Bioprocessing

Bioprocess engineering involves the comprehensive management of biochemical processes. These processes harness biological pathways and reactions mediated by living organisms such as animals, plants, microorganisms, or enzymes. They operate under controlled conditions to efficiently transform raw materials into various products, scaling production as needed through design, operation, control, and optimization. The product may be directly applicable as food, medicine, or industrial compounds or indirectly in the bioprocess without any direct product formation, such as in detoxifying industrial wastes or treating factory effluents.

1.0Definition of Bioprocessing

Bioprocessing involves biological or living systems or their components (e.g., enzymes, chloroplasts, etc.) and chemical engineering processes to obtain the desired products at the commercial level. Microorganisms have been used to produce fermented foods since the very past. Since then, new bioprocesses have been developed to produce various commercial products ranging from cheap to expensive speciality chemicals such as antibiotics, therapeutic proteins and vaccines. Bioprocess engineering is thus the backbone of the biotechnology industry that translates the research and development to the industries and is divided into three parts as per the processing

1. Upstream processing

2. Bioreactor and bioreactions

3. Downstream processing

2.0Operational stages of Bioprocess

A bioprocess consists mainly of two stages for converting raw material into the final product: upstream and downstream processing.

The upstream bioprocessing consists of four components, as detailed below: 

1. optimization of nutritional conditions in artificial media and formulation for culturing the living organisms, cells or their components 

2. sterilization of media, bioreactor and other additional tools and equipment 

3. production of pure, active and healthy inoculums in sufficient quantity

4. optimization of environmental conditions for growth and product formation 

On the other hand, downstream processing consists of two steps as detailed below: 

1. extraction, recovery and purification of product 

2. The disposal of effluents produced by the process

3.0Upstream Processing

The upstream processing of a bioprocess is the initial step in which microorganisms or cell lines are grown in bioreactors using basic steps such as inoculum preparation, media formulation, improvement of inoculum by genetic engineering, and growth kinetics optimization. The aim is to increase production levels and regulate temperature, pH, and pressure. 

Inoculum Development:

Inoculum development refers to the increase in the density of a viable number of microorganisms from a dormant stock stage to a final productive stage. Stanbury and Whitaker's criteria (1984) are important when considering a protocol for inoculum development. 

• Active and healthy state inoculum is required to reduce the lag phase 
• Sufficient large volumes are required with an optimum size of 0.1 – 10 % of the medium volume.
• Suitable morphological form. 
• Contamination free. 
• Possess product-forming capabilities.

Criteria for Selection of a Medium:

• Maximum product or biomass yield per gram substrate consumed. 
• Maximum purity of product or biomass. 
• Maximum product formation rate. 
• Low unwanted product yield. 
• Cheap, consistent quality and availability throughout the year. 
• Minimal troubleshoots in upstream and downstream processing. 
• The composition of a fermentation medium may be simple to complex, depending on the microorganism and its fermentation.

4.0Modes of Bioprocess Operation

One of the crucial decisions in developing a bioprocess is that the mode of bioprocess operations is to be applied to a particular bioprocess. Mainly, there are three different modes under which a bioprocess may be operated: 

1. Batch 

2. Fed-batch 

3. Continuous

Batch Fermentation

A batch fermentation system is a closed culture system containing an initial amount of nutrients and involves a number of biochemical processes. In batch cultures, the cell properties, such as the size of cells, nutrients, and metabolic function, vary considerably during the various growth phases. Showing cell biomass [X], rate of substrate consumption [Qs] and substrate concentration [S] in a batch culture.

Advantages of batch culture systems:

• Reduced risk of contamination or cell mutation as the growth period is short. 
• Lower capital investment. 
• Production of widely varying product/biological systems. 
• High conversion rate of raw materials.

Disadvantages of batch culture systems:

• Lower productivity levels related to more time consumption
• Increased focus on instrumentation. 
• Requirement of several subculture levels. 
• High costs for labour and process control.
• More susceptible to pathogenic microorganisms or toxins. 

Applications of Batch Cultures :

• Products involving low risk of contamination or mutation of microbes. 
• Production of small amounts of product required. 
• Employing proper product separation in batch or semi-continuous processes.

Fed-Batch Fermentation

A fed batch is a batch process always in a quasi-steady state. It is based on the non-toxic feeding of a growth-limiting substrate to culture without removing the fermentation broth. It is designed to accommodate increasing volumes with accelerated cell growth, resulting in high cell density.

Advantages of fed batch systems

• High cell yield  
• Almost at stationary state

Disadvantages

• Lower productivity levels due to consumption of time in preparation of fresh run in reactor  
• Higher costs for labour or process control

Applications 

• Fed-batch culture is ideal where a high concentration of substrate inhibits microbial growth.  
• Production of intracellular metabolites in the maximum amount.  
• Increased biomass production if the desired product is intracellular.

Continuous Fermentation

Continuous culture is an open process wherein exponential growth can be extended by the addition of fresh medium containing a limiting substrate. Fresh medium displaces an equal volume of spent medium along with the biomass. Thus, a steady state is reached. 

Advantages of continuous culture  

• Accurate information regarding kinetic constants, maintenance energy and growth yields. 
• Regulation of growth rates for extended periods by varying the dilution rates.  
• Enhances selectivity for thermophiles, osmotolerant or mutant organisms.  
• Results in higher productivity per unit volume 

Disadvantages

• Control of some non-growth-associated products takes work.  
• The wash-out phenomenon is caused by growth on walls, and cell accumulation results in an unsteady state.  
• Loss of original product strain over time if a faster-growing one overtakes it.  
• It is difficult to maintain filamentous organisms due to broth's viscosity and heterogeneous nature. Elongated growth periods increase the risk of contamination.

5.0Bioreactor

• Bioreactors are specially designed cylindrical vessels with hemispherical tops and bottoms built to provide suitable optimum environment conditions that allow the growth of the microorganisms for efficiently producing target product—cell biomass, metabolite and bioconversion. The design and operation of a bioreactor depend on the organism, operating conditions for desired product formation, and the value and scale of the product.
• The design also considers the unique aspects of biological processes, capital investment and operation cost. 
• Large-volume but low-value products require simple fermenters with no aseptic conditions. 
• High-value but low-volume products require sophisticated operation and proper maintenance of aseptic conditions.

Criteria of Bioreactor Design

A good bioreactor design should provide improved productivity and cost-effective validation of desired parameters for achieving consistent and higher-quality products. The criteria for bioreactor design are as follows-

• Microbiological and biochemical characteristics 
• Hydrodynamic characteristics 
• Mass and heat characteristics 
• Kinetics of cell growth and product formation 
• Genetic stability 
• Design related to sterilization 
• Control of bioreactor conditions 
• Design of downstream product separation 
• Capital and operating costs 
• Scale-up

Types of bioreactors

Based on the design or configuration, important types of bioreactors are discussed below: 

Stirred tank reactors—Stirred tank reactors are the most traditional type of bioreactor. They utilize agitators to mix nutrients, oxygen, and growing cells effectively. A key feature of these reactors is the agitator shaft. The impeller's design, shape, and size can vary depending on the specific bioprocess requirements.

Air-lift reactors - Air-lift reactors generate air movement using a draft tube. This creates a circulation pattern that keeps nutrients and oxygen well-mixed by lifting the fluid broth and cells up and down, both inside and outside the draft tube within the reactor.

Bubble column reactor - In a Bubble Column Reactor, nutrients and oxygen are mixed via air bubbles generated by a sparger jet. These reactors create a low-shear environment, which is crucial for some cell cultures, and achieve high oxygen transfer efficiency relative to the power input.

6.0Downstream Processing

• It refers to the separation and purification processes of fermentation/enzyme reaction into desired products. 
• Accounts for 60% of the total production costs.

Points to be considered while developing a downstream process:

• Nature of material to be processed 
• Location of the target product: e.g. intracellular, extracellular, inclusion bodies 
• Concentration of product 
• Relative properties of the product with respect to contaminants 
• Sensitivity to high shear rates, organic solvents, pH and temperature 
• The desired physical form of the final product, e.g. lyophilized powder, sterile solution, suspension 
• Percentage purity 
• Process economics

Operation of downstream processing on the basis of properties

• Filtration, membrane separation, centrifugation - Size  
• Centrifugation, sedimentation, floatation - Density 
• Membrane separation - Diffusivity 
• Extraction, chromatography, adsorption- Polarity 
• Extraction, precipitation, crystallization - Solubility 
• Adsorption, membrane separation, Electrophoresis - Electrostatic charge  
• Distillation, membrane distillation, pervaporation- Volatility 
• A RIPP (Recovery, Isolation, Purification and Polishing) strategy involves the initial use of low-resolution techniques such as precipitation, filtration, centrifugation, and crystallization to significantly reduce the volume and increase the overall concentration, followed by high-resolution techniques to obtain pure and polished finished products.

7.0Bioprocessing of Meat

The purposes of meat processing have evolved. Initially, the goal was to enhance shelf life, flavour, and nutritional value. Later, the focus shifted to transforming less desirable cuts into more appealing products. Today, the emphasis is on providing convenience and a variety of products. Consequently, low-value cuts are upgraded in meat processing to improve shelf life, flavour, taste, nutritional value, and convenience.