DEVELOPMENT OF FERMENTATION TECHNOLOGY COURSE AT UNIVERSITY MALAYA (1980 – 2006)

The article discusses the development of the fermentation technology course at the Department of Genetics and Cellular Biology, University Malaya from 1980 till 2006. It highlights the various problems in the development of the course from the development of syllabus, teaching and carrying out the laboratory practicals and the relevance of the course to the industrial demands

1. INTRODUCTION

Fermentation technology has always been one of the most important components in the study leading to the degrees of microbiology, biotechnology and bioprocess engineering. Most of our local universities offer the subject as a compulsory requirement for the degrees in biotechnology and microbiology. The fermentation technology course is considered a specialization subject and is usually offered during the advanced undergraduate years of the degree programme.

University Malaya is one of the earliest universities in Malaysia to offer this course to the students majoring in microbiology in 1980. This was due to the foresight of Prof Ho Coy Choke, the Foundation Professor in Microbiology who has the wisdom to foresee the importance of this subject in the coming years

The Fermentation Technology course in the University Malaya has evolved over 25 years of teaching and development from1980 till 2006. During that period the fermentation technology course has undergone many changes in response to the educational and industrial needs of the nation.

It is hoped that this article will give valuable insights to Universities which are planning to offer the subject and to provide direction for its development in the future





2 HISTORY OF FERMENTATION TECHNOLOGY IN UNIVERSITY MALAYA

The history of fermentation technology in University Malaya can be divided into two phases of development:

1 Period 1979 to 1995
2 Period 1995 to 2006

PERIOD 1979 to 1995

The initial period of the development of fermentation technology was the period of establishment of the course when it was initiated from zero beginning. It was a period of searching the direction for the development of the course and passes through many ups and downs in establishing the course.

In beginning the teaching of the course was jointly carried out by chemical engineering lecturers and genetic lecturers. This arrangement was proposed in the belief that the fermentation technology subject is the synergy between chemical engineering and microbiology components.

However, within a few years it was observed that such arrangement do not really work and it appears as if there are two separate courses were operating in independently rather than a single unified course in fermentation technology. There were poor communications between the microbiology and the chemical engineering departments to improve or strengthen the course

The weaknesses of inter departmental teaching of the subject could be seen in the following:

1 Over lapping of course content between microbiology and
engineering component
2 The engineering practicals were designed to suit to
chemical engineers and not microbiology students.
3 The practicals and lectures too mathematical and not
required by microbiologist and not directly relevant to
fermentation technology
4 The scope of the practicals are not in the maintenance and
operation of fermentors
5 The microbiology component was more biochemistry or
metabolism of biochemical pathways which are already
established in other components of the microbiology
course

In principle a proper fermentation technology course should be run in substance similar to the book by Stanbury and Whitaker (1989) and should involve the utilization of the fermentor. However during this early period of teaching fermentation technology there was little or no use of the fermentor for the practicals. This is possibly attributed to the lack of fermentors or these capital equipments are very costly to acquire. Even if fermentor was used, it is more for demonstration purpose with no hands on experience allowed for the students.

The course in fermentation technology was very microbiological in approach. Fermentation practicals were usually carried out using conical flasks and Petri dishes.

The practicals carried out in the chemical engineering department are more similar to A Level physics experiments. The practicals contributed to the fermentation technology course were more suited to chemical engineering majors.

The fermentation technology practicals carried out in the first phase if fermentation technology are as shown:

LIST OF FERMENTATION TECHNOLOGY PRACTICALS (1980-1995):
Anatomy of fermentor*,
Yeast fermentation*
Isolation and screening of microbes for microbial products*.
Growth curve studies*
Viscosity studies**
Reynolds numbers**
Partition coefficient**

Note:
* Contributed by Microbiology
** Contributed by Chemical engineering


REASONS FOR WEAKNESS OF FERMENTATION TECHNOLOGY

The main reasons for the weakness of the fermentation technology course were attributed to:
1 There were no experienced lecturers who were properly trained or specialized in the teaching of fermentation technology course
2 The syllabus used in fermentation technology were taken directly off from standard textbooks in biochemistry and physiology or from standard biochemical engineering text books such as Bailey & Ollis (1986) and Aiba et al (1965)

This state of affairs remained as stagnation for a few years until an opportunity arises with the injection of massive funding in 1995 under the new vice chancellor Dr Abdullah Sanusi who managed to secure a one off funding to over haul the standard of teaching in University Malaya.

This window of opportunity was exploited by the author for a major review and revamp of the fermentation technology to make the course more relevant to microbiology students and the fermentation industries.

PERIOD 1995 to 2006

During the second phase in the development of the fermentation technology course, the author took the sole responsibility of teaching the whole course completely to avoid duplication and irrelevant syllabus.

With the one off funding in 1995, we acquired:

1 Six units of two litre bench top teaching fermentors
2 One 20 litre in situ sterilization fermentor.
3 Renovating the laboratory to be a purpose built
fermentation Laboratory
4 Acquirement of support equipment for fermentation such
as laminar flow cabinets, large autoclaves among others

This opportunity has allowed the University Malaya to enter its “golden era” in fermentation technology whereby important changes are made in form and substance to improve the fermentation technology course to the international standard. Changes are made in the following areas:

PHILOSOPHY OF TEACHING
The study of fermentation technology encompasses complete coverage of all activities starting right from upstream, mid stream to downstream. However greater emphasis is given on the operation, maintenance of the fermentor which represents the heart of fermentation technology.(McNeil & Harvey,1990)

The applied aspects of fermentation such as the various techniques are given more priority.

Where and when possible the teaching of fermentation technology is carried out with the show and tell concept rather than just lectures. Teaching of the subject is carried out in the laboratory rather than lecture rooms

The mode of learning the course will be more from the point of apprenticeship approach in a programmed series of practicals where under careful supervision the students are guided through the various practical steps. At the end of a practical the students must show the ability to carry out the practical independently.


TEACHING PRIORITY
The priority of the above equipments is for general teaching of the fermentation technology course and not to be used in research gives the boost to the teaching of fermentation technology at undergraduate level. This will ensure that the students will get the full benefit of the course. In most universities the priority of the use of fermentors are more for research and post graduate use.

INCREASING STUDENTS INTAKE
For the first time in the history of the fermentation technology course large number of students are able to take the course and acquire hands on experience in operating fermentors. This is further enhanced by offering the fermentation technology course twice in the academic year to allow more students to have the opportunity of taking the course

SYLLABUS REVAMPED
After numerous visits and discussions with those involved in the fermentation industries and studies of various existing fermentation courses and workshops, a new teaching philosophy and a very rigorous syllabus for fermentation technology was adopted and implemented from 1995.

RIGOROUS TRAINING
The new revamped fermentation technology course will ensure that all the students will have undergone proper and complete training in fermentation technology and adapting to the stringent demands of the course

The new programme in Fermentation Technology has the following characteristics
1 More laboratory and hands on practicals
2 More man hours in the lab
3 More industrial fermentation components
4 More team work

GIVING MORE TIME
Fermentation technology is just not any ordinary microbiology discipline with laboratory practicals completed within a three hour practical session. Most fermentation practicals involved a lot of upstream and downstream preparations and the actual fermentation process is continuous running from hours into days.

As a subject, fermentation technology requires the handling of the fermentor itself. It is a subject which is learnt by handling and manually learning the essential techniques

FULFILLING INDUSTRIAL NEEDS
The fermentation technology curricula become more relevant to the need of the various fermentation industries by including the following aspects in the course:

1 Aseptic demand and integrity
2 Fermentation optimization
3 Trouble shooting and repair
4 Process validation
5 Quality control
6 Process monitoring
7 Scaling up
8 HACCP and GMP

A good example of industrial requirements in operating fermentors is the ability to thoroughly cleaned the fermentors by standard operating procedures and validating that the process of cleaning of the fermentors has been properly carried out to the expected level of cleanliness.

The course will require for the students to carry out proper Cleaning in Place (CIP) and Cleaning out of Place (COP) properly

Students doing the fermentation technology practicals worked as a group consisting of six students in each group. Each group will be provided with their own fermentor throughout the course. Each group will complete the whole set of fermentation practicals throughout the semester. The mode of examination is by continuous assessment.

Since some of the fermentation practicals may stretch over a few days, each group are allowed flexi time to complete their practicals

Students who do not complete each practical to the expectation of the lecturer will have to repeat the experiments until the practicals are properly executed or they have acquired sufficient ‘clinical’ skills in the operation of the fermentors

LIST OF FERMENTATION PRACTICALS (1995-2006)

The list of experiments carried out are as shown:

ANATOMY OF FERMENTOR (3 hrs)
Anatomy of fermentor whereby the students are required to dismantle and identify the various components of the fermentor and study the various systems making up the fermentor

CLEANING OF FERMENTOR (2 hrs)
Students are required to learn the importance of cleaning the fermentor properly and to carry out COP cleaning

ASSEMBLING AND FINAL PRESTERILIZATION CHECK OF FERMENTOR ( 1 hr)
Students are required to assemble the fermentor and to check that everything is in order prior to autoclaving or sterilization of fermentor

ANATOMY AND CALIBRATION OF FERMENTOR ELECTRODES ( 3 hrs )
Students also learn the anatomy and function of the various standard electrodes in the fermentor such as ph, Dissolved oxygen, foam probe, temperature probe. The students will learn the correct methods of calibrating the ph and DO probes

FERMENTOR FILTERS ( 3 hrs)
Students built their own cartridge filters using glass wool

RHEOLOGICAL STUDIES OF FERMENTATION BROTH ( 3 hrs)
Students carry out studies on rheology of the fermentation broth,
Students will carry out simple experiments to monitor change in viscosity of the fermentation broth

MIXINGS OF FERMENTATION BROTH ( 3 hrs)
Types of primary, secondary and tertiary mixings using dyes as tracers. Newtonian and Non Newtonian broth will be used for comparative studies

SETTING UP FERMENTATION CONSOLE ( 3 hrs)
Students will learn how to set up the fermentation console

POST STERILIZATION PROCEDURES ( 3 hrs)
Students learn how to carry out post sterilization procedures

WATER LOSS STUDIES IN STERILIZATION STUDIES ( 3 hrs)
Students carry out water loss studies from fermentor due to sterilizations

BLANK FERMENTATION RUNS ( 5 days)
Students learn to carry out blank runs to test the integrity of the fermentors

ASEPTIC TECHNIQUES IN INOCULATION OF FERMENTORS ( 3 hrs)
Students learn aseptic methods of inoculating the fermentor

ASEPTIC SAMPLINGS FROM FERMENTORS ( 3 hrs)
Students learn how to carry out aseptic samplings from the fermentor

TECHNIQUES TO DETERMINE MICROBIAL CONTAMINATIONS OF FERMENTATIONS ( Continuous assessments)
Students carry out various tests for detecting microbial contaminations

MICROBIAL SCREENING AND ISOLATIONS ( 1 week)
Students carry out microbial isolation and screening studies

MICROBIAL GROWTH CURVE STUDIES( 1 week)
Students carry out growth curve studies at different temperature and ph

AMPLIFICATION OF CELL CULTURES ( 1 week)
Students carry out microbial cell culture amplification

MONITORING OF FERMENTATION PROCESS ( 5 days)
Students carry out complete fermentation monitoring experiments using various parameters such as substrate, ph, biomass products etc using various chemical, instrumental techniques

OTR AND OUR IN FERMENTATION ( 3 hrs)
Students carry out OUR and OTR studies using the fermentors

DOWNSTREAM PROCESSING OF FERMENTATION PRODUCTS
Students carry out some down stream processing equipments

TROUBLE SHOOTING AND DIAGNOSTICS ( Continuous clinical observations)
Trouble shooting and diagnostics of the fermentor and fermentation process are continually carried out through out the whole fermentation practicals as part of their fermentation pathology exercise. Rapid physical, chemical, biochemical and microbiological techniques will be exposed

FINAL FERMENTATION PROJECT ( 2 weeks)
Where after learning all the above, the students will carry out detailed fermentation study of their choice incorporating all the lessons learned


3 CONCLUSIONS

Every university now aims to have fermentation technology course as part of their subjects offered to cope with the future demands in biotechnology. However in most cases the rush to establish the course has resulted in failure or inability to give the best due too poor planning or unforeseen circumstances

Fermentation technology as a subject is not as simple as just teaching microbiology or chemical engineering. It has its own unique identity. The subject of fermentation technology requires the complete understanding of upstream, midstream and downstream activities.

The key component in this subject is the hands on experience that must be learnt using real fermentors which is the essential component for any decent fermentation technology course

This paper has shown the problem and pitfalls and the journey to achieve a credible course in fermentation technology from expertise, laboratory facilities, hardware components and practical syllabus. It is hoped that that it will be a guideline to those wanting to offer or do research in the subject

REFERENCES

Aiba, S, Humprey, A, & Millis, ( 1965)
Biochemical Engineering
University of Tokyo Press, Tokyo

Bailey, J.E, & Ollis, J.F (1986)
Biochemical Engineering Fundamentals
Mc Graw Hill New York

McNeil,B & Harvey,L.M (Eds) (1990)
Fermentation: A Practical Approach
Oxford University Press, New York

Stanbury, P.F &Whitaker, A (1989)
Principles of Fermentation Technology
Pergamon Press, Oxford . UK


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MIXING AND SHEARING- THE GOOD, THE BAD AND THE UGLY

(STILL GOT TIME TO POST ANOTHER ARTICLE BEFORE LEAVING)
(PICTURE TAKEN FROM DRAGLIST.COM)

Everyone dealing with fermentors knows that mixing is one of the most important parameters that affect the fermentation process. Put it this way, in order to support the high growth of microorganisms and the production of high concentration of fermentation products, mixing is necessary.

Mixing is essential in order to provide homogenous condition throughout the fermentor required the optimum growth of the microorganisms. Homogenous conditions within the fermentor will allow efficient mass transfers of nutrients, heat and oxygen throughout the fermentor. It will also eliminate formation of gradients and built up of toxic products

However, even though mixing is crucial to the fermentation process, it needs to be properly executed as there are the good, bad and ugly sides of mixing which have impact on the fermentation process.

One of the sensitive sides of mixing is the effect of shearing. In fermentor as the impeller rotates it creates a flow. Shearing forces will be generated where there are velocity gradients between two points in the liquid flow. Differences in velocity gradients in shearing could occur between the same or different phases in the broth such as between liquid and liquid, liquid and gases and liquid and solids.

Under non turbulent or laminar flow, shearing still occur but would not be significant in fermentors as high rate mixing is often the order of the day.

In a simple model of visualization we could see shearing forces as forces that physically stretch, tear or wear out surfaces. Thus, in this context if uncontrolled shearing forces are damaging to the microorganisms and the products formed. The intensity of shearing are often related to the amount of physical force or mixing power exerted into the system. Higher turbulence would relatively lead to increased or more powerful turbulence.

There are many areas or zones in the fermentor which are strongly affected by the shearing forces generated by mixings. Most intense shearing is known to occur in the mixing zones of the impellers, and especially at the tip of rotating impellers. Shearing forces could also occur in the region of the baffles or where strong air sparging occurs.


THE GOOD- THINNING OUT BOUNDARY LAYER

Shearing may result in the reduction of the boundary layer. This will increase the rate of mass transfers across the boundary layer between the microbial cell and the environment

THE BAD -EFFECT OF SHEARING ON MICROORGANISMS IN FERMENTORS

Shearing can result in damages to the microorganisms and directly will affect the efficiency of the fermentation process. The effect of shearing on the microorganisms much depends on the morphology and growth forms of the microorganisms. Filamentous fungi will be easily tangled and damaged during mixing and shearing.

In the case of unicellular microbial cells the effect of shearing is not so much on the damage to individual cells but to the microbiological aggregates. Rarely do microorganisms occur as independent single cells in fermentors. Most, if not all occur as microbial aggregates such as microbial flocs, microbial mats.

Under these conditions the formation of the microbial aggregates is often the physiological response of the microorganisms to conditions of stress. These microorganisms will produce high amount of EPS around them in a protective response against damage.

Flocs are however not strong structures and studies in activated sludge system showed that larger flocs are easily broken down to smaller flocs under turbulence. Most of the damage by shearing occurs at the impeller tips.





UGLY SIDE-CONSEQUENCES ON DOWNSTREAM PROCESSING

Damages by shearing of the microbial cells will result in the release of metabolites which will complicate the fermentation process by increase in foaming and difficulty of recovery of fermentation products








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APOLOGIES

There will be a temporary cessation of blogging activities in this blog for the next two weeks as the author will be in South Korea visiting Myongji University, Seoul National University and Korea Institute of Science & Technology.

Blogging activities will resume after the period
Thank You


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CONTRACT MANUFACTURING IN PHARMACEUTICAL FERMENTATION INDUSTRIES- RISKS AND PROFITS

(PICTURE TAKEN FROM WWW.DSM.COM)

INTRODUCTION

Pharmaceutical industries are big bucks! Just see the volume of drugs produced by the giant pharmaceutical manufacturers and the record billions dollars profit shown every year. Yet these mega companies are always looking for profitability and finding ways to cut the costs of their production by trying to cut the costs of producing these drugs.

Some of these pharmaceuticals are produced by fermentation such as antibiotics, vaccines and biologics. The cost of production of these fermentation derived pharmaceuticals is sky high due to the high cost of capital investment in equipments and facilities. In view of this problem many of these giant pharmaceuticals companies tries to outsource their production or manufacturing by looking for contract manufacturing beyond their shores.

Asia has always been a very attractive site for contract manufacturing as it fulfills all the requirements needed for successful contract manufacturing ventures. It should be noted that outsourcing manufacturing capabilities is not a new phenomena as the Japanese were all ready at it as in the Fifties.

The most popular countries in out sourcing pharmaceutical contract manufacturing currently are India and China. Even Malaysia do not wants to be left out on this rich gravy train.

However, in pharmaceutical contract manufacturing is not all good news. Both the proprietor and the contract manufacturer face a lot of problems and issues. It may look good on paper, but in reality there are a lot of bumps to overcome and risks to be taken.

WHY BOTHER CONTRACT MANUFACTURING?

The first question that is often asked is why do the proprietary holders want to source out pharmaceutical contract manufacturing?

There is really one reason why pharmaceutical fermentation industries outsource their manufacturing or production, that is, to cut costs and increase their profitability. In this world of free market competition, the ability to reduce the cost of products will be crucial to the survival and profitability of the company. The advantages of foreign contract manufacturing include:

1 favorable tax benefits for the parent company, allowing them to
reduce overall tax liabilities and increase profits.
2 Other advantages over in-house manufacturing, including lower
costs, flexibility, access to external expertise and reduced capital.

The trouble is that the problem is not solved by simply carrying out contract manufacturing and just gets the benefits. As stated earlier contract manufacturing is not without risks.

There are many considerations to be taken before a suitable contract manufacturer can be hired such as:

1 Have they got the proper industrial facilities to carry out the manufacturing?
2 Do they have the right people and expertise to ensure the smooth running of the contract manufacturing?
3 Can the contract manufacturer produce the products to the expected quality and standards?
4 Have the contract manufacturer sufficient industrial experience and track records to carry out such production?
5 Can they justify the correct costing and tenders to compete with other contract manufacturers?

It is a well known observation that most of the experts in the fermentation of pharmaceuticals are new and have very little experience in running industrial level contract manufacturing and scale up productions. Most of them are probably so new and are in their baby steps phase of the learning curve to pharmaceutical fermentation contract manufacturing. Most of the experts are perhaps new lecturers in universities who are too academic inclined and not exposed to the practicals of production floors. Their levels of fermentation are perhaps still at the level of Mickey Mouse projects!

True! We do have facilities for fermentation, but most are just pilot plant scale and not really geared for industrial fermentations. This is in the case of the Universities fermentation suite or pilot fermentation unit.

Even though we may have companies that supposedly have the capacity to do contract manufacturing, but in reality they do not function as professional pharmaceutical fermentation contract manufacturer. All they can show are a few big size shiny fermentors and a few staff in gleaming white lab coats. In fact they are more involved in the propagandistic public exhibitions of what they can promise. A visit to their website will expose a lot of weakness and where tons of money is poured down the drain with no returns

A facility for proper industrial scale fermentation pharmaceutical contract manufacturing must be able to show its ability and capacity to produce industrial scale production. The facilities must be adaptable enough to be used for various types of pharmaceutical fermentations. In that sense the company should not show the priority of exhibition booths to fulfill the visiting school students

These companies should be more opened to the public of the successful tenders that have been executed and not just reports of visits and exchange of MoUs.


CONTRACT MANUFACTURING IS NOT WITHOUT ITS LIABILITIES

As for the country or company that offer contract manufacturing burdened by high capital costs and need for specialist educated force. Also the demand for facilities by companies must be high to sustain the company and make sufficient profits out of the venture.

They must be able to produce products to the specifications of the clients.

Their facilities and equipment must be adaptable to various specific demands of clients and they must have the capacity to carry out large scale production. Everything is ok if contract is profitable and booking books are full and you have long term contracts. If not you are going to have many stainless steel white elephants! And a lot of research staff and laboratory coated technicians walking aimlessly at costs!

Poor marketing is another problem. As they say it even if you have poor products good marketing will generate sales. If you have good product but poor marketing no one will buy your products

But out sourcing is not without risks as there are many factors to consider before choosing which country to invest or outsource. In certain cases, if no careful considerations are given the whole outsourcing exercise could be a failure with serious economic, legal and social consequences.

PHARMACEUTICAL FERMENTATION

Pharmaceutical fermentation contract manufacturing is not as simple as contract manufacturing for garments and shoes where all you need are simple production lines, simple machinery and thousands of workers. Far from it, pharmaceutical fermentation is very technologically complex that require well qualified experts and very sophisticated equipments and facilities. It is a very costly operation which put the investor into a lot of possible financial and even regulatory risks. Its products affect human health and safety directly.

Pharmaceutical fermentation is not as simple as food or beverage fermentations. In biologics fermentation aseptic and clean room fermentation is mandatory. Downstream processing steps are more sensitive

THE WAY OUT
The only way out of this predicament is to swallow our pride and work with other manufacturing contractors by:

1 Use facilities that are already in use to produce biologics or facilities by established companies in the region
2 Build new facilities by established contractors not by fermentor sellers or their sales men
3 Carry out joint ventures with other contractors as we did with Mitsubishi and proton cars and hoped that they do carry out technology transfers in the real sense!
Maybe the only significant advantage out of these arrangements is that we have our rights to add “ Made in Malaysia” on every products produced. He he





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DEMYSTIFYING KOJI

I just cannot understand it. At times I feel the way the whole world discovers or respect Japanese products seems to be out of perspective. At times they regarded their products as far superior or even have the magical elixir of life!
One good example is their fermented product or koji which is used widely to seed or culture their various fermented food and beverages such as miso,soy sauce and sake. There is really nothing special about it or the great hype being associated with it. Koji is just the fermenting agent! koji can refer both to a fungus and to a food ingredient that is commonly made using rice, barley, or soybeans.
Seed koji refers the spores of the fungus Aspergillus oryzae. The fungus is a type of mold that is native only to the humid Southeast and East Asian regions.
MAKING KOJI
Koji is made by sprinkling seed koji over steamed rice, barley, or soybeans and cultivating the fungus under temperature conditions suitable for its growth. As the fungus propagates, enzymes break down the grains' starch and proteins into sugars and amino acids.
DIFFERENT KOJI FOR DIFFERENT FERMENTATION
The main factors determining are type of koji and type of substrate used. Different types of koji are used for different type of fermented food. Even for sake there are various types of koji seeds used.
Although the main microorganism for koji is Aspergillus, koji are known to contain about fifty types of enzymes. The most important enzymes in a koji are alpha amylase, glucoamylase and acid protease.
The poor reception for koji type of fermentation is probably due to the western obsession with SF than SSF


UNPOPULARITY OF KOJI IN INDUSTRIES
Using koji has now grown out of popularity because modern fermentation industries are always looking for automated fast fermentation under the mantra of "faster, cheaper, and more." Mass production of widely used foods by fast fermentation led to the emergence of national brands in the fermentation industry. This lead to cheaper fermented food and beverages. However the drawback is that fermentation is not simply a process you can just speed up the fermentation time and volume produced. This has the effect of affecting the quality of the fermentation products in terms of subtle flavors and rich nutrients of naturally fermented products. In trying to short cut the fermentation extraneous enzymes and additives are added. This of course will affect the fermentation process and the quality of the fermented products.

A GAME OF TEMPERATURE, TIME AND HUMIDITY

Making koji is often a family secret. Generally it takes three days
Rice- soaked in water overnight - steamed 40 minutes- cool down, scarred to facilitate fungal growth-- sprinkled with seed koji--- transfer to fermentation room
If we look at the whole koji preparation it is more an intimate play of temperature, time and humidity. Different types of temperatures are exploited and controlled to get the right koji and enzyme activities. There is a frequent use of cloth and straw wrapping to insulate the koji temperature



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LOOKING DEEP INTO SOLID SUBSTRATE FERMENTATION

INTRODUCTION
Somehow, when we talk about fermentation, almost everybody conjures up the image of submerged fermentation (SF) as carried out in the big stainless steel fermentors or the fermentation vats. Not many are aware of the other type of fermentation called as solid substrate fermentations (SSF).

In the East, SSF is used in traditional food fermentations and it has always stayed in the back burners of the fermentation popularity. The ‘unpopularity of SSF does not mean SSF is inferior. In certain ways SSF has more advantages to SF depending on the situation.

It is only in the last few decades that the West is interested in exploiting SSF in the production of microbial enzymes and other microbial products. It is interesting to point here that in terms of diversity of fermentation products the SSF is more restricted compared to SF

The unpopularity of SSF compared to SF is more attributed to the poor understanding and control of the SSF fermentation compared to SF which is well established in the West. The popularity of SF in the west is more because of the rich diversity of fermentation products that can be obtained through SF compared to SSF. This does not mean that there are no SSF in the West. The diversity of cheeses produced by molds in SSF testifies to this.

SF is more established in the west is probably initiated attributed to their fascination of alcoholic beverages. This is followed by the interest in the West to scale up or carry out industrial production through which they have through science and technology to understand and control the process. They would have recognized early that in terms of fermentation products SSF is more efficient compared to the passive SF

The jumpstart in SF is perhaps single handedly contributed by the antibiotics fermentation industries during the World wars and see the legitimacy of Industrial Microbiology.

The poor understanding and limitations of SF is probably the main reason why SF always remain in the back burners of fermentation

It is comparatively, easier to carry out scale up of SF for industrial fermentations compared to SSF.

In a way it is wrong for us to regard SSF and SF as two different types of fermentations. Both types of fermentations are carried out by the microorganisms.

The main differences between SSF and SF are that:
1 SSF uses little water
2 The substrates for the microorganisms are significantly in solid
forms and not in solution

These two factors have important consequences in influencing SSF and giving SSF its unique characteristics

LITTLE WATER IN SSF
When we say SSF uses little water in fermentation it could mean literally in terms of very low volume of water used in the fermentation or very little free or available water occurred in the fermentation.

This is a significant difference compared to SF where the aqueous phase is the dominant component in the fermentation process.

In situation where little water are used in SSF, water is still required for the metabolism of the fermentative microorganisms but it occur surrounding the substrate particles as a thin film of water. A good example is in tapai fermentation.

In Sauerkraut fermentation even though large volumes of water are used for the fermentation, these water are not free or easily available to the microorganisms due to competition with the salt ions.

Where very low volume of water are used in SSF, we could regard the SSF as dry fermentation as exhibited in tempe SSF where fungal mycelial growth covered the surfaces of the wet soya beans.

In such dry SSF, there are porous spaces around the sold substrate to allow for easy mass transfer of heat and oxygen in the substrate matrix

IMPACT OF AMOUNT OF WATER IN SSF

As discussed above water is the critical issue in SSF and SSF operate in very little water environment. Water is important in SSF as
1 It is the medium where nutrients required by the microorganisms
are dissolved and transported to the microorganisms.
2 It is required by the microorganisms to carry out the various
metabolic and biochemical reactions to grow

The right amount of water is critical in any SSF. One good example is in the SSF of tempe. Higher amount of water could result in a more vigorous growth of the mycelia resulting in the clogging of the pores between the solid substrate and hampering the mass transfers of oxygen as well increase in the metabolic heat. This would result in a very hard and compact tempe which is not popular compared to the soft fluffy tempe.

With the decrease in substrate porosity and the resulting decrease in oxygen may even increase the risks of bacterial contamination. Low amount of water on the other hand may result in poor accessibility of nutrients which will result in poor growth of the mycelia.

Low amount of water in SSF means that the fermentation products in solution are not significantly diluted and will occur in high concentration. There is also the side benefits of low volume effluent generated in SSF compared to SF
SUBSTRATES IN SOLID PRESENTATIONS
In SF, the nutrients are usually dissolved, well mixed and dispersed in the fermentation broth. Good mixing will ensure good mass transfer of nutrients, oxygen and heat throughout the fermentor.

In SSF, the conditions are significantly different as the substrates occur dominantly in particle or solid forms and are not easily available to the microorganisms. These solid substrates are often static or fixed and not easily mixed or dissolved. SSF microorganisms have to actively colonized the surface of the substrates, dissolved the solid substrates powered by extracellular enzymes to release the nutrients needed for the growth

The advantages of this dry SSF are that there are large surface areas available and easy aeration. Its setback is however low heat transfer capacity. However often at times it is important in finding the correct size of solid particles in SSF. Too big a paricle might yield to surface area t volume ratio thus limiting the efficiency for microbial action. Yet at the same time using large particles will allow large void space to facilitate oxygen, water and heat mass transfer needed in aerobic SSF.

In small particles SSF even though there is a higher surface area to volume ratio to optimize microbial action, it often however hinders the efficiency of the various mass transfer processes needed to support SSF

AERATION IN SSF

We are discussing aeration in the context of aerobic SSF. Aeration serves the following main functions in SSF.
1 to maintain aerobic conditions,
2 to desorb carbon dioxide,
3 to regulate the substrate temperature and
4 to regulate the moisture level.

In SSF that involves the formation of a thin film of water around the substrate the efficiency of oxygen mass transfer is very high in the case of dry SSF such as tempe. However in tapai SSF due to the high sugar concentration resulting in the thick viscous film of liquid around the SSF, there is a very poor mass transfer of oxygen thus allowing alcohol fermentation to proceed




HEAT DISSIPATION IS CRUCIAL ISSUE IN SSF

One of the main characteristics in dry SSF such as in tempe and composting is the high generation of heat generated by the aerobic metabolism. To make the conditions worst in SSF the solid materials used in SSF are often have low thermal conductivities. This would easily result in built up of heat.

The effective dissipation of heat is often related to aeration of the SSF system. Aeration not only brings in oxygen but also help in the removal of heat from the SSF.

The amount of heat produced and managed in SSF is very crucial to the fermentation process. High uncontrollable heat is not good in SSF as it affects the composition of microorganisms, its physiology of growth and even product formation.

MICROORGANISMS IN SF AND SSF

In both type of fermentations, the microorganisms are the agents of change. Differences might differ in the type of microorganisms involved and the behaviour and physiological requirements of these microorganisms. The function of the fermentors is to support the growth of the microorganisms as such the type and configuration of SF or SSF will be dictated by the nature of the microorganisms involved

In SSF the nature of microorganisms generally differ from SF as it involves:
1 Mixed cultures or diversity of microorganisms
2 That it does not rely on single or pure culture fermentations
These two characteristics are consequences of using complex and solid substrate which can only be effectively utilized by the combined action of mixed microorganisms

Since it depend on mixed culture of microorganisms which are in most cases natural inocula, aseptic is not really required in the SSF. Exclusivity of microorganisms in SSF in most cases are imposed by the phenomena of natural protection whereby not many organisms can grow in the environment such as the very low availability of free water or very high salt or sugar concentration
This does not mean to say SSF cannot go bad or contaminated. Failure to provide the ideal conditions in SSF can result in spoilage of fermented food

MICROBIAL GROWTH FORMS

There are two main microbial growth forms commonly encountered in SSF.

1 Mycelial mat by fungi
2 Microbial film by bacteria


MYCELIAL GROWTH

In aerobic SSF mycelial growth form on the surfaces of the substrates is common. In this growth form the main unit of growth of the filamentous fungal is the hyphae. The hyphae growth is characterized by apical growth. A mass of hyphae constitute the mycelium or mat growing on the surface of SSF

The key point is that active growth occurs only at the tip of the hyphae as it actively seek new source of substrate. Extracellular hydrolytic enzymes are secreted at the apical tip to breakdown the substrate.

Its impossible for the hyphal to keep on growing deeper to search for nutrients and transport it backwards to other hyphae. Sooner the back zones of the mycelia will die or sporulation occurs.

BIOFILM GROWTH

The formation of biofilm is usually characterized in SSF where bacteria growth is dominant. The development of biofilm in SSF followed a sequential development whereby the surfaces of the SSF is colonized and development of the biofilm will result in the growth of high number of cells on the surfaces trapped within the EPS matrix

In terms of structure the biofilm offer different challenges in mass transfer compared to mycelial growth of SSF.

The function of the biofilm is in fact a barrier to mass transfers between the microorganisms and the environment. This situation would easily result in the formation of various physical and chemical gradients from the surface to the inner depth of the biofilm. Aerobic and anaerobic zones capable of supporting different physiological groups of microorganisms would be formed easily

EVOLUTION OF SSF FERMENTORS
Technologically, the SSF fermentors have not really evolved and are still stuck with the concept of racks or tray fermentors with little or no changes from the past. Many designs have been published that belong to the first category including static and agitated models but only few models are used in commercial production

It is SF fermentors with its submerged fermentation that has undergone much technological evolution in size, design and function. This was really triggered by the birth of industrial microbiology spurred by the world wars.

Even today, bioprocess engineers are coming up with newer and unorthodox design for submerged fermentation in fermentors for the production of new metabolites from different type of cells. But seriously speaking the high yield of fermentation products such as penicillin is more due to the successful account of strain development rather than significant improvement in bioreactor design

This development of SF over SSF does not mean that SSF is irrelevant. It is more a reflection of the poor understanding and limitations of the various physical, chemical and biological processes to be controlled in any SSF process. It could also mean that there is a poor appreciation of the potential of SSF in the fermentation industries.

The main limitations of engineering design of SSF are:
1 Problem of heat dissipation
2 Mixing

The main method of heat dissipation is by evaporative cooling. In this process the loss of heat is correlated with loss of water from SSF. Thus it is crucial while there is a need to cool the heat there is also the need to retain the moisture for proper SSF to procee

While good mixing is important to obtain good mass transfers in SSF in drum type fermentors, the product of SSF might not be suitable as in the case of tempe fermentation where the final presentation of the tempe is important in itself




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