Topic 1: Introduction to Bioproducts and Bioseparations

I. BioproductsClassification and characteristics of bioproductsCharacteristic of fermentation broth on downstream processing.Broad classification of bioproducts.Primary and secondary metabolite.

II. BioseparationsIntroduction to bioseparations.Stages of downstream processing.Basic principles of engineering analysis.Criteria for process development.

OBJECTIVES

What is bioproducts:Chemical substances made by living things ranging from small molecules to higher molecules (macromolecules).Derived by extraction from original host or by synthesis in bioreactor containing cells or enzymes.They are sold for their chemical activity;

BIOPRODUCTS: Classification of Bioproducts

CompoundFunctionMethanolSolventEthanolFuel and pharmaceutical usePenicillinAntibacterial agentTaxolAnticancerSteptokinaseBlood dissolving activityWhole cell Bacillus thuringienisisInsecticide

Range of Characteristics of Bioproducts:

3 major categories: 1. Very high value, low volume (therapeutic proteins and enzymes, interferone, factor VIII, urokinase).etc. of high purity, produced in the range of grams to kg.

2. High value, low volume and high purity products (diagnostic enzymes, human growth hormon, monoclonal antibodies and insulin)etc. produced in tens or hundreds kg.

3. Bulk industrial product of relatively low purity (antibiotic, amino acids, organic acids, ethanol, proteases and amylases) etc. produced in hundreds of kgs to tons in quantity.

Correlation between market volume or concentration of desired product and selling price or purity requirements:

Different separation principles and mechanisms for isolation and purification depends on: Molecular mass (50 to 2,000,000) Charge distribution Hydrophobicity Structure and immunogenic structure Specific activity

Most are fragile; bioactivity and stability affected by factors such as pH; temperature, ionic strength and the nature of solvent; shear force during stirring and presence of denaturing agents such as surfactants, metal ions and other chemicals.

BIOPRODUCTS: Characteristics of Bioproducts

In classical biotechnology, products produced naturally by the living cells as of penincillin by molds, ethanol by yeast and organic acids and enzymes by bacteria; conventional separation via chemical engineering unit operations are used:Unit operations: filtration, centrifugation, sedimentation, adsorption and liquid-liquid extraction are well described mathematically and easy scaled-up, subsequently pilot plant study.

In modern biotechnology, the microorganisms are induced under specific conditions.

1. The type of microorganisms and their morphological features (size and shape) size for microbial, plant and animal cell and their agglomerates bacteria and yeast. Slimy mass affect the viscosity fungi forms agglomerates (100-4000 um) 2. Concentrations of cells, products and byproducts and (see following table 3. Physical and rheological characteristics. dried biomass density of 1400 kg/m3, but broth is lower 110 kg/m3 (70-80% water) pellet and floc 1030 kg/m3 due to water entrapped in the cells

BIOPRODUCTS: Characteristic of Fermentation Broth on Downstream Processing

Concentration of biomass and product in fermentation broth:

Bioproducts can be broadly classified into the following categories 1. small molecule: consisting of fine chemicals antibiotic hormones, amino acids and vitamins;

2. large molecule: proteins, polysaccharides and nucleic acids;

3. and particulate products: cells, apores, liposomes and subcellular particles or organelles.

BIOPRODUCTS: Broad Classification of Bioproducts

Small biomolecules in fermentation processes and of important commercial products, include naturally occurring compounds and metabolites such as citric acid, vitamins, amino acids and antibiotics. Small biomolecules can be divided into two categories: 1. Primary metabolites and 2. Secondary metabolitesPrimary metabolites:That is formed during the primary growth phase of the organism. Figure 1. 2 shows the key central metabolic intermediates of biosynthetic pathways in heterotrophs, organism that use organic compounds such as carbon source.

BIOPRODUCTS: Primary and Secondary Metabolites

The intermediates shown in this overview are used in catabolism, the processes by which microorganisms obtain energy from organic compounds, and as well as in biosynthesis, also called anabolism (synthesis of protein, fats and other cell constituents)

Refer to Harrison et al., 2003 for further reading of some examples of small biomolecules and their importance in the fermentation industries.

Secondary metabolites:

Secondary metabolites are not produced the primary growth phase of a micro-organism, but at or near the beginning of the stationary phase.Antibiotics are the best known and most extensively studied secondary metabolites. Eg antibiotic synthesized by fungi as a means of competing with bacteria (and sometimes other fungi) for unrestrained growth in natural (dirty) environments.Primary metabolites are the raw material for the synthesis of secondary metabolites and the cells energy charge are important modulators of the pathways leading to a number of different secondary metabolites and their general routes of synthesis are shown in Figure 1.10 (Harrison et al., 2003)

Secondary metabolites made by plants and fungi have found many roles in human culture. Apart from steroid hormones, hallucinogens, and other psychoactive drugs, there are numerous cytotoxic secondary metabolites that have found their way into cancer therapy, cell and biochemical research and physiological applications.

Modern bio-products involving genetic manipulation or processes is dependent upon biochemical engineering, is divided into two disciplines, upstream engineering (fermentation) and downstream engineering (purification, or bioseparations).

Purification of bioproducts involves a long sequence of steps, and each step requires the use of one or more unit operations, such as sedimentation, adsorption and drying.

BIOSEPARATIONS: Introduction to Bioseparations

For bioseparation purposes, important data include; Thermal stability Solubilities Diffusivities Charge Isoelectric pH Reaction rate constants Separation thermodynamics

Four Stages: 1. Removal of solids (or recovery) 2. Isolation of products 3. Purification 4. PolishingUnit operations are applied in varying combinations for each stage.The unit operations can be divided by function into; Liquid-liquid separations (dewatering, concentration, particle removal) Solute-solute separations (isolation, purification) Solid-liquid separation (polishing)

BIOSEPARATIONS: Stages of Downstream Processing

BIOSEPARATIONS: Basic Principles of Engineering PrinciplesAim in engineering analysis, is to determine how much and how fast: Develop equations for product capacity, processing rate, and possibly product purity Three principal ingredients of engineering analysis: 1. Equilibria 2. Material Balance 3. Flux (or transport phenomena)

Process and Product Quality:

Product quality due to processing are purity, fold purification, specific activity and yield.Product purity is defined as follows:

Purity is a strictly quantitative measure, therapeutic protein can be 99.99% pure but still unacceptable if any pyrogenis present. A pyrogen is any substance that produces a fever

Fold purification is the ratio of the purity at any stage in the process to the purity at the start of the purification process;

-where units of biological activity are assayed by means of a biological test, such as moles of substrate converted per second per liter or fraction of bacterial cells killed. -For proteins (the mass), is usually in total protein; on this basis, the specific activity reaches a constant value when the protein is pure.Yield is given by;

BIOSEPARATIONS: Criteria for Process DevelopmentThe following criteria should be used in evaluating and developing a bioseparation process: Product purity Cost of production as related to yield Scalability Reproducibility and ease of implementation Robustness with respect to process stream variables

Examples of Bioproseparation:

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