SEALS IN FERMENTORS

One of the most stringent requirements in the design of a fermentor is the ability of the fermentor to maintain strict aseptic integrity throughout the fermentation process. Of course this is applicable in monoseptic fermentation involving a single pure culture fermentation. For such requirements, theretically there should be complete isolation or barrier between the fermentor content and the surrounding environment. This would literalloy mean that the fermentor is completely closed by wall or structure to the environment with no openings or holes that expose the content of the fermentor to the environment.

In reality this is difficult to achieve as fermentors need holes or ports that allow important connections for the insertion of impellers, electrodes, inlet gas and outlet gas and even sampling ports. If this is so then the design of the fermentor must reach a compromise where while on one hand it maintains its aseptic integrity and on the other hand it allows port holes for the necessary components to enter or leave the fermentor but by not compromising its aseptic integrity

The solution to this dilemma is by:
1 Providing seals
2 Maintaining the seals continually aseptic

The seal is especially crucial at the stirrer or agitator entrance to the fermentor. The stirrer is the essential component in any fermentor as it is involves in the mixing and homogennization of the contents of the fermentor. Any seal that is used in the agitator system will have to fulfil various requirements such as:

1 It should be able to maintain the state of aseptic integrity of the fermentor while functioning
2 It should be able to withstand the stress of repeated sterilizations of the fermentor such as SIPs
3 It should be able to withstand the various CIP procedures
4 It should be able to sustain the pressure developed within the fermentor
5 If the fermentor is involved in food and pharmaceutical fermentations, it should be built of sanitary materials
6 The seal components will not contaminate the fermentation process
7 It should be able to isolate the contents inside the fermentor from the external environment

In any seal there is usually a need for:
1 Packing structure within the seal
2 Lubricant to smoothen the rotation of the shaft within the seal
3 Steam sterilization of the seal to ensure no contaminations occurring through the seal

There are other seals within the fermentor which is made up of the O - rings that help in the close air tight sealing between two surfaces. We have discussed this section in previous blogs Read more!

BOTTLED MICROBIAL CULTURES- DOES IT REALLY WORKED?

If you go through the internet, you will be seeing a lot of advertisements trying to sell their cultures which are supposed to the ultimate solution to solving almost anything from pollution to bioremediation and even to composting among others. The vendors claimed to have discovered or concocted mixtures of species of microorganisms that seems to do the trick.

The question is does it really do the work as promised by defying the laws of microbiology itself.

Let us look at a simple example whereby a mixture of microorganisms are effective in reducing pollution of a heavily polluted water body

Questions that should be asked are;

1 Have these microbial cultures been successfully adapted to using the wastes in the polluted wastewaters
2 How are these cultures adapted to degrading the wastewaters?
3 Are there proven laboratory or feasibility studies supporting this claim?
4 Are the findings reported in well known peer reviewed journals

Throughout my experience in microbial degradation, it has always been the principles of microbiology that it is the substrate that chooses the microorganisms and not vice versa.
Adaptation involves various stages of not only adapting to toxic conditions but also developing the necessary enzymes to utilize the complex substrate. Microbes are not stupid! Given the choice they will use the easily assimilated carbon in the wastewaters before going after the difficult carbon.

Wastewaters especially the polluted and complicated wastewaters are not only toxic but contain a variety of carbon which are difficult to degrade. I cannot see how by just pouring hundreds of buckets of these 'special microorganisms' into the moving wastewaters such as rivers and streams will solve the pollution problem. Up to now I have only heard claims upon claims of these magical cultures doing wonders...Where are the proof in properly carried out experiments published in eminent scientific journals. I don't think it is wise or safe to take the opinion from salesmen selling the cultures, laymen or farmers

Often when they carry out the 'experiments' they monitor it months and years after the event

One thing I do agree the media which they used for building up the cultures are mainly mplass based which are rich in sugars and vitamins which support the growth of many microorganisms and are good fertilizers. So is the so called success in the use of these microorganisms more because of the 'magical bacteria' or because of the rich nutrients in the molasses? These molasses solution are often viscous and acidic which resulted in lowering the ph of the water and precipitating the colloids and chemicals. But will it be lasting in the long run and on a bigger scale? We need more research and experiments properly carried out before validating these products. If not a lot of people will get tricked and lose a lot of their money over the magical property of these microbes Read more!

SCIENTIFIC APPROACH TO DETERMINE PROBLEM AREAS IN BIOLOGICAL WASTEWATER TREATMENT PLANTS

Most, if not all, biological wastewater treatment plants are either not working or are not functioning efficiently to their expectations. This sad state of affairs gets worst with time due to changes in the operating parameters of the WWTP or due to poor monitoring and servicing of the WWTP.

The main contributing factor to this poor state of WWTP probably arises out of the attitude of the companies regarding the WWTP as a liability in that it does not contribute to the profits of the company. It is not surprising therefore that they will not invest more money in operating and maintaining the WWTP efficiently. In the first place they build the WWTP more as an excuse to get the necessary operating license to carry out their manufacturing activities

The WWTP has always been situated at the back of their manufacturing premise and not in front of their minds. The full realization and impact of poor housekeeping in maintaining an operating WWTP only comes too late when Department Environment officers slap them with compounds and threatening imprisonments and forced transfer of their manufacturing facilities

FAILURE OF WWTP

The failure of WWTP could be attributed to many factors such as design oversights, poor operation, poor maintenance and servicing of the WWTP. These various failures will ultimately affect the performance of the microbes in the WWTP which will ultimately affect the performance of the WWTP. After all a biological WWTP is nothing more than a bioreactor that support the growth and function of high concentration of microorganisms

NATURE AND CLUE FROM FAILURES

WWTP failures reflect both the state of the pathology of the WWTP and give us vital clues to the where and why the WWTP fails. We should try to study the nature of these failures scientifically so that it will aid us in our decision whether we can repair, replace or improved the WWTP process. Failing to do this we might end up in the future building or renovating the WWTP which will end up in similar predicament years later

MAIN TYPES OF FAILURES

There are generally failure two main types of failures:

1 Sudden failure

2 Gradual failure

Sudden failure of WWTP is a failure that is not anticipated and usually occurs immediately beyond our control. Examples of sudden failure are electrical outage, blower burnt out, toxic chemicals or sudden hydraulic loading due to heavy rain. The impact of sudden failure is often very serious which can throw out the WWTP out of its function

Gradual failure of a WWTP occurs slowly and show cumulative deterioration with time. It is usually reflective of slow breakdown of system. Example of gradual failure of WWTP is exemplified by gradual clogging of the porous filters used in supplying air to the WWTP. Such failures could have been averted as the signals of impending failure are there. Such failures could have been avoided if remedial actions such as repair taken before turning to disaster

Previous records if kept could have indicated clues of impending failure, when the performance of the WWTP is slowly spreading out of optimum band state of operations. We should be able to see the loss in treatment efficiency of the WWTP over time in terms of BOD, COD and TSS reduction

WHY MOST WWTP FAILURES GO UNDETECTED?

In reality most failures go undetected because there are no staffs that are trained to monitor the performance of the WWTP. By the time the WWTP completely broke down, it is already too late to take preventive actions

Even if monitoring of the WWTP is carried out, it usually involves ‘end of pipe’ analyses and not ‘up pipe’ or ‘along pipe’ analyses. In most cases the analyses just involved the basic regulatory requirement parameters such as BOD, COD and TSS. These parameters are insufficient to show the state of health of the WWTP and you do not really know which unit process is facing the problem?

IMPORTANCE OF ADDITIONAL PARAMETERS MONITORING

There are other parameters besides BOD, COD and TSS to give us the clues on the exact state of health of the WWTP. Any changes in the operating parameters of the WWTP will be reflected by the gradual change in the composition and properties of the microorganisms of the WWTP. There will also be changes in the chemical; parameters in the mixed liquour due to the changes in the microbial consortium in the WWTP.

The failure for nitrification could be indicated by very poor DO and high NH3 content of the wastewaters. High biomass washouts could be attributed presence of pin flocs within the WWTP are some of the examples of how additional parameters monitoring help to determine WWTP failures

SCALEDOWN EXPERIMENTS

To confirm the observations there might be the need to carry out scale down studies to pin point the cause of the WWTP failure. Samples of the mixed liquour from the WWTP are removed and transferred to various smaller vessels to study the effect of mixing, aeration or oxygen uptake. From the studies we can conclude if enough mixing or aeration are the culprit of the WWTP failure.

Additional respirometric experiments may be carried out to determine if the microorganisms are viable or if the wastewater is biodegradable.

NEED TO DO PROFILE STUDIES OVER THE SERIES OF UNIT PROCESSES IN WWTP

Wastewater treatment in a WWTP usually involves the flow of the wastewater through a series of unit processes which include physical and biological treatment

Analyses of various parameters as the wastewater enter and leave the various unit processes from the influent inlet to the treated effluent outlet will be able to tell us:

1 Which unit processes are most effective in reducing the pollution load?

2 Which unit processes are the rate limiting steps and controlling the bottleneck of the process?

3 Which unit process are the ones having problems?

This information would be useful to allow us to improve the efficiency of the unit process in mind or even to increase the optimum rate to the limit for existing functioning unit processes

IN SITU AND ON SITE MEASUREMENTS
Simple measurements can be conducted by using ph and DO probe with long cables, together with simple tracer studies to see the problems of flow and circulations within the WWTP. Any short circuiting or improper mixings of the WWTP will be easily noticed

STITCH IN TIME
The success in determining the cause of plant failure and inefficiency will allow rapid action to remedy the situation or even to optimize the rate limiting steps in the wastewater treatment process. This will allow the factory to save time and costs.

If the failure is not detected immediately and the WWTP is allowed to deteriorate until whole system backfire then it will be difficult to pinpoint source of error and to repair it early.

Read more!

FERMENTATION PRODUCTS- CHEMICAL VERSUS MICROBIOLOGICAL SYNTHESIS

The common thing shared by both chemical synthetic industries and the microbial fermentation industries is that both are involved in the synthesis and production of chemical compounds. That is where the similarity ends.

Chemical industries produced both organic and inorganic chemicals in more varieties by carrying out the chemical reactions which utilize more energy and pressure compared to the fermentation industries. Microbial fermentation industries carry out the synthetic reactions under normal ambient temperature and pressure using microorganisms and enzymes to catalyze the changes. Their biological reactions do not require high energy or extreme conditions.

Fermentation products formed by the microorganisms are however more labile and occur in lower concentrations or in very dilute solutions. These characteristics have created problems not only in the production of the chemicals but also in the downstream isolation, concentration and purification of the fermentation products compared to the chemical industries.

It is interesting to note that most of the chemical downstream processing units for product recovery are also used in the fermentation industry downstream processing of chemical oroducts. However, in the case of downstream processing of fermentation products additional downstream processing units are added in the purification and concentration of fermentation products. Read more!

PROBLEMS OF CULTIVATION OF MAMMALIAN CELLS IN FERMENTORS

In the past few decades there have been great interests in the cultivation of mammalian cells, including human cells in fermentors for the production of biologics for diagnostic and therapeutics used in human health care.
This is not a new direction in the progression of fermentation technology but a natural anticipated progress in the field of fermentation technology.

Historically speaking the field of fermentation technology has been based on the cultivation of microorganisms such as bacteria to carry out the transformation and production of fermentation products of great economic and industrial importance. It seems then that the power of the microbes are limitless in producing any kind of metabolites. Great strides in the development in microbial fermentation technology were made with parallel development in the study of microbiology, physiology, biochemistry and genetics with similar development in the design and construction of fermentors by bioprocess engineers.

The culmination of this progress is in a nutshell the fourth definition of fermentation by the engineers which defined fermentation being the study of the cultivation of high concentration of microorganisms in bioreactors microorganisms. The fermentor is seen as a special vessel which provides all the optimum conditions necessary to support the growth of high concentration of microorganisms and allow the transformations to occur.

With the development in plant biotechnology especially in the generation of tissue cultures, it then becomes a natural extension or progression to culture single plant cells in fermentors as there is really not much difference between microorganisms and plant cells when they occur as single plant cells.

Much knowledge, experience and breakthroughs in microbial cultivation in fermentors could be similarly applied to single plant cells. Well, at least superficially they seems similar, but in reality there are many problems faced in the cultivation of single plant and mammalian cells compared with the cultivation of microbial cells

THE PROBLEMS..
----------------------------

As we have said earlier the common factor shared between microorganisms and single plant and mammalian cells is that all the cells for the cultivation in the fermentors are SINGLE cells. Beyond it are more differences than similarities.

We can see the main differences between the cultivation of microbial cells and mammalian cells in the following:

1 Larger size
2 Longer time to grow
3 Fragility of cells
4 Lower oxygen consumption
5 Tendency to form multicellular aggregates
5 Complex medium requirements
6 The cells might end up not producing the desired products efficiently

The above factors are crucial when one consider cultivating the mammalian cells in fermentor.

The fragility of mammalian cells means that these cells are easily broken up by the various shear forces generated in the fermention broth such as effect of mixing by impellers, liquid shears and bubbles cavitation

Due to the long growth time means that it takes very long period for the mammalian cells to reach optimal concentration in the fermentors. This situation would make the fermentation process more susceptible to microbial contaminations. There is a stringent requirement to prepare clean room standards for mammalian cell cultivation.

The cultivation of mammalian cells require complex media which is more costly adding to the costs of the fermentation

In mammalian cell cultivation the issue is not so much about using cell substrate for the highest yield or productivity but more of:
1 instability protein expressions which might deteriorate with time
2 protein produced require extensive post translational modifications especially glycosylation of protein such as in production of monoclonal antibodies

Bacterial systems are poor choice in the production of the biologics because bacteria cannot carry out complex post translational modifications of proteins.They can only produce simple proteins such as insulins.

These proteins formed too are not easily secreted by the bacterial system and tend to accumulate inside the bacterial cells as crystalline deposits. Their release from the cells would enticed complicated downstream processing which are costly and there is also the danger of endotoxins being produced if gram negative bacteria are involved

The released proteins might require resolubilization and refolding to bring it to its effective state. This does not consider percentage losses of products during downstream activities

FERMENTOR REQUIREMENTS FOR MAMMALIAN CELL CULTIVATION
------------------------------------------------------------------------------
In view of the above considerations, any fermentor to be used for mammalian cell cultivation must be:

1 Exhibit minimum shearing forces either liquid to liquid shearing, liquid to impeller shearing or
bubbles shearing
2 Provide durable aseptic integrity throughout the fermentation duration

As we already know the CSTR is the most widely used fermentor in industrial fermentations and its engineering principles and reliability well established. Modifications can be made to the CSTR to make it able to cultivate mammalian cells

The fermentation vessel should be rounded at the bottom and equipped with baffles to prevent vortex which could damage the cells.

Strong air sparging is avoided and laminar hydrodynamic flow is encouraged. Micro bubbles are generated in a laminar flow mixing for oxygenation. Since the mammalian cells require low amount of oxygen supply of oxygen minimum aeration to maintain DOT values of about 20% saturation

Strong aseptic adaptations might be included with increasing the number of seals to prevent contamination. The sampling port should be equipped with direct steam sterilization to prevent contaminations during samplings







to be continued.... Read more!

USING MICROORGANISMS FROM TYPE CULTURES COLLECTION FOR FERMENTATION

This may be considered the most easy, convenient way to start a fermentation by obtaining the cultures directly from culture collection centres. Superficially it seems that you save time and money by not trying to search and screen microbial isolates from various habitats to provide microorganisms for the fermentation process. But is it so?

While it is true certain groups of microorganisms can produce antibiotics, and obtaining these microorganisms should be economical and we are still assured of its antibiotic producing capabilities. It must be noted however that these microorganisms in the Culture Collections are just kept for reference and taxonomic purposes and studies. They are not industrial strains or high yielder. Logically if the strain is a super producer it would be kept under tight security under lock and key:))

The microorganisms maintained at the Culture Collections are products of isolation and identification years ago which are perpetually maintained by periodic sub culturings. Maybe these microorganisms have "lost" some of its important genetic traits?

It is better to search for new strains from exotic habitats in the hope of finding a super producing strain or a new metabolite Read more!

STERILITY CHECK IN FERMENTOR OPERATIONS

One of the most important aspects before and while running a fermentation study using a fermentor is to monitor whether that fermentor is effectively sterilized and/ or that during the whole fermentation run that the fermentor maintain its aseptic integrity. It is indeed very sad in my personal experience meeting with various fermentation technologists especially those trained from an engineering background that the question of sterility and contamination have never been seriously considered. What is important to them that the fermentor is 'functioning mechanically' and is yielding growth of microorganisms or producing tons and tons of data. Did they ever think for all the efforts, time, energy and costs that their data could be wrong or useless? In fermentation studies, wrong experiments can still yield data....wrong and error filled data!!?? And to make things worst they send their students on a wild goose chase analyzing these wrong data and reaching definitive conclusions? This must be the case of abuse of statistics and computer! After all if its rubbish data coming in you will get rubbish data or conclusions coming out

Most fermentation technologists take lightly to the concept of microbial contamination. This is not so in the stringent pharmaceutical fermentation industries where cGMP are always applied and validation of sterilizations and aseptic conditions are sustained as routine procedures. Any contamination would be a very heavy consequence in such industries.

Most fermentation technologists in universities, colleges and research institutes I met seemed to be in the state of denial thinking that:

1 Their fermentors are 100% sterile or can maintain aseptic integrity
2 They dont bother doing blank sterility test runs periodically to check if their fermentors are up to the sterilty validation
3 They do not periodically remove samples during fermentation runs to check for microbial contaminants

It is true that those fermentors before they are delivered are tested for sterility check for about 5 days equivalent of fermentation run. But these fermentors must be continually checked by conducting blank runs using rich broth and operated without any organism inoculated

It is impirtant to know if contamination occurs to determine whether it is due to:
1 Fermentor structural weakness
2 Operator failure
3 Feed or what ever Read more!