Acetone-Butanol Fermentation

An example of anaerobic fermentation as well as a mixed fermentation where different products are obtained by using different species of Clostridium. The production of butanol by butyric acid bacteria was first observed by Louis Pasteur in the 19^th century. Before World War-I processes involving microorganisms were developed for the production of butadiene which is required for the production of synthetic rubber. Cham Weizmann reported that Clostridium acetobutylicum is capable of producing acetone, butanol and ethanol in an economically feasible quantity.

During World War-I, acetone was in great demand to manufacture the explosive trinitrotoluene (TNT). Hence, the acetone-butanol fermentation rapidly expanded. But after war, the demand for acetone decreased and butanol increased, as it was required as a solvent for the rapid drying of nitrocellulose paints in automobile industry. But after World War II petroleum based processes replaced biological fermentation processes of acetone-butanol production, which lead to the closure of many industries. Now,  Acetone-Butanol is produced predominantly from petroleum based raw materials.

However, the fermentative production of acetone-butanol is still being carried out in certain countries where the carbon source material, specially, starchy material are available at cheaper rate. 

Riboflavin/Vitamin B₂ is produced as a byproduct in this fermentation process.

Uses of Acetone-Butanol:

1. Butanol is extensively used in brake fluid, antibiotic recovery procedures, urea, formaldehyde resins, amines for gasoline additives and as ester in the protective coating industry.

2. Butanol is also used for the synthesis of butadiene which is used in the preparation of synthetic rubber.

3. Acetone is used as a universal organic solvent and also in the preparation of explosives like trinitrotoluene.

 Microorganism: Clostridium spp.

Raw material: Starch, molasses, sucrose, wood hydrolysates

Products: Butyric acid, Butanol, Acetone, Isopropanol and some acetic acid, H₂, CO_2.

The relative proportion of the products depend on bacterial strain used and fermentation conditions.

Biosynthesis of acetone-butanol

 Fermentation Process of Acetone-Butanol:

Acetone-butanol fermentation process has the following phases:

(i) Production of inoculum

(ii) Fermentation process

(iII) Harvest and recovery

     Two species of Clostridium, Clostridium acetobutylicum and Cl. saccharoacetobutylicum are generally employed for acetone-butanol fermentation. They differ slightly in their nutritional requirements and fermentation factors. For Clostridium acetobutylicum, potato/corn medium is used and for Cl. saccharoacetobutylicum, molasses medium. Inoculum growth and fermentative production of the solvents are carried out at 31° to 32°C for Cl. saccharoacetobutylicum and at approximately 37°C for Cl. acetobutylicum.

(a) Production of Inoculum

Inoculum of Cl. saccharoacetobutylicum is developed in media with molasses, calcium carbonate, ammonium sulphate or phosphate and sometimes corn-steep liquor.

Clostridia are spore formers and are easily maintained as soil stocks in contrast to the vegetative cells. The spores are not very sensitive to oxygen. However, prolonged storage of these spores leads to decrease in the acetone butanol production.

Spores from soil stocks are initially added to deep tubes of semisolid potato-glucose medium for molasses cultures. As the spores are added to the bottom of these tubes they along with soil particles sink to the bottom of the tubes. The submerged location of the spores can protect the vegetative cells from oxygen after germination of spores.

Inoculated tubes are heat shocked and rapidly cooled to incubation temperature to select heat resistant spores. The tubes are then incubated at 31° to 32°C for 20 hours. The growth that occurs in the tubes are used as inoculum for larger batch of molasses medium present in inoculum tanks. The inoculum is grown for 24 to 26 hours before addition to production tank.

Large volumes of inoculum are produced by successive transfers of inoculum by volume to larger media with incubation period of 20 to 24 hours for each batch.

With Cl. acetobutylicum, Stages of inoculum preparation are generally similar to that using Cl. saccharoacetobutylicum except:

1. Spores of soil stock are added to the deep tubes containing corn medium

2. The tubes are incubated at 37°C for 20 hours.

Corn medium is more prone to contamination so sterile conditions should be ensured, periodically.

    Various inoculum stages, particularly the last stage, before the inoculum is transferred to production tank, are checked for contamination by testing :

1. pH

2. Density of the inoculum

3. Rate of gas evolution

4. Presence of facultative anaerobe tested by aerobic plating on agar medium

5. Microscopic observation for contamination by hanging drop method

6. Determination of titratable acidity is done with corn medium in addition to the above tests.

 Building up of inoculum over different stages ensure:

ü  Greater resistance to contamination

ü  Shorter fermentation times

ü  Greater yields

ü  More substrate utilization

 At each of these stages of inoculum transfer, anaerobic conditions are produced/ ensured by:

1. The reducing condition of the medium

2. Immediate use of freshly sterilized and cooled medium before air becomes incorporated

3. Evolution of fermentation gasses

4. By filling the head space of the inoculum tank with sterile inert gas 

Acetone-butanol fermentation process can be described under the following phases:

(i) Production of inoculum

(ii) Fermentation process

(iii) Harvest and recovery 

Medium:

(a) Molasses Medium:Molasses (black strap) which is a by-product in the sugar industry, is used a 5-7% concentration. Nitrogen source is added in the form of ammonium sulphate. In addition, calcium carbonate and sometimes corn-steep liquor are also added. pH of the medium is 5.5 to 6.5 for molasses medium.

Calcium carbonate is added to prevent development of gross acidity in the medium. Excess addition of this salt lowers the production of the solvents. Ammonium sulphate is added at 18 to 24 hours of fermentation.

(b) Corn Medium: Corn meal obtained after corn oil extraction is added at 8% to 10% to water with or without stillage (residue from the preceding fermentation). It is then heated for 20 minutes at 65°C to gelatinize the starch before sterilization of the medium. The pH of the medium is 5.0 to 6.5 for corn medium.

The media are then sterilized and employed in fermentation depending upon the type of Clostridium species used in the fermentation. 

Stillage, that is, residue formed in the previous fermentation is added to the medium approximately at 30% to 40%. It results in the addition of certain nutrients like proteins, carbohydrates and minerals to the freshly prepared medium. It may contain some toxic compounds since it is the result of a previous fermentation, so excess amounts should not be added.                         

(ii) Fermentation Process:

Fermentation is carried out under anaerobic conditions. Production tanks of the capacity of 50,000 to 2.5 lakhs gallons are used in the fermentation. The incubation period is 36 h (2 to 2 ½ days). If the freshly steamed molasses medium is employed, approximately 2 to 4% of inoculum is needed, while for freshly steamed corn medium slightly less inoculum is employed. The inoculum is added first to the production fermenter followed by the addition of medium. This ensures thorough mixing of inoculum with the medium and maintaining anaerobic condition. Before and after inoculation, fermenters gassed with CO₂

 Fermentation generally passes through three phases:

(a) First Phase (0-18 h):

In this phase rapid growth of the bacterium and formation of acetic acid and butyric acid in large amounts along with the production of large quantities of carbon dioxide and hydrogen gases. The pH of the medium which was initially 5.0 to 6.5 for corn medium and 5.5 to 6.5 for molasses medium, decreases to 5.2. This phase, lasts for approximately 13 to 17 hours of incubation. The titratable acidity increases to a maximum and adaptive enzymes are produced which convert acids to neutral solvents.

(b) Second Phase (Next 18 h):

A sharp decrease in the titratable acidity due to conversion of more acids into acetone and butanol. As acids are metabolized to neutral solvents, acetone and butanol, there is an increase in pH, this process is called as acid break. It gets delayed if there is contamination. The rate of gas formation reaches maximum after acid break. It gradually slows as the fermentation process proceeds further.

(c) Third Phase:

The rate of solvent production decreases along with decreased rate of gas formation.  A final pH of 4.2 to 4.4 in the corn medium and 5.2 to 6.2 in the molasses medium. Many cells undergo autolysis at this point resulting in the release of riboflavin into the medium. Products should be recovered before this. 

Anaerobic conditions ensured by:

1. Immediate use of freshly sterilized and cooled medium before air becomes incorporated
2. Active evolution of fermentation gasses 
3. Addition of sterile inert gas/fermentation gasses derived from parallel fermentationsto the headspace of the fermenter
4.  Gassing with carbon dioxide before and after inoculation

Yield:

The ratio of yield of acetone, butanol and ethanol differ slightly depending on the fermentation medium. But, generally the yield is approximately equal to 30% conversion of carbohydrate to solvents.

In a corn medium the ratio of butanol, acetone and ethanol are 6:3:1 respectively, but in molasses medium the ratios of butanol, acetone and ethanol are 6:5:3.

Apart from these solvents, carbon dioxide and hydrogen are also produced as byproducts of the fermentation.

(Gases produced form approximately half of the sugar medium. Total weight of the gases will be one and half times more than the solvents

The acetone-butanol fermentation yields several by products in addition to the gases - include isopropanol, formic acid, acetic acid, butyric acid, acetyl methyl carbinol and yellow oil, which is a mixture of higher alcohols and acids, which are industrially very important.   

 (iv) Harvest and Recovery:

The individual solvents present in the solvent mixture are separated by fractional distillation. Acetone and butanol are collected in separate fractions. Ethanol and isopropanol are collected as a single fraction and sold as a general solvent.

Carbon dioxide converted to solid CO2/dry ice. The residue contains riboflavin and other B vitamins as well as considerable quantity of bacterial cells. The residue is concentrated and dried and used as vitamin feed supplement. 

A beer still is used for the recovery of the products from the fermentation broth. 

Drawbacks/To be taken care of

1)   Contamination due to bacteriophages and Lactobacillus is a common problem which can be prevented by undertaking absolute sterilization.

2)      Maintenance of absolute sterility

3)      Product inhibition

4)      High costs of distillation

5)      Mixture of fermentation products- add to the cost of recovery

 (1 gallon=3-4 L)

MICROBIAL EXAMINATION OF FOOD- Viable colony count, Examination of fecal Streptococci

 Microbiological examination of a food is required in order to estimate its shelf-life or its suitability for human consumption. 

The total viable count or more often, the numbers of a particular component of the total flora such as moulds in a cereal, psychrotrophic bacteria in a product to be stored at low temperature, anaerobes in a vacuum- packed food, or yeasts in a fruit beverage may be needed. Or it may be required to ensure that a food meets established microbiological criteria.

The total mesophilic plate count is widely used as an indication of the microbiological quality of foods unless they are known to contain large numbers of bacteria as a result of their preparation such as fermented milk and meat products.

Microbiological analysis is also required in case of spoilage or foodborne illness where the presence of a pathogen is implicated. The methods for determining an estimate of the total mesophilic count are different from those for identifying the presence of a pathogen, or its isolation for further study.

The isolation of specific pathogens, which may be present in very low but significant numbers in the presence of larger numbers of other organisms, often requires quite elaborate procedures. It may involve enrichment in media which encourage growth of the pathogen while suppressing the growth of the accompanying flora, followed by isolation on selective diagnostic media, and finally the confirmatory tests. Microbiological criteria or the investigation of an outbreak of foodborne illness thus require the monitoring of certain products for specific pathogens, but the difficulties in detecting low numbers of pathogens make it impracticable as a routine procedure.

Indicator organism

An alternative to monitoring for specific pathogens is to look for an associated organism present in much larger numbers – an indicator organism. This is a concept developed originally for pathogens spread by the faecal-oral route in water. A good indicator organism should always be present when the pathogen may be present. It should be present in relatively large numbers to facilitate its detection. It should not proliferate in the environment being monitored and its survival should be similar to that of the pathogen for which it is to be used as an indicator.

Escherichia coli is a natural component of the human gut flora and its presence in the environment, or in foods, generally implies some history of contamination of faecal origin. In water microbiology E. coli meets these criteria very well and is a useful indicator organism of faecal pollution of water which may be used for drinking or in the preparation of foods.

There are, however, limitations to its use in foods where there appears to be little or no correlation between the presence of E. coli and pathogens such as Salmonella in meat, for example. E. coli cannot may not grow in water but, it can grow in the richer environment provided by many foods.

            Thus, routine microbial examination of food mainly relies on viable colony count, and examination of fecal streptococci.

v Viable colony count

Viable colony count/Plate count is used to estimate the number of viable cells that are present in a sample. The most common procedure for the enumeration of bacteria is the viable plate count.

*       It relies on bacteria growing a colony on a nutrient medium. A viable cell count allows one to identify the number of actively growing/dividing cells in a sample.

*       In this method, serial dilutions of a sample containing viable microorganisms are plated onto a suitable growth medium. The suspension is either spread onto the surface of agar plates (spread plate method), or is mixed with molten agar, poured into plates, and allowed to solidify (pour plate method). The plates are then incubated under conditions that permit microbial reproduction.

*       It is assumed that each bacterial colony arises from an individual cell that has undergone cell division. Therefore, by counting the number of colonies and accounting for the dilution factor, the number of bacteria in the original sample can be determined.

Drawbacks

*       Traditional plate counts are expensive in Petri dishes and agar media

*       The major disadvantage is that it is selective and therefore biased.

*       The nature of the growth conditions, including the composition and pH of the medium used as well as the conditions such as temperature, determines which bacteria in a mixed population can grow.

*        Since there is no universal set of conditions that permits the growth of all microorganisms, it is impossible to enumerate all microorganisms by viable plating.

Key points

*       Selective procedures for the enumeration of coliforms and other groups can be designed using selective media/conditions.

*       For routine laboratory investigations, it is a good and sensitive method of detecting the presence of a viable bacterium.

*       This is the basis of the traditional pour plate, spread plate or Miles and Misra drop plate and is widely used in microbiology laboratories.

*        In the pour plate method, a sample (usually 1 ml) is pipetted directly into a sterile Petri dish and mixed with an appropriate volume of molten agar.

*       Even if the molten agar is carefully tempered at 40 – 45^oC, the thermal shock to psychrotrophs may result in them not producing a visible colony.

*       The spread-plate count avoids this problem and also ensures an aerobic environment but the sample size is usually limited to 0.1 ml

 

*       Since we usually have no idea of how many bacteria are in a sample, it is almost always necessary to prepare a dilution series to ensure that we obtain a dilution containing a reasonable number of bacteria to count.

*        Dilutions in the range 10^-1(1/10) to 10^-8 (1/100,000,000) are generally used, although with particular types of samples the range of dilutions can be restricted.

^*             For example, for water that is not turbid, the dilution needed can be 10^{− 6}

The Miles and Misra drop count and spiral plater have been developed to reduce costs of traditional plating methods.

v Miles–Misra technique

The Miles and Misra Method (or surface viable count) is used to determine the number of colony forming units in a bacterial suspension or sample. The technique was first described in 1938 by Miles, Misra and Irwin.

*       The inoculum / suspension is serially diluted. 

* A calibrated dropping pipette, or automatic pipette, delivers drops of 20μl on petri dishes containing nutrient agar or other appropriate medium.

*       Three plates are needed for each dilution series- an average of at least 3 counts are needed.

*       The surface of the plates need to be sufficiently dry to allow a 20μl drop to be absorbed in 15–20 minutes.

*       Plates are divided into equal sectors (it is possible to use up to 8 per plate). The sectors are labelled with the dilutions.

*       In each sector, 20 μl of the appropriate dilution is dropped onto the surface of the agar (without touching the surface of the agar with the pipette) and the drop allowed to spread naturally.

*       The plates are allowed to dry before inversion and incubation at 37 °C for 18 – 24 hours (or appropriate incubation conditions considering the organism and agar used).

*       Each sector is observed for growth, high concentrations will give a confluent growth over the area of the drop, or a large number of small/merged colonies.

*       Colonies are counted in the sector where the highest number of full-size discrete colonies can be seen (usually sectors containing between 2-20 colonies are counted).

*       The following equation is used to calculate the number of colony forming units (CFU) per ml from the original aliquot / sample

Some disadvantages are that the rate of absorption of drops onto the surface of agar depends upon the environmental conditions like temperature and humidity. 

And, a skilled microbiologist is needed to perform serial dilution and to follow aseptic techniques.

 

v Spiral plater

A method for determining the number of bacteria in a solution by the use of a machine which deposits a known volume of sample on a rotating agar plate in a decreasing amount in the form of an Archimedes spiral. After the sample is incubated, different colony densities are apparent on the surface of the plate. By counting an appropriate area of the plate, the number of bacteria in the sample is estimated.

When compared to the pour plate procedure, the spiral plate method gave counts that were higher than counts obtained by the pour plate method. The time and materials required for this method are substantially less than those required for the conventional aerobic pour plate procedure.

 
 *       The spiral plater has a mechanical system which dispenses 50 ml of a liquid sample as a spiral track on the surface of an agar plate-known volume of sample is inoculated, from the center to the periphery of the plate.

*       The system is designed so that most of the sample is deposited near the centre of the plate with a decreasing volume applied towards the edge. There is a progressive decrease in the concentration of the sample.

*       This produces an effect equivalent to a dilution of the sample by a factor of 10³ on a single plate, thus saving on materials as well as saving the time required in preparing and plating extra dilutions.

Comparison of raw and analysed spiral plate images

Spiral platers are used extensively in food safety testing, as well as the pharmaceutical industry. The spiral plating method can save time and resources by cutting down on additional dilution and plating steps.

The system is not suitable for all food samples, as particulate material can block the hollow stylus through which the sample is applied.

 Advantages of spiral plating

• Reduces the number of serial dilutions needed for microbiological testing-3 dilutions obtained in 1 plate
• Reduces the number of Petri dishes needed-fewer the number of dilutions, fewer Petri dishes are required.
• The Spiral distribution enables a reduction of the counting procedure. In most cases, the count is only needed to be performed on one portion of the agar.

 

Fecal Streptococci

The fecal streptococci and enterococci are members of the genera Streptococcus and Enterococcus.  These bacteria are spherical, gram positive, grow in chains, are facultative anaerobic and ferment sugars.  The enterococci and many fecal streptococci are normal flora of the gastrointestinal tract, where they are present at high concentrations.  However, they can sometimes spread from the gastrointestinal tract and cause bacteremia, wound infections, urinary tract infections and endocarditis, more likely in new borns and in the elderly. 

Enterococci are now a concern in nosocomial infections as well as infections of non-hospitalized populations because of growing antibiotic resistance, especially to antibiotics such as vancomycin.

The streptococci and enterococci are relatively fastidious in their growth requirements because they have lost the ability to synthesize many essential nutrients. Therefore, they require several amino acids and the B vitamins to be grown in the laboratory.

Faecal streptococci are recovered from faeces in significant numbers. These species are S. faecalis, S. faecium, S. avium, S. gallinarum, S. bovis, and S. equinus. In the water microbiology, fecal streptococci are indicators of recent fecal water contamination.

*       S. bovis and S. equinus are predominately found in animals

*       S. faecalis and S. faecium are more specific to the human gut.

Streptococci can be classified on the basis of Lancefield (carbohydrate of the cell wall) antigens (A, B, C, D, E-T). Lancefield divided the streptococci into serological groups based on the presence/absence of certain carbohydrate antigens. Lancefield's Group D possesses the antigen glycerol teichoic acid.

Streptococci that posses the Group D antigen are those generally considered to be faecal streptococci—S. faecalis, S. faecium, S. avium, S. gallinarium, S. bovis, and S. equinus.

The fecal streptococci and enterococci belong to serogroup D. 

Enterococci

The enterococci include S. faecalis, S. faecium, S. durans and related biotypes. Enterococci

• grow at both 10°C and 45°C;

• survive at 60°C for at least 30 min;

• grow at pH 9.6;

• grow in the presence of 6.5% sodium chloride;

• reduce 0.1% methylene blue in milk.

They also

• exhibit resistance to the antibiotic vancomycin;

• react with streptococcal Group D antiserum;

• are bile-esculin positive;

Faecal streptococci along with total and faecal coliforms, are generally regarded as the most useful indicators of faecal pollution, and their use has been recommended internationally.

Detection of Faecal Streptococci

Methods for enumeration of fecal streptococci

*       Most probable number (MPN) method

*        Membrane filtration

*       Plate count methods

*       Fluorescent antibody techniques

*       Biochemical species identification systems

*       DNA-based identification systems

The methods most commonly used to assess faecal streptococci in water are the most probable number (MPN) and the membrane Filltration (MF) techniques. 

The MPN technique is applicable primarily to raw and chlorinated waste water and sediments, and it can be used for fresh and marine waters. The disadvantages of the MPN technique are that it requires both time and materials, requiring low-cost labour. 

The MF method may also be used for fresh and saline samples, but it is unsuitable for highly turbid waters. Water with high levels of colloidal or suspended materials can clog the membrane filter pores and prevent filltration. Moreover, suspended materials cause spreading colonies that may interfere with target colonies and thereby prevent accurate counting.

The pour plate technique (PP), is more suitable for enumerating faecal streptococci densities in polluted waters. The pour plate method is a simple technique familiar to water bacteriologists; it is less time-consuming than MPN counts and cheaper, but it is as accurate as membrane filter counts.

Mediums for the growth of fecal streptococci used in a multiple tube (most probable number; MPN) method, with a membrane filter (MIF) procedure, or by the agar pour plate technique are

*       Azide dextrose broth

*       SF medium (Streptococcus Faecalis medium)

*       BAGG broth - Buffered Azide Glucose Glycerol Medium

*       Ethyl violet azide broth/ (EVA) broth

*       KF streptococcal medium (Kenner faecal) streptococcus broth

*    Enterococcus agar

 

The World Health Organisation recommends the use of Enterococcus agar for use with pour plates, spread plates, or membrane filtration techniques for enumerating faecal streptococci and incubation at 44°C for 48 h.

 

The MPN or multiple tube dilution (MTD) method can be used to enumerate faecal streptococci .

1.1  Presumptive isolation in azide dextrose broth (AD)

*       The AD-EVA procedure involves inoculation of dilutions of sample into replicate tubes of azide dextrose AD broth.

*        Positive tubes exhibit turbidity following 24-48 h incubation at 35-37°C.

*       These tubes are confirmed by transfer to Ethyl violet azide broth/ (EVA) broth, where a positive reaction is indicated by the formation of a purple "button", or dense turbidity, after 48 h incubation at 35-37°C.

*       The faecal streptococcus count is estimated from the positive EVA tubes using an MPN table.

*  Various other broths have been recommended for the recovery of faecal streptococci, including Kenner faecal streptococcus (KF) broth and Pfizer selective enterococcus (PSE) broth

*       However, the AD-EVA method is probably the most commonly used liquid medium system, and is recommended by WHO.

1.2  Presumptive isolation in AD broth for 24-48 h is followed by confirmation by streaking a portion of growth to plates of PSE (Pfizer selective enterococcus) agar that are then incubated for 24 h at 35°C.

 

2. Membrane filtration involves drawing a known volume of liquid sample through a membrane filter with a 0.45 um pore size.

*       Bacteria present in the liquid are retained on the filter surface and the filter is then transferred to a selective medium for colony development.

*       The number of colonies counted on the membrane is taken to be equivalent to the number of streptococcal cells present per volume of sample.

*        Compared to the most probable number method, membrane filtration produces results more quickly, allows a larger volume of sample to be tested, allows colonies to be counted directly, and allows the immediate biochemical testing of isolated colonies.

*       However, filtration may not be used for highly turbid samples and is not recommended for chlorinated effluent.

*       KF broth/agar (Kenner faecal streptococcus) was probably the most commonly used faecal streptococci membrane filtration medium.

*        Faecal streptococci appear on this medium as pink to dark red colonies after incubation at 35°C for 48 h.

*    When the membrane is overlaid with PSE medium, faecal streptococci colonies can be distinguished by the presence of dark brown halos indicative of esculin hydrolysis.

 

3.  The bile-esculin test is a biochemical test performed to differentiate Enterococci and group D Streptococci from non-group D viridans group Streptococci on the basis of their ability to hydrolyze esculin.

• Esculin is a glycosidic fluorescent compound, and its hydrolysis can also be observed by the loss of the fluorescence.
• The blackening of the medium and  loss of the fluorescence demonstrates a positive test. The lack of color change demonstrates a negative tube test.

·  Many organisms are capable of hydrolyzing esculin, but only a few of them can do so in the presence of bile (4% bile salts or 40% bile.). This property is utilized to identify organisms of Enterobacteriaceae, genera Streptococcus and Listeria etc.

·  The Bile-esculin test is performed on a selective differential agar; bile esculin agar, which consists of bile as well as esculin.

·  The agar contains different bile salts that inhibit the growth of other Gram-positive organisms and allows the selective isolation of Enterococci and Group D Streptococci.

·  Nowadays, bile-esculin disks are available that are commonly used for the rapid differentiation between Group D Streptococci and non-Group D Streptococci.