Upstream and Downstream Processing
Dr. Mamat Kandar
Microbes in Industrial Processes • Several types of industrial processes where microorganisms are used
to produce desirable end products which have well defined industrial uses and applications. These may be broadly classified into the following groups : • Food, beverages, food additives and supplements • Health-care products • Microbial enzymes • Industrial chemicals and fuels
Microbial Natural Product Discovery Process
The term of “Fermentation” Physiological sense • A process that produces energy by
breaking down energy-rich compounds under anaerobic conditions
• The basis of food fermentation, which is commonly used in the food industry to produce diverse food products and also as a food preservation method
Biotechnological context • A process in which microorganisms,
cultured on a large scale under aerobic or anaerobic conditions, convert a substrate into a product that is useful to human
Fermentation in biotechnological context
• Fermentations are also broadly classified according to the organization of the biological phase : • For suspended growth, the cells are freely dispersed in the growth
medium and interact as individual or flocculated units • In supported growth, the cells develop as a biofilm, normally on an inert
support material and result in the formation of a complex interacting community of cells
Example of scheme of entire fermentation process
UPSTREAM PROCESS DOWNSTREAM PROCESS
Upstream Processing • The upstream process à the entire process from early cell isolation and
cultivation, to cell banking and culture expansion of the cells until final harvest (termination of the culture and collection of the live cell batch) • Upstream processing includes :
• preparation and formulation of the fermentation medium • inoculum preparation • Fermentation process and fermenter design
The Fermentation Media
• Fermentation media must satisfy all the nutritional requirements of the microorganism and fulfill the technical objectives of the process • The nutrients should be formulated to
promote the synthesis of the target product, either cell biomass or a specific metabolite
• The main factors that affect the final choice of individual raw materials are as follows : • Cost and availability: ideally, materials should be inexpensive, and of consistent quality and year
round availability • Ease of handling in solid or liquid forms, along with associated transport and storage costs, e.g.
requirements for temperature control • Sterilization requirements and any potential denaturation problems • Formulation, mixing, complexing and viscosity characteristics that may influence agitation, aeration
and foaming during fermentation and downstream processing stages • The concentration of target product attained, its rate of formation and yield per gram of substrate
utilized • The levels and range of impurities, and the potential for generating further undesired products
during the process • Overall health and safety implications
Elements of Media • Carbon Source à e.g. molasses, malt exract, starch, cellulose, whey, fats, oils
etc. • Nitrogen Source à corn steep liquor, yeast extracts, peptones and soya
meal. Purified amino acids are used only in special situations, usually as precursors for specific products
• Minerals à cobalt, copper, iron, manganese, molybdenum, and zinc, etc. • Vitamin and Growth Factors à Many bacteria can synthesize all necessary
vitamins from basic elements • Precursors à e.g. phenylacetic acid or phenylacetamide added as side-chain
precursors in penicillin production • Inducers and Elicitors à e.g. Genetically Modified Microorganisms (GMMs) • Inhibitors à e.g. sodium bisulphites which is used in the production of
glycerol by S. cerevisiae • Cell permeability modifiers à e.g. penicillin and surfactants • Oxygen à e.g. pure oxygen • Antifoams à e.g. plant oils
Inoculum Preparation The Producer Microorganism • The specific product resulting from fermentation is
determined by the type of microorganism carrying on the process and the substance in which the fermentation occurs
• Microorganisms are used extensively to provide a vast range of products and services because : • the ease of their mass cultivation, • speed of growth, • use of cheap substrates (which in many cases are wastes) • the diversity of potential products • Their ability to readily undergo genetic manipulation
• Fermentation industries often prefer to use established GRAS (generally regarded as safe) microorganisms
Industrial Strains • Irrespective of the origins of an industrial microorganism, it
should ideally exhibit: 1. genetic stability; 2. efficient production of the target product, whose route of
biosynthesis should preferably be well characterized; 3. limited or no need for vitamins and additional growth factors; 4. utilization of a wide range of low-cost and readily available carbon
sources; 5. amenability to genetic manipulation; 6. safety, non-pathogenicity and should not produce toxic agents,
unless this is the target product; 7. ready harvesting from the fermentation; 8. ready breakage, if the target product is intracellular; 9. production of limited byproducts to ease subsequent purification
problems
Strain Improvement • Strain improvement has been accomplished using : • Conventional methods :
• natural methods of genetic recombination à which bring together genetic elements from two different genomes into one unit to form new genotypes (e.g. conjugation, transduction and transformation)
• Mutagenesis à Mutations result from a physical change to the DNA of a cell, such as deletion, insertion, duplication, inversion and translocation of a piece of DNA, or a change in the number of copies of an entire gene or chromosome by physical and chemical mutagen
• Modern Method à Genetic Engineering • Recombinant DNA technology à allowed specific gene sequences to be
transferred from one organism to another and allows additional methods to be introduced into strain improvement schemes. This can be used to increase the product yield by removing metabolic bottlenecks in pathways and by amplifying or modifying specific metabolic steps
Conventional Methods Mutagenesis
The Fermentation Process
The fermentation process can also be divided into three basic systems depending on the feeding strategy of the culture, namely: • batch, • continuous, or • fed-batch
(a) Batch culture: a non-steady-state culture with cells at different stages of their growth cycle. Growth rate changes with time in accordance with the classic growth curve, and available nutrient concentrations change with time. Metabolism operates in a non-steady state.
(b) Chemostat culture: a growth rate-limiting substrate is added at a constant flow rate (f) in the inflowing medium. The volume (V) of the culture is kept constant by overflow of effluent through a side port. Metabolism operates in a steady state.
(c) Turbidostat culture: a continuous flow culture in which biomass density is measured and controlled at a fixed value (x) by automatic adjustment of the inflow rate (f) of the complete medium. Metabolism operates in a pseudo-steady state
The process can also depending on the amount of free water in the medium that be categorized as : • solid-state fermentation (SSF) à the medium
contains no free-flowing water. The organisms are moistened e.g. “koji” fermentation of soybeans, production of amylase and protease by Aspergillus oryzae on roasted soybeans and wheat, bioremediation, and detoxification of agroindustrial wastes • submerged fermentation (SmF) à
microorganisms grow submerged in a liquid medium where free water is abundant
(up) solid-state fermentation, (down) submerged fermentation)
Categories of fermentation: Type I • Type I à When the product is formed
directly from the primary metabolism used for energy production
• Growth, energy metabolism, and product formation almost run in a parallel manner
• Exponential and stationery stages are not separated from each other
• Example : production of ethanol, gluconic acid, and single-cell protein
• It can be represented as:
Categories of fermentation: Type II
• Type II à The product is also formed from the substrate used for primary energy metabolism. However, the product is produced in the secondary pathway, as illustrated below:
• The exponential and stationery phase are separate
• Example : production of some amino acids, citric acid, and itaconic acid
Categories of fermentation: Type III • Type IIIà There is a clear distinction
between the primary metabolism and product formation because they occur at separate times • Substrate consumption and rapid growth
occur in the first phase and product formation occurs in the second phase • The product is formed from amphibolic
metabolic pathways and not from primary metabolism • Example : production of vitamins and
antibiotics
Fermenter Design and Construction • The heart of the fermentation process is the
fermenter à The main function of a fermenter is to provide a suitable environment in which an organism can efficiently produce a target product that may be cell biomass, a metabolite or bioconversion product
• Types of Fermenters : • Simple fermenters (batch and continuous) • Fed batch fermenter • Air-lift or bubble fermenter • Cyclone column fermenter • Tower fermenter • Other more advanced systems, etc
Cross section of a fermenter for Penicillin production
Downstream Processing
Downstream processing (DSP) refers to: • the recovery and purification of
biosynthetic products • Process development à the
establishment of platform technologies and high-throughput methods optimized using experimental approaches based on quality by design and design of experiment
Optimization Fields in Downstream Processing
Complexity of Downstream Process
• The complexity of DSP is determined by the required purity of the product at minimum recovery costs • A holistic approach is
required when developing a new industrial purification strategy
Complexity DSP
Microbial morphology
Flocculation characteristics
Downstream Processing
Components Concentration Formulation and
Microbial Biomass Separation
Liquid–solid separation of fermented broth involves the separation of two phases:
• solid à microbial biomass
This type of separation is used in many processes for :
(1) recovery of biomass carrying valuable target metabolite (solid component), with the liquid being discarded
(2) liquid recovery (the solids being discarded)
(3) recovery of both solids and liquid
Consideration of selection of downstream fermentation processes and the design of separation system
difficult-to-filter slurries à enabling them to be filtered more easily
Pretreatment Filtration to make concentrated biomass à discard unused liquid from culture
Biomass Concentration and separation making
improvements to the quality of the solid or liquid products
Post- treatment
Cell Disruption and Release Intracellular Components
• Many of microbial products are intracellular à need to disrupt cell
• The effectiveness of a cell disruption method based on biological materials
• The microorganisms or other cells can be disintegrated or disrupted by physical, chemical or enzymatic methods
Suitability of cell disruption methods for various sample types
Concentration
• It is considered to be the most expensive part of process production
• Concentration includes: • Separation of target metabolites after filtration
(purification) • De-wateringà If low amount of product is found in
very large volume of spent medium, the volume is reduced by removing water to concentrate the product
• The commonly used techniques for concentrating biological products are evaporation, liquid-liquid extraction, membrane filtration, precipitation and adsorption
• A recently developed economic downstream process for biomolecule recovery is the aqueous two-phase system (ATPS)
evaporator Liquid-liquid extraction
Aqueous two-phase system (ATPS)
• The aqueous two-phase system (ATPS) is a clean alternative to traditional organic– water solvent extraction systems • The distribution of the desired
product is based on its surface and ionic character and the nature of the phases • the type of phase forming
components depend on: • pH • ionic strength • temperature
Formulation and Polishing • Formulation is a crucial link between production and application
and dictates economy, shelf life, ease of application, and enhanced field efficacy • Formulation broadly refers to the maintenance of activity and
stability of a biotechnological products during storage and distribution
Biological Product Formulation
concentrating them through removal of most of the water (drying)
High molecular weight products (antibiotics, etc.)
involves crystallization by adding salts
Protein Certain stabilizing additives can be added to protein formulations to prolong their shelf life
Drying Process
large volumes of liquids. • Small droplets of liquid containing
the product are passed through a nozzle directing it over a stream of hot gas
Freeze-drying • Lyophilization is based on the
principle of sublimation of a liquid from a frozen state • The liquid containing the product is
frozen and then dried in a freeze- dryer under vacuum
Generally, before a fermentation-based product can be incorporated into the supply chain it must be free of moisture to prevent deterioration and maintain stability
The overall scheme for upstream (USP) and downstream (DSP) processes
Thank You J