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= Solid separation of fermentation product =

Importance of recovery and purification
If a fermentation broth is analyzed at the point of harvest, it is likely that a specific product may be present at a low concentration (typically 0.1–5 g dm−3, depending upon the initial substrate concentrations taken) in an aqueous solution that contains solid constituents like intact microorganisms, cell fragments, soluble and insoluble medium components, and other metabolic products. The product might also be intracellular, heat labile, and easily broken down by contaminating microorganisms. All these factors tend to increase the difficulties of product recovery. To ensure good recovery or purification, speed of operation may be the overriding factor because of the labile nature of a product. The processing equipment or fermenter therefore must be of the correct type and also the correct size to ensure that the harvested broth can be processed within a satisfactory time limit.

Note that each step or unit operation in downstream processing involves the loss of some product as each operation will not be 100% efficient and product degradation may have occurred.

Criteria for the recovery process
The recovery process is based on the following criteria:

1. The intracellular/extracellular location of the product.

2. The concentration of the product in the fermentation broth/product.

3. The physical and chemical properties of the desired product (to select separation procedures).

4. The intended use of the product obtained.

5. The minimal acceptable standard of purity.

6. The magnitude of biohazard of the product or broth.

7. The impurities present in the fermented broth.

8. The marketable price for the product.

The main objective of the first stage in the recovery of an extracellular product is the removal of large solid particles and microbial cells using the technique of centrifugation or filtration. In the next stage, the broth is fractionated into major fractions using ultrafiltration reverse osmosis, adsorption/ion-exchange/gel filtration or affinity chromatography, liquid–liquid extraction, two phase aqueous extraction, supercritical fluid extraction, or precipitation. Subsequently, the product containing fraction is purified by fractional precipitation, followed by more precise chromatographic techniques and crystallization toobtain a product, which is highly concentrated and free from impurities. Other products are isolated using modifications of this flow-stream. Finally, the finished product might require drying.

Stages in the Recovery of Product From a Harvested Fermentation Broth
It is possible to modify the processes of the broth so that it can be handled faster with equipment that is simple, making use of a number of techniques :

1. Selecting microorganisms that do not produce pigments or undesirable metabolites.

2. Modifying the fermentation conditions in order to reduce the production of undesirable metabolites.

3. Precise and efficient time of harvest.

4. pH control post harvest.

5. Temperature treatment after harvesting.

6. Addition of flocculating agents.

7. Use of enzymes to attack cell walls.

'''Fermentation and product recovery are integral processes. There are interactions between the two parts and thus, neither stage should be developed independent of the other''' as this might result in problems and unnecessary expense or costs. Topiwala and Khosrovi (1978), when considering water recycle in biomass production stated that the interaction between the different unit operations in a recycle process made it imperative that commercial plant design and operation should be viewed in an integrated fashion. The parameters to consider include time of harvest, pigment production, ionic strength and culture medium constituents.

Illustration of the range of techniques used in microbiological recovery processes. Citing the process of flow of recovery and partial purification of Penicillin G.

Illustration of the range of techniques used in microbiological recovery processes:
 * Harvest broth from fermenter
 * Chill to 5–10°C
 * Filter off P. chrysogenum mycelium using rotary vacuum filter
 * Acidify filtrate to pH 2.0–2.5 with H2SO4
 * Extract penicillin from aqueous filtrate into butyl acetate in a centrifugal counter-current extractor (treat/dispose aqueous Phase)
 * Extract penicillin from butyl acetate into aqueous buffer (pH 7.0) in a centrifugal counter-current extractor (recover and recycle butyl acetate)
 * Acidify the aqueous fraction to pH 2.0–2.5 with H2SO4 and re-extract penicillin into butyl acetate as in stage 5
 * Add potassium acetate to the organic extract in a crystallization tank to crystallize the penicillin as the potassium salt
 * Recover crystals in a filter centrifuge (recover and recycle butyl acetate)
 * Further processing of penicillin salt

Additional considerations

One of the major problem currently faced in product recovery is large-scale purification of biologically active molecules. For a process to be economically viable, large-scale production is required, and therefore large-scale separation, recovery, and purification. Subsequent requirements include the transfer of small-scale preparative/analytical technologies (eg, chromatographic techniques) to the production scale while maintaining efficiency of the process, bioactivity of the product and purity of the product so that it conforms with the safety legislation and regulatory requirements. This has been shown in the study done by Pyle (1990).

Removal of microbial and insoluble constituents
Microbial cells and other insoluble materials are typically separated from the harvested broth by filtration or centrifugation. Due to the small size of many microbial cells, considering the use of filter aids to improve filtration rates, while heat and flocculation treatments are employed as techniques for increasing the sedimentation rates in centrifugation.

Flocculation
Flocculation can also be utilized in different downstream processing operations to aid in product recovery. In a study by Hao, Xu, Liu, and Liu (2006), the use of the flocculants chitosan and polyacrylamide on cell debris and soluble protein in the fermentation broth, to enhance the recovery of 1,3-propanediol by reactive extraction and distillation, helps us visualise the importance of flocculation.

Bowden, Leaver, Melling, Norton, and Whittington (1987) have significantly  contributed in cell recovery. With the use of electrophoresis and dielectrophoresis with an intention to exploit charged properties of microbial cells, additionally, ultrasonic treatment to improve flocculation characteristics and magnetic separations. Although not necessarily for the removal of cells, other downstream operations, which involve the application of an electrical field are quite promising. One such process is electrodialysis involving the transfer of ions from a dilute solution to a concentrated one through a semipermeable membrane on application of an electrical field (Moresi & Sappino, 2000). Similarly, Lopez and Hestekin (2013) report the use of electrodialysis in the separation of organic acids in aqueous solution wherein the product, sodium butyrate, was successfully transferred from the aqueous phase into an ionic liquid phase by electrodialysis. A whopping recovery rate of 99% was obtained in addition to reduced energy input compared to traditional processing.

Foam separation (Floatation)
The recovery of surface active products is clearly an important potential application of this technique. While developing the method of separation, the important variables, that require experimental investigation are pH, airflow rates, surfactants, and [http://colligend-collector%20ratios. colligend-collector ratios.]

Foam separation depends on use of methods, that exploit differences in surface activity of materials and molecules (may be whole cells or molecular such as a protein or colloid, that would be selectively adsorbed/attached to the surface of gas bubbles that are rising through a liquid, to be concentrated or separated and finally removed by skimming. It is possible to make some materials surface active by the application of surfactants such as long-chain fatty acids, amines, and quaternary ammonium compounds. Materials that have been made surface active and collected are termed colligends whereas the surfactants are termed collectors. Davis, Lynch, and Varley (2001) provide experimental evidence on the use of foam separation in the recovery, citing its importance of the lipopeptide biosurfactant surfactin from B. subtilis cultures. Their findings show that improved surfactin recovery can be achieved when foaming was simultaneous with the fermentation stage rather than as a nonintegrated semibatch process.

Precipitation
Precipitation is one process that could be used at various stages of the product recovery process as it allows enrichment and concentration in one step, inturn reducing the volume of material for further processing.

It is possible to revive some products (or to remove certain impurities) directly from the broth by precipitation, or use the technique after a crude cell lysate has been obtained.

Typical agents used in precipitation help in rendering the compound of interest insoluble, including:

1. Acids and bases to change the pH of the solution until the isoelectric point of the compound is reached and pH equals pI, so when there is then no overall charge on the molecule and its solubility is therefore decreased.

2. Salts like ammonium and sodium sulfate are used for the recovery and fractionation of proteins. The salt removes water from the surface of the protein revealing hydrophobic patches, which come together causing the protein to precipitate. The most hydrophobic proteins will thereby precipitate first, therefore allowing fractionation to take place. This technique is also termed “salting out.”

3. Organic solvents. Dextrans can be precipitated out of a broth by the addition of a small amount of methanol. Chilled ethanol and acetone can also be used in the precipitation of proteins mainly due to changes in the dielectric properties of the solution4. Nonionic polymers like polyethylene glycol (PEG) can be used in the precipitation of proteins and are similar in behavior to organic solvents.

5. Polyelectrolytes can be used in the precipitation for a range of compounds, in addition to their use in cell aggregation.

6. Protein binding dyes (triazine dyes) bind to and precipitate certain classes of protein [http://(Lowe%20&%20Stead,%201985). (Lowe & Stead, 1985).]

7. Affinity precipitants are an area of much current interest in that they are able to bind to, and precipitate, compounds selectively [http://(Niederauer%20&%20Glatz,%201992). (Niederauer & Glatz, 1992).]

8. Heat treatment as selective precipitation and purification step for multiple thermostable products and in deactivation of cell proteases [http://(Ng,%20Tan,%20Abdullah,%20Ling,%20&%20Tey,%202006). (Ng, Tan, Abdullah, Ling, & Tey, 2006).]

Theory of filtration
A simple filtration apparatus is one which consists of a support covered with a porous filter cloth. A filter cake gradually builds up as filtrate passes through the filter cloth. As the filter cake increases in thickness, the resistance to flow will gradually increase.

Therefore, if the pressure applied to the surface of the slurry is kept constant the rate of flow will gradually diminish. Alternatively, if the flow rate were to be kept constant the pressure will gradually have to be increased. The flow rate would also be reduced by blocking of holes in the filter cloth and closure of voids between the particles, if the particles are soft and compressible. When particles are compressible, it might not be feasible to apply increased pressure.

Flow through a uniform and constant depth porous bed may be represented by the Darcy equation:


 * $$\text{Rate of flow} = \frac{dV}{dt} = \frac{KA\ \Delta P}{\mu L}$$

where μ, liquid viscosity ; L, depth of the filter bed; ∆P, pressure differential across the filter bed; A, area of the filter exposed to the liquid; K, constant for the system. K itself is a term which depends on the specific surface area s (surface area/unit volume) of the particles making up the filter bed.

Filtration which is almost used in almost all scales of operation in order to separate suspended particles from a liquid/gas, using a porous medium which retains the particles but allows the liquid or gas to pass through. It is possible to carry out filtration under a variety of conditions, but several factors will influence the choice of the most suitable type of equipment to meet the specified requirements affordable, these include:

1. The properties of the filtrate, especially its viscosity and density.

2. The nature of the solid constituents, particularly their size and shape, the size distribution and packing characteristics.

3. The solids:liquid ratio.

4. The need for recovery of the solid or liquid fraction or sometimes both.

5. The scale of the operation.

6. The need for batch or continuous operation.

7. Aseptic conditions.

8. The importance of pressure or vacuum suction to ensure an adequate flow rate of the liquid.

Use of filter aids
It is quite common to use filter aids when filtering bacteria or other fine or gelatinous suspensions that prove slow to filter or partially block a filter. Kieselguhr (diatomaceous earth) is the most widely used material. In some processes such as microbial biomass production, filter aids cannot be used and cell pretreatment by flocculation or heating must be taken into account. Additionally, it is not practical to use filter aids when the product is intracellular and when it’s removal would present a further stage of purification.

The steps include:

1. A thin layer of Kieselguhr is applied to the filter to form a rather precoat prior to broth filtration.

2. The appropriate quantity of filter aid is then mixed with the harvested broth. Filtration is started, to build up a satisfactory filter bed. The initial raffinate is returned to the remaining broth done prior to starting the true filtration.

3. When vacuum drum filters are to be used which are fitted with advancing knife blades, then a thick precoat filter is initially built up on the drum.

Plate and frame filters
A plate and frame filter is a pressure filter in which the simplest form consists of plates and frames arranged alternately. The plates are covered with filter cloths or filter pads. The plates and frames are then assembled on a horizontal framework and held together by means of a hand screw or hydraulic ram so that there is no leakage between the plates and frames, forming a series of liquid-tight compartments.

Pressure leaf filters
There are a number of intermittent batch filters usually called by their trade names. Pressure leaf filters incorporate a number of leaves, each consisting of a metal framework of grooved plates, that is covered with a fine wire mesh, or sometimes a filter cloth and often precoated with a layer of cellulose fibre is present.

Vertical metal-leaf filter
The vertical metal-leaf filter consists of a number of vertical porous metal leaves mounted on a hollow shaft in a cylindrical pressure vessel. The solids from the slurry gradually build up on the surface of the leaves and the filtrate is removed from the plates via the horizontal hollow shaft.

Horizontal metal-leaf filter
In a horizontal metal-leaf filter, the metal leaves are mounted on a vertical hollow shaft within a pressure vessel. Often, only the upper surfaces of the leaves are porous. Filtration is continued until the cake fills the space between the disc-shaped leaves or when the operational pressure has become excessive.

Stacked-disc filter
An example of this type of filter is the Metafilter. This is a very robust device and because there is no filter cloth and the bed is easily replaced, labor costs are often low. It comprises of a number of precision-made rings, which are stacked on a fluted rod.

Rotary vacuum filters
The Large rotary vacuum filters are commonly used by several industries, which produce large volumes of liquid which need continuous processing. The filter consists of a rotating, hollow, segmented drum covered with a fabric or metal filter, which is partially immersed in a trough containing the broth to be filtered.

A number of rotary vacuum drum filters are manufactured, which differ in the mechanism of cake discharge from the drum:

1. String discharge.

2. Scraper discharge.

3. Scraper discharge with precoating of the drum.

In the filtration processes, the flow of broth was perpendicular to the filtration membrane. Consequently, blockage of the membrane led to the lower rates of productivity and/or the need for filter aids to be added, and these were unfortunate serious disadvantages. Thus, a cross flow filtration method was employed.

The benefits of cross-flow filtration are :
1. High Efficient separation of >99.9% cell retention.

2. Closed system for the containment of organisms with no aerosol formation.

3.The separation was independent and media densities.

4. No need of addition of filter aid (Zahka & Leahy, 1985) The major components of a cross-flow filtration system are a media storage tank (or the fermenter), a pump, and a membrane pack. The membrane is usually in a cassette pack of hollow fibers or flat sheets in a plate and frame type stack or a spiral cartridge (Strathmann, 1985). In this way, and by the introduction of a much convoluted surface, large filtration areas can be attained in compact devices. Two types of membrane may be used; microporous membranes (microfiltration) with a specific pore size (0.45, 0.22 μm etc.) or an ultrafiltration membrane with a specified molecular weight cut-off (MWCO).

Centrifugation
Microorganisms and similar sized particles can be removed from a broth by use of a centrifuge when filtration is not quite a satisfactory separation method. Even though a centrifuge may be expensive compared to a filter it may be useful when:

1. Filtration is slow or difficult.

2. The cells or other suspended matter should be obtained free of filter aids.

3. Continuous separation to a high standard of hygiene is a requirement.

Noncontinuous centrifuges are of extremely limited capacity and therefore not suitable for large-scale separation. The centrifuges used in harvesting fermentation broths are operated on a continuous or semicontinuous basis. Some centrifuges can be used for separating two immiscible liquids yielding a rather heavy phase and light phase liquid, as well as a solids fraction. They may also be used for the breaking of the emulsions. In accordance with Stoke’s law, the rate of sedimentation of spherical particles suspended in a fluid of Newtonian viscosity characteristics is proportional to the square of the diameter of the particles.

Cell aggregation and flocculation
Following an industrial fermentation, it is quite common to add flocculating agents to the broth to help in dewatering .The use of flocculating agents is widely practiced in the effluent-treatment industries for the removal of microbial cells and suspended colloidal matter (Delaine, 1983). It is well known that aggregates of microbial cells, although they have the same density as the individual cells, will sediment faster because of the increased diameter of the particles (Stokes law). This sedimentation process could be achieved naturally with selected strains of brewing yeasts, particularly if the wort is chilled at the end of fermentation, and therefore leads to a natural clearing of the beer. Microorganisms in solution are usually held as discrete units in three ways. First, their surfaces are negatively charged and therefore repulse each other. Second, because of their generally hydrophilic cell walls a shell of bound water is associated with the cell which acts as a thermodynamic barrier to aggregation. Finally, due to the irregular shapes of cell walls (at the macromolecular level) steric hindrance is inevitable.

During flocculation, one or more mechanisms besides temperature can induce cell flocculation:

1. Neutralization of anionic charges, primarily carboxyl and phosphate groups, on the surfaces of the microbial cells, thus allowing the cells to aggregate. These include changes in the pH and presence of a range of compounds, which alter the ionic environment.

2. Reduction in surface hydrophilicity.

3. The use of high molecular weight polymer bridges. Anionic, nonionic, and cationic polymers can be used, though the former two even require the addition of a multivalent cation.

Range of Centrifuges
Several centrifuges will be described which vary in their manner of liquid and solid discharge, their unloading speed and their relative maximum capacities. When choosing a centrifuge for a specific process, it is important to ensure that the centri- fuge will be able to perform the separation at the planned production rate, and operate reliably with minimum manpower. Largescale tests may therefore be necessary with fermentation broths or other materials to check that the correct centrifuge is chosen.

Basket centrifuge (perforated-bowl basket centrifuge)
Basket centrifuges are useful for separating mould mycelia or crystalline compounds. The centrifuge is most commonly used with a perforated bowl lined with a filter bag of nylon, cotton etc. A continuous feed is used, and when the basket is filled with the filter cake, it is possible to wash the cake before removing it. The bowl may suffer from blinding with soft biological materials so that high centrifugal forces cannot be used.

Tubular-bowl centrifuge
The tubular-bowl centrifuge is a centrifuge to consider using for particle size ranges of 0.1–200 μm and up to 10% solids in the in-going slurry. The figure shows an arrangement used in a Sharples Super-Centrifuge. The major component of the centrifuge is a cylindrical bowl (or rotor) (A), which may be of a variable design depending on the application, suspended by a flexible shaft (B), driven by an overhead motor or air turbine (C). The inlet to the bowl is via a nozzle attached to the bottom bearing (D). The feed which may consist of solids and light and heavy liquid phases is introduced by the nozzle (E). During operation solids sediment on the bowl wall while the liq- uids separate into the heavy phase in zone (G) and the light phase in the central zone (H). The two liquid phases are kept separate in their exit from the bowl by an adjust- able ring, with the heavy phase flowing over the lip of the ring. Rings of various sizes may be fitted for the separation of liquids of various relative densities. Thus the centrifuge may be altered to use for:

1. Light-phase/heavy-phase liquid separation.

2. Solids/light-liquid phase/heavy-liquid phase separation.

3. Solids/liquid separation (using a different rotor)

The solid-bowl scroll centrifuge (decanter centrifuge)
This type of centrifuge is used for continuous handling of fermentation broths, cell lysates and coarse materials such as sewage sludge. The slurry is fed through the spindle of an archimedean screw within the horizontal rotating solids bowl. Typically the speed differential between the bowl and the screw is in the range 0.5–100 rpm Multichamber centrifuge.

Ideally, the solid-bowl scroll centrifuge  for a slurry of up to 5% solid particle of size 0.1–200 μm diameter. In the multichamber centrifuge, a series of concentric chambers are mounted within the rotor chamber. The broth enters via the central spindle and then takes a circuitous route through the chambers. Solids collect on the outer faces of each chamber. The smaller particles collect in the outer chambers where they are subjected to greater centrifugal forces (the greater the radial position of a particle, the greater the rate of sedimentation).

Although the vessels can have a greater solids capacity than tubular bowls and there is no loss of efficiency as the chamber fills with solids, their mechanical strength and design limits their speed to a maximum of 6500 rpm for a rotor 46cm diameter with a holding capacity of up to about 76 dm. Because of the time needed to dismantle and recover the solids fraction, the size and number of vessels must be of the correct volume for the solids of the batch run.

Disc-bowl centrifuge
The Disc-bowl centrifuge relies for its efficiency on the presence of discs in the rotor or bowl. A central inlet pipe is surrounded by a stack of stainless-steel conical discs. Each disc has spacers so that a stack can be built up. The broth to be separated flows outward from the central feed pipe, then upward and inward between the discs at an angle of 45 degrees to the axis of rotation. The close packing of the discs assists rapid sedimentation and the solids then slide to the edge of the bowl, provided that there are no gums or fats in the slurry, and eventually accumulate on the inner wall of the bowl. Ideally, the sediment should form a sludge which flows, rather than a hard particulate or lumpy sediment. The main advantages of these centrifuges are their small size compared with a bowl without discs for a given throughput. Some designs also have the facility for continuous solids removal through a series of nozzles in the circumference of the bowl or intermittent solids removal by automatic opening of the solids collection bowl. The arrangement of the discs makes this type of centrifuge laborious to clean. However, recent models such as the Alfa Laval BTUX 510 (Alfa Laval Sharpies Ltd, Camberley, Surrey, U.K.) system are designed to allow for cleaning in situ.

Subsequent to centrifugation, cell disruption is employed wherein the method of disruption must ensure that labile materials are not denatured by the process or hydrolyzed by enzymes present in the cell.