger.

Orifice sparger. A perforated pipe.

Nozzle sparger. A partially closed pipe.

7. TEMPERATURE CONTROL:

Usually the heat produced by microbial activity and mechanical mixing is not enough and

external must be provided or the heat is too much and it must be removed from the system. This

can be done by silicone heating coils or the heating jacket through which the water is circulated.

For the large scale fermenters, internal coils and cold water circulation are preferred because of

the increase in surface area.

OPERATION OF A FERMENTER SYSTEM

Industrial fermentation processes may be carried out as batch fermentations, fed-batch operations

or continuous fermentations. Batch and fed-batch operations are quite common, continuous

fermentations being rare. The mode of operation is, to a large extent, dictated by the type of

product being produced.

BATCH FERMENTATION

Batch fermentation system is a closed culture system which contains an initial, limited amount of

nutrient. In batch processing, a batch of culture medium in a fermenter is inoculated with a

microorganism (the ‘starter culture’) and incubation is allowed to proceed under optimal

physiological conditions. In the course of the entire fermentation, nothing is added except

oxygen, antifoam and acid/base

to control the pH. Composition of the culture medium, biomass, and metabolite

concentration change constantly as a result of the cell metabolism. The fermentation proceeds for

a certain duration (the ‘fermentation time’ or ‘batch time’) and the product is harvested.

zThe inoculated culture will pass through a number of phases, as shown in figure. After

inoculation there is a phase during which it appears that no growth takes place; this period is

referred to as the lag phase and considered as a time of adaptation. Following a period during

which the growth rate of the cells gradually increases, the cells grow at maximum rate and this

period is known as log phase or exponential, phase. After the substrate is exhausted, the growth

ceases and this is called stationary phase. The depletion of substrate for maintenance and the

presence of toxic substances cause the cell death, called death phase.

Batch fermentations typically extend over 4-5 days, but some traditional food fermentations may

last months. Most beer breweries use batch processes commercially.

FED-BATCH FERMENTATION

A fed-batch is a biotechnological batch process which is based on feeding of a growth limiting

nutrient substrate to a culture. The fed-batch strategy is typically used in bio-industrial processes

to reach a high cell density in the bioreactor. Mostly the feed solution is highly concentrated to

avoid dilution of the bioreactor. The controlled addition of the nutrient directly affects the

growth rate of the culture and helps to avoid overflow metabolism (formation of side

metabolites, such as acetate for Escherichia coli, lactic acid in cell cultures, ethanol in

Saccharomyces cerevisiae), oxygen limitation (anaerobiosis).The volume of fermenting broth

increases with each addition of the medium, and the fermenter is harvested after the batch time.

CONTINUOUS FERMENTATION

In continuous fermentations, sterile medium is fed continuously into a fermenter and fermented

product is continuously withdrawn, so the fermentation volume remains unchanged .Typically ,

continuous fermentations are started as batch cultures and feeding begins after the microbial

population has reached a certain concentration. In some continuous fermentations, a small part of

harvested culture may be recycled, to continuously inoculate the sterile feed medium entering the

fermenter. ‘Plug flow’ fermentation devices, such as long tubes that do not allow back mixing,

must be inoculated continuously . Elements of fluid moving along in a plug flow device behave

like tiny batch fermenter. Hence, true batch fermentation processare relatively easily transformed

into continuous operations in plug flow fermenters, especially if p H control and aeration are not

required. Continuous cultures are particularly susceptible to microbial contamination, but in

some cases the formation conditions may be selected (e.g. low pH, High Alcohol or salt content)

to favor the desired microorganisms compared to potential contaminants.

TYPES OF FERMENTER SYSTEMS

Most commercially useful fermentations may be classified as either submerged cultures or

solid state fermentations. These are the two basic types of fermenter systems, which will be

discussed in detail.

Solid-state and submerged fermentations may be each subdivided- into oxygen requiring aerobic

processes, and anaerobic that must be conducted in the absence of oxygen. Examples of aerobic

fermentations include submerged-culture citric acid production by Aspergillus Niger and solid

state Koji fermentation (used in production of soya sauce). Fermented meat products such as

bologna sausage (polony) dry sausage, pepperoni and salami are produced by solid state

anaerobic fermenations utilizing acid-forming bacteria, particularly lactobacillus, Pediococcus

and mircoococcus species. A submerged culture anaerobic fermentation occurs in yogurt

making.

SUBMERGED CULTURE FERMENTER SYSTEM

Submerged fermentation is the cultivation of microorganisms in liquid nutrient broth. Submerged

fermenter systems may use a dissolved substrate e.g. sugar solution, or a solid substrate

suspended in a large amount of water to form a slurry. Submerged fermentations are used for

pickling vegetables, producing yoghurt, brewing beer and producing wine and soay sauce.

The process involves growing carefully selected microorganisms in closed vessels containing a

rich broth of nutrients (the fermentation medium) and a high concentration of oxygen. As the

microorganisms break down the nutrients, they release the desired enzymes into solution.

Fermentation takes place in large fermenter with volumes of up to 1,000 cubic metres. The

fermentation media sterilizes nutrients based on renewable raw materials like maize, sugars and

soya. Parameters like temperature, pH, oxygen consumption and carbon dioxide formation are

measured and controlled to optimise the fermentation process. Firstly, in harvesting enzymes

from the fermentation medium one must remove insoluble products, e.g. microbial cells. This is

normally done by centrifugation. As most industrial enzymes are extracellular (secreted by cells

into the external environment), they remain in the fermented broth after the biomass has been

removed. The biomass can be recycled as a fertiliser, but first it must be treated with lime to

inactivate the microorganisms and stabilise it during storage.

Advantages:

Measure of process parameters is easier than with solid-state fermentation.

Bacterial and yeast cells are evenly distributed throughout the medium.

There is a high water content which is ideal for bacteria.

Disadvantages:

High costs due to the expensive media

Large reactors are needed and the behaviour of the organism cannot be predicted at times.

There is also a risk of contamination

A typical submerged culture vessel has the features shown in the following figure:

SUBMERGED CULTURE FERMENTER DESIGN: (1)Reactor Vessel (2)Jacket

(3)Insulation (4)Protective Shroud (5)Inoculum Connection (6)Ports of sensors of pH,

temperature and dissolved O2 (7)Agitator (8)Gas Sparger (9)Mechanical Seal (10)Reducing

Gearbox (11)Motor (12)Harvest Nozzle (13)Jacket Connection (14)Sample valve with steam

connection (15)Sight Glass (16)Connections of acids, alkalis and antifoam agents (17)Air

inlet (18)Removable Top (19)Medium Feed nozzle (20)Air exhaust Nozzle -connect to

condenser, not shown (21)Instrumentation ports for foam sensors pressure gauge and other

devices (22)Centrifugal foam beaker (23)Sight glass with light-not shown and steam

connection (24)Rupture disc nozzle

DIFFERENT TYPES OF SUBMERGED CULTURE FERMENTERSThe major types of submerged cultures fermenter systems are as follows:

1. Stirred tank fermenter

2. Air lift fermenter

3. Bubble column fermenter

4. Fluidized-bed fermenter

5. Trickle-bed fermenter

1. STIRRED TANK FERMENTER

A stirred tank fermenter is the simplest type of fermenter system.  It is composed of a reactor and

a mixer such as a stirrer, a turbine wing or a propeller. This is a cylindrical vessel with working

height to-diameter ratio (aspect ratio)of 3-4. A central shaft supports three to four impellers ,

placed about 1 impellers diameter that direct the flow axially(parallel to shaft) or

radially(outwards from the shaft). Sometimes axial- and radial flow impellers are used on the

same shaft. The vessel is provided by four equally spaced vertical baffles, that extend from near

the walls in to vessels. Typically, the baffle width is 8-10% of the vessel diameter.

This reactor is useful for substrate solutions of high viscosity and for immobilized enzymes with

relatively low activity. However, a problem that arises is that an immobilized enzyme tends to

decompose upon physical stirring. This system is generally suitable for the production of rather

small amounts of chemicals.

2. BUBBLE COLUMN FERMENTER

A bubble column fermentation system is an apparatus used for gas-liquid reactions first applied

by Helmut Gerstenberg. This is a cylindrical vessel with a working aspect ratio 4-6. The

introduction of gas takes place at the bottom of the column and causes a turbulent stream to

enable an optimum gas exchange. In this way, the compressed gas provides agitation. It is built

in numerous forms of construction. The mixing is done by the gas sparging and it requires less

energy than mechanical stirring. The liquid can be in parallel flow or counter-current.

Bubble column reactors are used in various types of chemical reactions like wet oxidation, or as

Algae bioreactor. Although simple, it is not widely used because of its poor performance relative

to other systems. It is not suitable for very vicious broths or those containing large amount of

solids.

3. AIR LIFT FERMENTER

Air-lift bioreactors are similar to bubble column reactors, but differ by the fact that they contain

a draft tube. The draft tube may be an inner tube (called "air-lift bioreactor with an internal

loop”) or an external tube (called "air-lift bioreactor with an external loop”) which improves

circulation and oxygen transfer and equalizes shear forces in the reactor. In the internal-loop

designs the aerated riser and unaerated down corner are contained in small shell. In the external-

loop configuration, the riser and the down comer are separate tubes that are linked near the top

and the bottom. Liquid circulates between the riser (upward flow) and the down comer

(downward flow). The working aspect ratio of airlift fermenters is 6 or greater.

Generally, these are very capable fermenters, except for handling vicious broths. The ability to

suspend solids and transfer O2 and heat is good. The hydronamic shear is low. The external loop

design is relatively little-used in industry.

4. FLUIDIZED-BED FERMENTER

These are similar to bubble columns with an expanded cross section near the top. Fresh or

recirculated liquid is continuously pumped into the bottom of the vessel, at a velocity that is

sufficient to fluidize the solids or maintain them in a suspension.These fermenters need an

external pump. The expanded section slows down the local velocity of the upward flow, such

that the solids are not washed out of the bioreactors.

In this type of fermenter, a fluid (gas or liquid) is passed through a granular solid material at high

enough velocities to suspend the solid and cause it to behave as though it were a fluid. This

process, known as fluidization, imparts many important advantages to the FBR. As a result, the

fluidized bed reactor is now used in many industrial applications.

5. TRICKLE-BED FERMENTER

These consist of a cylindrical vessel packed with support material (e.g wood chips, rocks, plastic

structure). The support has large open spaces, for the flow of liquid and gas and the growth of

microoraganisms on the solid support. A liquid nutrient broth is sprayed onto the top of support

material, and trickles down the bed. Air may flow up the bed, countercurrent to the liquid flow.

These fermenters are used in vinegar production, as well in other process. These are suitable for

liquids with low viscosity and few suspended solids.

SOLID STATE FERMENTER SYSTEMS

SSF involves the growth of micro organisms on moist solid substrate where there is little water

in the spaces between the substrate molecules and a continuous gas phase. In the beginning it

was thought that liquid state fermentation or submerged fermentation (SLF) has more advantage

over SSF. But recent studies in the West have shown SSF to be the cheapest and more

environmentally friendly relative to SLF in the production of value added industrial based

products such as enzymes, bio fuels and the likes. However in the East it is still in the back

burners of the fermentation popularity just due to poor understanding and control of SSF.

Traditional uses of SSF systems include the production of fermented foods, pigments, and koji in

the Far East. Within the past decade, the production of other, higher-value microbial metabolites

such as antibiotics ,biopesticides , aromas, gibberellic acid and bacterial amylase to name a few,

have been evaluated with highly promising results. Bread, sausages, and soy sauce are also some

familiar products of SSF.

Some of the advantages of SSF are listed below:

The use of little moisture may facilitate the production of some specific compounds

which can’t be produced in SLF.

The products obtained in SSF are more thermo tolerant relative to those produced in SLF.

The low availability of water reduces the possibilities of contamination by bacteria and

yeast. This allows working in aseptic conditions in some cases.

Simply designed reactors with few spatial requirements can be used due to the

concentrated substrates.

DIFFERENT TYPES OF SS FERMENTERSThe design of the fermenter is very important for a fermentation process. Solid substrate

fermentation fermenters vary in technical sophistication from the very primitive banana leaf

wrappings to highly automated machines used mainly in Japan. There are many different types

of fermenters used for SSF. A few are explained below:

1. TRAY FERMENTERThis fermenter is one of the simplest and widely used fermenters. Its basic part is a wooden,

metal, or plastic tray, often with a perforated or wire mesh bottom to improve air circulation. A

shallow layer of less than 0.15 m

deep, pretreated (e.g., steamed)

substrate is placed on the tray for

fermentation. Temperature and

humidity-controlled chambers are

used for keeping the individual trays

or stacks. A spacing of at least one

tray height is usually allowed between stacked trays. Cheesecloth may be used to cover the trays

to reduce contamination, but strict monosepticity is not attempted. Inoculation and occasional

mixing are done manually, often by hand.

Small- and medium-scale koji operations in Asia mostly use this technology.

Disadvantages:

Despite some automation, tray fermenters are

labor intensive

require a large area

Difficulties with processing hundreds of trays limit their scalability

2. STATIC BED AND TUNNEL FERMENTERSThese are the modification of tray fermenter employing a single, larger and deeper, static bed of

substrate with forced aeration through the bed. The substrate is located in an insulated chamber.

Tunnel Fermenter:

In the tunnel fermenter, the bed of solids may be quite long but is usually no deeper than 0.5 m.

Tunnel fermenters may be highly automated with mechanisms for continuous feeding, mixing

inoculation and harvest of substrate.

3. ROTARY- DISK

FERMENTERSRotary disk fermenters are used in large scale koji fermentations in Japan. They consist of a

upper and lower chambers, each with a circular perforated disk to support the substrate. A

common central shaft rotates the disks. Inoculated substrate is introduced in the upper chamber

and slowly moved to the transfer screw. The upper screw transfers the partly fermented solids

through a mixer to the lower chamber where further fermentation occurs. The mixer breaks up

the partly fermented substrate–mycelium aggregates halfway through the fermentation process.

Fermented substrate is eventually harvested using the lower transfer screw. Both chambers are

aerated with humidified,

temperature-controlled air. Rotating-disc

contactors have been used in effluent

treatment. They utilize a growing microbial

film on slow rotating discs to oxidize the

effluent.

AUTOMATIC ROTARY KOJI FERMENTER

4. TOWER FERMENTORTower fermenter is simple in design and easy to construct. It is similar in concept to rotary koji

fermentor, consisting of a long cylindrical vessel with an inlet at the bottom, an exhaust at the

top, and a jacket to control temperature. A stack of several tray chambers form the tower. It does

not require agitation hence there are no shafts, impellers or blades. Tower fermentors are used

for continuous fermentation of beer, yeast and SCP. In 1955 these were used in brewing industry.

TOWER FERMENTOR

Disadvantages:

Despite of being simple and agitation free (to keep yeast cells in suspension) the tower

fermentors have following drawbacks :

1. Long start up

2. Technical complexity

3. Skilled personnel Required

4. No product consistency

5. AGITATED TANK FERMENTORHelical ribbon-stirred tank fermentors have been employed for solid-state culture of fungi such

as Chaetomium cellulolyticum on wheat straw. Other similar designs have utilized multiple

helical screws for agitation of large rectangular tanks.

6. CONTINUOUS SCREW FERMENTORA screw fermentor is used for continuous fermentation process. Sterilized, cooled, and

inoculated substrate is fed at the inlet. The screw moves the fermenting solids toward the harvest

port. The fermentation time depends on the length of the screw and the rotational speed.

As the device is not aerated, therefore only anaerobic or microaerophilic fermentations may be

done.

SCREW FERMENTOR

7. AUTOCLAVE FERMENTOR

Most fermentors are sterilized by autoclaving, or hot steam under pressure. For small laboratory

fermentors they are sterilized in autoclaves. In the case of large fermentors, most if not all are

equipped with in situ sterilization facilities built into the fermentor system.

For most autoclaving sterilizations in both small and large fermentors, the accepted autoclaving

conditions is at 121 degrees Centigrade at pressure of 15 to 20 psi. The autoclaving holding time

is about 15 to 20 minutes.

There are certains point which should be kept in mind while autoclaving,they are as follows:-

i. To be efficient in autoclaving it is very important to drive off any air pockets that might

be present in the autoclave. Air is a poor heat conductor. If the air is not driven out it will

be difficult to bring the right temperature in all the autoclave. Let the autoclave heat and

steam up and release the hot steam through an escape valve before closing the valve and

starting the sterilization process.

ii. Ensure that the temperature recorded in the autoclave chamber is uniform throughout the

whole chamber. Make sure the temperature stated on the panel outside the autoclave is

the real temperature inside the autoclave chamber. We do not want under heating and

overheating to occur. Place thermo probes to measure the real temperature of the

autoclave and repair if needed.

iii. Do not overload the autoclaving chamber. This might lead to poor degree of sterilization

being achieved.

iv. Ensure that the autoclave is not leaking or suffering from leak in pressure as it will affect

the sterilization process.

APPLICATIONS OF FERMENTORS IN INDUSTRY

Industrial fermentation is the intentional use of fermentation by microorganisms such as

bacteria and fungi to make products useful to humans. Fermented products have applications as

food as well as in general industry.

FOOD FERMENTATION

Ancient fermented food processes, such as making bread, wine, cheese, curd, dosa etc., can be

dated to more than 6000 yr ago. They were developed long before man had any knowledge of the

existence of the microorganisms involved. Fermentation is also a powerful economic incentive

for semi-industrialized countries, in their willingness to produce bio-ethanol.

PHARMACEUTICALS AND THE BIOTECHNOLOGY INDUSTRY

There are 5 major groups of commercially important fermentation:

1. Microbial cells or biomass as the product, e.g. single cell protein, bakers

yeast, lactobacillus, E. coli, etc.

2. Microbial enzymes: catalase, amylase, protease, pectinase, glucose

isomerase, cellulase, hemicellulase, lipase, lactase,streptokinase, etc.

3. Microbial metabolites :

Primary metabolites – ethanol, citric acid, glutamic

acid, lysine, vitamins, polysaccharides etc.

Secondary metabolites-- all antibiotics fermentation

4. Recombinant products: insulin, hepatitis B vaccine, interferon, granulocyte colony-

stimulating factor, streptokinase

5. Biotransformations: phenylacetylcarbinol, steroid biotransformation, etc.

NUTRIENT SOURCES FOR INDUSTRIAL FERMENTATION

Growth media are required for industrial fermentation, since any microbe requires water,

(oxygen), an energy source, a carbon source, a nitrogen source and micronutrients for growth.

Carbon & energy source + nitrogen source + O2 + other requirements → Biomass + Product +

byproducts + CO2 + H2O + heat

PRODUCTION OF INDUSTRIAL ENZYME USING DIFFERENT

FERMENTORS

NEUTRAL PROTEASE:

Neutral protease is produced at indrustial level using agro-industrial residues as substrate e.g.

wheat bran, rice husk, rice bran, spent brewing grain, coconut oil cake, palm kernel cake, sesame

oil cake, jackfruit seed powder and olive oil cake etc. while developing a production medium it is

very important to monitor the cost-effectiveness of the medium so these agro-industrial residues

mentioned above are are very cheap and easily available. Among all substrates wheat bran is the

best. Seven fungal cultures, i.e. three strains of Aspergillus oryzae and four strains of

Penicillium species e.g. P. funiculosum, P. funiculosum, P. pinophilum, P. aculeatum were

evaluated using a plate assay for enzymeproduction, which showed a strain of A. oryzae NRRL

1808 as the most useful culture. Protease enzyme is produced in two fermentor systems, in solid-

state fermentors (SSF) and sub-merged fermentors (SmF).

PRODUCTION OF NEUTRAL PROTEASE IN SSF AND IN SMF AND

THEIR COMPARISON:

In SSF a medium having an initial moisture content of 43.6%, when inoculated with 1  ml of

spore suspension (8 × 108 spores) and incubated at 30 °C for 72 h (31.2 U enzyme per gram of

fermented substrate – U/gds) is used while in SmF medium (pH 7.5) containing 2% (w/v) wheat

bran, when inoculated with 3 ml of spore suspension and incubated at 30 °C and 180 rpm for

72 h gave maximum enzyme yield of 8.7 U/gds is used. SSF gives best result comparative to

SmF because of 3.5-fold more enzymeproduction in SSF and it clearly demonstrating the

superiority of SSF over SmF.

Biesebeke et al. compared the molecular and physiological aspects of the fungus in submerged

and solid-statefermentation. He observed a number of differences correlated with the different

growth conditions. SmF has advantages in process control and easy recovery of extracellular

enzymes, mycelia or spores. However, the products are dilute and enzymic extracts might be less

stable than those from SSF. SSF has been developed and described for fungal enzymeproduction

and its advantages include simplicity, lower production costs, high enzyme yields and low

wastewater output. SSF has the added advantage since it is a static process without mechanical

energy expenditures, although problems such as temperature and pH control are encountered.

GLUCOAMYLASE

PRODUCTION OF GLUCOAMYLASE IN SSF:

Aspergillus sp. A3 is used for the production of glucoamylase under solid state fermentation.

Different substrates like wheat bran, green gram bran, black gram bran, corn flour, barley flour,

jowar flour, maize bran, rice bran and wheat rawa are the best substrate and give best results

among all these wheat bran showed the highest enzyme activity. The maximum enzyme activity

under optimum conditions was 247 U/g of wheat bran. The optimum conditions are fructose as

additive 1% w/w, urea as additive 1% w/w, incubation time of 120 h, incubation temperature at

30 °C, 2:10 (v/w) ratio of salt solution to weight of wheat bran, inoculum level 10% v/v,moisture

content of solid substrate 80%, 1:50 ratio of substrate weight to flask volume and pH 5.0.

SSF holds tremendous potential for the production of enzymes. In case of crude fermented

product SSF is of special interest, may be used directly as enzyme source. For the SSF processes

Agro-industrial residues are generally considered the best substrates.

PRODUCTION OF GLUCOAMYLASE IN SMF:

Currently, glucoamulase enzyme is also produce in submerged fermentation (SmF), generally

employing genetically modified strains. Comparative to SSF in SmF the cost of production is

high and is uneconomical.

So as a result the SSF should be considered as an attractive method and it has has many

advantages over submerged fermentation for the production of enzyme.

ALGAE BIOFUELS PHOTOBIOREACTOR

Ability of microalgae to mitigate CO2 emission and produce oil with a high productivity show

that it has the potential for applications of producing the third-generation of biofuels. For

microalgae biofuel production there is a need of identification of preferable culture conditions

for high oil productivity, development of effective and economical microalgae cultivation

systems, as well as separation and harvesting of microalgal biomass and oil.

Chisti in 2007 proposed that under suitable culture conditions, some microalgal species are able

to accumulate up to 50–70% of oil/lipid per dry weight. And Gouveia and Oliveira in 2009

proposed tha the fatty acid profile of microalgal oil is suitable for the synthesis of biodiesel.

Chisti also proposed the major reason of using microalgal oil for biodiesel production which is

the tremendous oil production capacity by microalgae, as per hectare, they could produce up to

58,700 L oil, which is one or two magnitudes higher than that of any other energy crop.

However, it also faces a number of technical hurdles that render the current development of the

algal industry economically unfit. In addition, it is also necessary, but very difficult, to develop

cost-effective technologies that would permit efficient biomass harvesting and oil extraction.

Nevertheless, since microalgae production is regarded a feasible approach to mitigate global

warming, it is clear that producing oil from microalgal biomass would provide significant

benefits, in addition to the fuel. Photobioreactor could be effective to grow microalgae by using

favorable light source and reactor configuration. Collection and concentration of microalgal

biomass from cultivation systems contribute heavily to the operation cost of the overall process.

METHANE FERMENTATION SYSTEM FOR FOOD RECYCLING

Keeping in mind the applicability of food waste leachate (FWL) in bioreactor landfills or

anaerobic digesters to produce methane as a sustainable solution to the persisting leachate

management problem and this research was made in Korea. Taking into account the climatic

conditions in Korea and FWL characteristics, the effect of key parameters, i.e. temperature,

alkalinity and salinity on methane yield was investigated. The monthly average moisture content

and the ratio of volatile solids to total solids of the FWL were found to be 84% and 91%,

respectively. The biochemical methane potential experiment under standard digestion conditions

showed the methane yield of FWL to be 358 and 478 ml/g VS after 10 and 28 days of digestion,

respectively, with an average methane content of 70%. Elemental analysis showed the chemical

composition of FWL to be C13.02H23.01O5.93N1. The highest methane yield of 403 ml/g VS was

obtained at 35 °C due to the adaptation of seed microorganisms to mesophilic atmosphere, while

methane yields at 25, 45 and 55 °C were 370, 351 and 275 ml/g VS, respectively, at the end of

20 days. Addition of alkalinity had a favorable effect on the methane yield. Dilution of FWL

with salinity of 2 g/l NaCl resulted in 561 ml CH4/g VS at the end of 30 days. Considering its

high biodegradability (82.6%) and methane production potential, anaerobic digestion of FWL in

bioreactor landfills or anaerobic digesters with a preferred control of alkalinity and salinity can

be considered as a sustainable solution to the present emergent problem.

FWL is a mechanically pretreated, easily soluble substrate, which can be handled by

environmentally friendly biological practices such as landfilling and anaerobic digesters to

obtain both economic and environmental co-benefits. The lab-scale BMP test showed the

methane yield of FWL to be 478 ml/g VS at 35 ± 2 °C after 28 days of digestion. Methane gas in

the digestion process accounted for over 70% (v/v) of the total biogas produced. Methane yield

was highest (403 ml/g VS) at a mesophilic temperature of 35 °C in a period of 20 days.

Alkalinity addition had positive effect on the methane yield. Dilution of FWL with salinity of

2 g/l NaCl resulted in 561 ml CH4/g VS at the end of 30 days. Taking into account its elemental

composition C13.02H23.01O5.93N1 and high biodegradability (82.6%), FWL can be used as a highly

desirable feedstock for methane production in bioreactor landfills or anaerobic digesters and can

be treated as a sustainable solution to the present emergent problem for a clean and renewable

energy resource.

The Bioenergy Co. of Japan has decided to construct a power plant in Tokyo Bay to recycle food

waste as part of its methane fermentation power generation project, which is part of the "Super

Eco-Town Project" promulgated by the Tokyo Metropolitan Government. The plant will come

on stream in fiscal 2005.

Under the Food Recycling Law enacted in 2001, all business entities in the food industry are

obliged to reduce or recycle food waste by more than 20 percent by 2006. Methane fermentation

power generation is recommended in the law as one way to recycle food waste.

BUBBLE COLUMN FOR CITRIC ACID PRODUCTION

Citric acid is produced from the cells of the yeast Candida guilliermondii in a bubble column,

that have been immobilized by adsorption onto sawdust. At a dilution rate of 0.21 h−1 in a

nitrinogen-limited medium containing glucose, a reactor productivity of 0.24 g l−1 h−1 has been

achieved which is twice that observed in a batch fermenter culture using freely suspended cells.

The corresponding specific production rate was 0.024 g citrate g−1 biomass h−1 while the yield

was 0.1 g citrate g−1 glucose utilized. These latter values were lower than those observed using

freely suspended cells, indicating that further improvements can be made to the operation of the

reactor. In comparison with literature reports describing other cell immobilization techniques,

adsorption onto sawdust allows similar reactor productivities while being cheap and permitting

simple immobilization and reactor operation.

KOJI FERMENTATION SOLID STATE

Aspergillus oryzae has two glucoamylase-encoding genes, glaA and glaB, their patterns of

expression are different. Expression of the glaB gene is marked in solid-state culture (koji), but

low in submerged culture. To elucidate the induction mechanism of the glaB promoter in solid-

state culture (koji), a fusion gene system using the glaA or glaB promoter and the Escherichia

coli uidA gene encoding β-glucuronidase (GUS) is employed. The expression of glaB-GUS was

induced by starch or maltooligosaccharides in a similar manner to that glaA-GUS, but other

physical factors were found to be required for the maximal expression of the glaB gene in solid-

state culture (koji). The time-course of glaB-GUS expression in solid-state culture (rice-koji

making) suggested that its expression is induced by low water activity (Aw) of the medium and

high temperature. When mycelia grown on a membrane under standard conditions were

transferred to low-Aw and high-temperature conditions (membrane-transfer culture,

MTC), glaB expression was markedly induced, but that of glaA was not. Additionally, glaB-

GUS production was induced in MTC using a membrane with smaller pore size, suggesting that

a physical barrier against hyphal extension could regulate glaB expression. Under conditions

found to induce glaB expression, namely, starch, low-Aw, high-temperature and physical

barriers, approximately 6400 U/mg-protein was obtained, equivalent to that in solid-state culture

(koji). In conclusion, glucoamylase production under these induction conditions achieved in

MTC reached 274 U/ml-broth, which was equivalent to the level observed in solid-state culture

(koji). Northern blot analysis indicated that glaB expression was induced at the level of

transcription 4 h after the transfer to the inducible conditions described above.

MUREE BREWERY : AN INDUSTRIAL VISIT TO STUDY FERMENTOR

SYSTEMS:

AN INDUSTRIAL OVERVIEW

“Murree Brewery” is an ISO 14001 Certified Company established in the year 1860 in the

British era is leading beer industry in Pakistan. It has two leading manufacturing units one

located in Rawalpindi and other in Hattar (KPK), Pakistan. It was established for the ever

increasing demand of the beer by the personal British Raj .it is the oldest venture in Pakistan for

beer market up till now. The Murree Brewery at Ghora Galli was among the first modern beer

breweries established in Asia. The virtues of beer brewed from barley malt & hops as a light

alcoholic beverage were not lost on the local population who rapidly became avid consumers. By

the turn of the 20 century, the name "Murree" was famous for its beer in keg and bottle in the

bars, beer halls and army messes of British India. Murree Beer was first awarded a medal for

product excellence at the Philadelphia Exhibition in 1876, followed by numerous awards over

the past 150 years, Murree brewery has potential distributors with high quality beers throughout

globe. The industry has expanded its business beyond Pakistan and is available to rest of the

world.

MUREE BREWERY PRODUCTS

Murree brewery has a wide range of products which are categorized into alcoholic and non

alcoholic products but the main product of export is the beer and its different types produced

there Our Premium products include

NON ALCOHOLIC PRODUCTS:

Apple malt

Peach malt

Lemon malt

Strawberry malt

Murree Sparklets (mineral water)

Cindy Malt

Malt 79

ALCOHOLIC PRODUCTS:

Whisky

Malt whisky

Beer

Millennium beer(7% alcohol)

Classic (alcohol 5-3%)

Murree beer(4.8-4.9%)

Vodka

Twelve years old Single Malt Whiskies

Vintage with a blend of a Scotch Grain Whisky,

Silver Top Gin,

Bolskaya Vodka

Doctor's Brandy.

INDUSTRIAL VISIT OF GROUP THREE:

BEER PRODUCTION BY FERMENTATION

Group three visited Murree brewery on October 12 , 2012 to study the fermentor systems there

and studied the beer fermentation process and each and every step involved in detail. The visit

was organized by Mr.khalil who was very warm and welcoming briefed us each and every step

of the beer production in detail and made us possible to study the fermentor system and its

aspects in detail. In order to understand the process we must look in to account the concept of

brewing that is beer fermentation from barley which can be defined as Fermentation in brewing

is the conversion of carbohydrates to alcohols and carbon dioxide or organic acids using yeasts,

bacteria, or a combination thereof, under anaerobic conditions. A more restricted definition of

fermentation is the chemical conversion of sugars into ethanol. The equation is as follows:

C6H12O6 → 2 C2H5OH + 2 CO2

BEER FERMENTATION THE PROCESS

The basic ingredients of beer are water, barley(starch source) able to be fermented (converted

into alcohol),brewer's yeast to produce the fermentation and flavoring agents such as hops.

Barley seeds of particular quality are imported from Australia and are graded for the production

of a particular product they are categorized as grade A, grade B, grade C.

The first place in the brewery which we visited was the brew house to observe the malting which

is broken into three steps germination of barley and its mashing and germination. The process is

categorized into four steps:

FIG 1 and 2 depict the germinating barley in the

brew house of Murree brewery in the first step of

beer production (malting)

MALTING:

The process of making barley grains ready for the process of brewing is known as malting.

STEEPING:

Malting Of Barley In The Muree Brewery Brew House The First Step Of Malting

The seed is soaked in water in a vat and aerated for 1 to 1.5 days for sprouting to activate the

enzyme at a low temperature from 10-12`C. barley seeds are graded as A, B and C depending

upon the type of moisture(40-45% moisture ) grade A(beer) and grade B seeds (malt)

production.

GERMINATION:

The germination of seeds is carried out in brew house for five days where barley seeds are

aerated and maintained at particular temperatures during day 1 and 2(18-19`C). After

germination is done the seeds converted to malt by the process of chitting .In chitting the seends

are germinated with sprouts on them .

KILINING:

The germinated seeds are taken to the kiln and the sprouted seeds are roasted at 180`C in a kiln.

The kiln at Murree brewery was really of old and antique style .kilning basically modifies the

germinating seeds by enhancing flavors and modification of barley seeds for malt and other

products production.

MASHING:

Mashing is the process of combining a mix of milled grain (typically malted barley with

supplementary grains such as corn, sorghum, rye or wheat), known as the "grain bill", and water,

known as "liquor" which is used to crush malt in a vessel called a "mash tun" with the help of

heating. Mashing allows the enzymes in the malt to break down the starch in the grain into

sugars, typically maltose to create a malty liquid called wort. Mashing of barley produces

wort .hard water is maintained at 180`C and the crushed malt is transferred to a lautering unit.

LAUTERING PROCESS:

Lautering is the separation of the wort (the liquid containing the sugar extracted during mashing)

from the grains which is done in a lauter turn at 75`C temperature .The seed is separated from the

extract and un dissolved extract is removed.

WORT BOILING:

Boiling of malt extracts is called as wort. It basically ensures its sterility, and thus prevents a lot

of infections. During the boil hops are added, which contribute bitterness, flavor, and aroma

compounds to the beer. The wort is boiled at 100`C and unsuspended phenols and extract is

pumped to the boiler. The boil lasts between 15 and 120 minutes, depending on its intensity, the

hop addition schedule, and volume of water the brewer expects to evaporate. The wort is

separated on the basis of specific gravity and it is clarified aerated and cooled then transferred to

the fermentor for beer production.

Fig1(a) Lautering turn fig1(b) wort separator. Fig1(C)Temperature And Pressure Control Unit Fig1(D) Wort Boiler

BEER FERMENTATION

BATCH FERMENTATION:

Murree brewery uses the open batch fermentation process to ferment beer from yeast. The yeast

strain of saccharomyces cerevisiae imported from Germany which is grown in the form of stock

solutions to be added to the fermentor. The yeast stock solution is made in 100ml beakers and

their serial dilutions are made from 1to 10 liters the inoculums is then transferred to the

Fermentors in a batch. Yeast require one day for activation which is maintained at the

temperature of (14-16`C). The process is known as yeast publication .fermentation produces beer

of different categories depending upon the wort density.

FERMENTOR DESIGN:

Murree brewery had 24 multiple open batch bubble column fermentor systems in their

fermentation unit for beer production. Fermentors installed at Murree brewery was a cylindro-

conical vessel or CCVs, with a conical bottom and a cylindrical top lined inside with

thermocouple to maintain the temperature and temperature sensor at the bottom and a pressure

regulating unit as well. The cone's aperture of the batch fermentor was typically around 60°at

this angle it allows the yeast to flow towards the cone's apex, but is not so steep as to take up too

much vertical space. This type of confirmation handles both the fermenting and conditioning in

the same tank. Fermentor was made up of stainless steel lined with a layer of concrete with a size

of one hectoliter (100liters). At the end of fermentation, the yeast and other solids which have

fallen to the cone's apex can be simply flushed out of a port at the apex. Fermentation takes place

of the wort in the presence of yeast to beer. After fermentation beer is obtained depending upon

the density of the wort. The beer with 12% alcohol content is used to make classic beer and

millennium beer, with (6-7%) and light or Murree beer has low content of alcohol.

BEER CONDITONING AND STORAGE

After initial or primary fermentation, the beer is now conditioned, matured or aged in one of

several ways, which can take from 2 to 4 weeks, several months, or several years, depending on

the type of beer. The beer is usually transferred into a second container, to a trub which is free

from dead yeast, secondary metabolites and other undesirable flavors that are a result of primary

fermentation .

PACKAGING:

Packaging is putting the beer into the containers in which it will leave the brewery. Typically,

this means putting the beer into bottles, aluminium cans and kegs, but it may include putting the

beer into bulk tanks for high-volume customers.

RESEARCH PLANNING AND DEVLOPMENT LAB

We visited research planning and quality control lab of Murree brewery as well our team met

their quality control manager Mr Muhammad Sohail he briefed us about the fermenting

problems., batch , new product ranges , future products and quality control problems. The

monitoring of the products at each step to avoid any contamination. The yeast culture is checked

at the end of every batch of beer and malt before packaging. Random sampling and OCU are

done to check the quality of their products. The whole batch is pasterurized at 20`c then 40 `C -

70`C for thirty seconds and labeled in their specialized packaging unit.

FUTURE PRODUCTS:

Big peach pineapple malt

Cola whisky leechi malt

Fig 1. Batch fermentor systems 2. An open batch fermentor 3. Group 3 with Fermentors

4. Yeast publication unit

PACKAGING AND STORAGE:

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Fig1(C)Temperature And Pressure Control Unit Fig1(D) Wort Boiler