Downstream processing

Industrial fermentations has both upstream processing (USP) and downstream processing (DSP) stages. USP involves all factors and processes leading to, and including, the fermentation, and consists of three main areas.

 1.      First aspect of USP deals with the producer microorganism. They include obtaining a suitable microorganism, industrial strain improvement to enhance productivity and yield, maintenance of strain purity, preparation of a suitable inoculum and the development of selected strains to increase the economic efficiency of the process.

2.      Second aspect of USP involves fermentation media, especially the selection of suitable cost-effective carbon and energy sources, along with other essential nutrients. Media optimization is a vital aspect of process development to ensure maximum yield and profit.

3.      The third component of USP relates to the fermentation, which is usually performed under controlled conditions. The ultimate aim is to optimize the growth of the organism or the production of a target microbial product.

 DSP includes all processes following the fermentation. It aims at efficient, safe and reproducible recovery of the target product with biological activity, purity, etc. The target product may be recovered by processing the cells or the spent medium depending upon whether it is an intracellular or extracellular product. Maximum recovery yield and minimum costs are therefore important to be ensured.

Two critical factors to be remembered during DSP

·         The level of purity of product which is usually determined by the specific use of the product.

·         The product’s biological activity is very important and must be retained.

 Fermentation factors affecting DSP include

·         The properties of microorganisms, particularly morphology, flocculation characteristics, size and cell wall rigidity. These factors have major influences on the filterability, sedimentation and homogenization efficiency.

·         The presence of fermentation by-products, media impurities and fermentation additives, such as antifoams, may interfere with DSP steps and accompanying product analysis.

·         Choice of fermentation substrate influences subsequent DSP. A cheap carbon and energy source containing many impurities may provide initial cost savings, but may require increased DSP costs. Overall cost savings may be achieved with a more expensive but purer substrate.

·         The physical and chemical properties of the product, along with its concentration and location, are key factors as they determine the initial separation steps and overall purification strategy. It may be the whole cells themselves that are the target product or an intracellular product, possibly located within an organelle or in the form of inclusion bodies. Alternatively, the target product may have been secreted into the periplasmic space of the producer cells or the fermentation medium.

·         Stability of the product also influences the requirement for any pretreatment necessary to prevent product inactivation and/or degradation. 

·         Adopting methods that use existing available equipment may be more cost-effective than introducing more efficient techniques which may need new facilities.

Steps in DSP 

DSP can be divided into a series of distinct unit processes linked together to achieve product purification.

The number of steps is kept to a minimum to reduce cost. Also, even though individual steps may obtain high yields, the overall losses of multistage purification processes may be huge. So, lesser the treatment steps, lesser the overall loss and cost.

The specific unit steps chosen will be influenced by the economics of the process, the required purity of the product, the yield attainable at each step and safety aspects.

In many cases, integration of fermentation and DSP is preferred to increase productivity, decrease the number of unit operations and reduce both the overall time and costs.

The fermentation broth at the time of harvesting is a complex heterogeneous mixture of cell debris, metabolic products, and unused portions of the medium. The required product usually forms a small proportion of the broth. The product required could be the cells themselves such as in yeast manufacture, or within the cells (such as in streptomycin or some enzymes) or free in the medium as with penicillin.

In a few cases no separation takes place such as in the acetone butanol fermentation, where the entire beer is used. In most cases, however, the separation methods used are filtration, centrifugation, decantation, and foam fractionation. Where the required fraction is in the cells then much of the impurities are removed with the filtrate after the cells have been isolated. 

The various methods used in solids removal are discussed below with the approximate level of purification obtained in each stage is shown below. The procedure followed within each stage depends on the material being extracted.

 

Step Process	Recovery/Purity (%)
1a. Cell harvesting	0.1-1.0 (if product is soluble)
	90-99 (if product is cell such as yeasts)
Filtration
Centrifugation
Decantation
1b. Disruption of cells
2. Primary isolation of the product	1-10
Adsorption and/or ion exchange
Solvent extraction
Precipitation
Ultracentrifugation
3. Purification	50-80
Fractional precipitation
Chromatography (adsorption, partition, ion exchange, affinity)
Chemical derivatization
Decolorization
4. Final product isolation	90-100
Crystallization
Drying
Solvent removal

Conventional steps followed in DSP for the purification of products in the soluble portion of fermented broth

Whole-broth Treatment

1. Whole-broth Treatment

In some fermentations such as the acetone-butanol fermentation, the whole unseparated broth is stripped of its content of the required product. In the antibiotic fermentations, resins are used to directly absorb the antibiotics streptomycin (using cationic-exchange resin) and novobiocin (anionic resin.) The antibiotics are eluted from the resins and then crystallized. This process saves the capital and recurrent expense of the initial separation of solids from the broth.

2. Cell harvesting

The first step in the downstream processing of suspended cultures is a solid–liquid separation to remove the cells from the spent medium. Each fraction can then undergo further processing, depending on whether the product is intracellularly located, or has been secreted into the periplasmic space or the medium. Choice of solid–liquid separation method is influenced by the size and morphology of the microorganism (single cells, aggregates or mycelia), and the specific gravity, viscosity and rheology of the spent fermentation medium. These factors can influence the transfer of the liquid through pumps and pipes.

 1.1  Broth conditioning

Broth conditioning techniques are mostly used in association with sedimentation and centrifugation for the separation of cells from liquid media. They alter or exploit some property of a microorganism, or other suspended material, such that it flocculates and usually precipitates. This uses the ability of some cells to adsorb to the gas–liquid interfaces of gas bubbles and float to the surface for collection, which occurs naturally in traditional ale and baker’s yeast fermentations.

 Certain floc precipitation methods are also used at the end of many traditional beer and wine fermentation processes, where the addition of finings (egg albumen, isinglass, etc.) may be employed to precipitate yeast cells.

Major advantages of these techniques are their low cost and ability to separate microbial cells from large volumes of medium.

Some organisms naturally flocculate, which can be enhanced by chemical, physical and biological treatments. Such treatments can also be effective with cells that would not otherwise form flocs.

Coagulation, the formation of small flocs from dispersed colloids, cells or other suspended material, can be promoted using coagulating agents (simple electrolytes, acids, bases, salts, multivalent ions and polyelectrolytes).

Subsequent accumulation of these smaller flocs into larger settleable particles, called flocculation, is done by adding inorganic salts (e.g. calcium chloride) or polyelectrolytes. These are high molecular weight, water soluble, anionic, cationic or non-ionic organic compounds, such as polyacrylamide and polystyrene sulphate.