INTRODUCTION TO BIOSEPARATIONS

Bioproducts are products extracted from plants, animals, and microorganisms to sustain life
and promote health, support agriculture and chemical enterprises, and diagnose and remedy
disease. From the bread, beer, and wine produced by ancient civilizations using fermented
yeast, the separation and purification of biological products (bioproducts) have grown in
commercial significance to include process-scale recovery of antibiotics from mold, which
began in the 1940s, and isolation of recombinant DNA and proteins from transformed
bacteria in biotechnology protocols initiated in the 1970s.

1.1 Bioproducts
To identify features that allow selection and specification of processes to separate
bioproducts from other biological species 1 of a host cell, it is useful to classify biological
species by their complexity and size as small molecules, biopolymers, and cellular
particulates (Table 1.1).

Table 1.1 : Products of Bioseparations

1
Bioproducts are sold for their chemical activity eg:

 methanol for its solvent activity,

 ethanol for its neurological activity (or as a fuel),
 penicillin for its antibacterial activity,
 streptokinase (an enzyme) for its blood-clot-dissolving activity,
 hexose isomerase for its sugar-converting activity, and
 whole Bacillus thuringiensis cells for their insecticide activity.
The choice of separation method depends on the nature of the product, as well as purity,
yield, and most importantly activity requirements. Bioproducts have unique properties. For
bioseparation purposes, important properties include thermal stability, solubility, diffusivity,
charge, and isoelectric pH, among others.
A considerable amount of process planning is based on the lability, or susceptibility to
change, of most bioproducts. Temperature, pH, and concentration must be maintained within
specific ranges to assure product bioactivity.
Purification of bioproducts by a bioseparations process typically involves a long sequence of
steps, and each step requires the use of one or more unit operations, such as filtration,
extraction, chromatography, and drying.

Developing a sequence of bioseparations

The development of a flowsheet for the recovery and purification of a biological product is a
creative process that draws on the engineer’s experience and imagination. Experienced
engineers rely heavily on certain rules of thumb, or heuristics, for putting together the
skeleton of a recovery and purification process.
A few such heuristics include:

 Remove the most plentiful impurities first.

 Remove the easiest-to-remove impurities first.
 Make the most difficult and expensive separations last.
 Select processes that make use of the greatest differences between the properties of
the product and those of its impurities.
 Select and sequence processes that exploit different separation driving forces.
Figure 1 depicts the bioseparation steps that may be involved in a product recovery process.
The primary recovery stages are mainly concerned with separating the product from cells or
cell debris; these stages follow the rule of removing the most plentiful impurities first
(usually water).
The intermediate recovery stages concentrate the product by an operation (e.g., ultrafiltration,
evaporation, reverse osmosis, etc.) that depends on the nature of the product. Occasionally, it
is necessary to perform a protein-refolding step if the product is a protein formed within the
cells’ inclusion bodies (IBs), which are insoluble aggregates sometimes formed when
recombinant proteins are produced by cells. The final purification stages follow the rule of
doing the most difficult and expensive separations last; these stages sometimes require the
use of separation driving forces that are different from each other (e.g.,charge difference, size
difference, or solubility difference) to attain the required purity. Selection and sequencing of

2
unit operations is based on the properties of the product, the properties of the impurities, and
the properties of the producing micro-organisms, cells, or tissues.
Most bioprocesses, especially those employed in the production of high-value, low-volume
products, operate in batch mode. Conversely, continuous bioseparation processes are utilized
in the production of commodity biochemicals, such as organic acids and biofuels. Two of the
most common unit operations in bioseparation processes are filtration and liquid
chromatography.

Figure 1: A bioproducts recovery process typically involves some combination of these

steps and operations
A series of bioseparation steps are commonly required upstream of the bioreactor (e.g.,
filtration of incoming gases and culture media), after the bioreactor (i.e., downstream or
recovery processes), and during (e.g., centrifugal removal of spent media) fermentation and
cell culture operations. A general sequence of biorecovery steps is designed to remove
solvent, insolubles (e.g., particle removal), unrelated soluble species, and similar species. A

3
nondenaturing protein recovery process, for example, consists of consecutive steps of
extraction, clarification, concentration, fractionation, and purification. The performance of
each purification step is characterized in terms of product purity, activity, and recovery,
which are evaluated by:
𝑏𝑖𝑜𝑝𝑟𝑜𝑑𝑢𝑐𝑡 𝑚𝑎𝑠𝑠
𝑝𝑢𝑟𝑖𝑡𝑦 =
𝑏𝑙𝑖𝑜𝑝𝑟𝑜𝑑𝑢𝑐𝑡 𝑚𝑎𝑠𝑠 + 𝑖𝑚𝑝𝑢𝑟𝑖𝑡𝑒𝑠 𝑚𝑎𝑠𝑠

𝑢𝑛𝑖𝑡𝑠 𝑜𝑓 𝑏𝑖𝑜𝑙𝑜𝑔𝑖𝑐𝑎𝑙 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦

𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 =
𝑏𝑖𝑜𝑝𝑟𝑜𝑑𝑢𝑐𝑡 𝑚𝑎𝑠𝑠

𝑏𝑖𝑜𝑝𝑟𝑜𝑑𝑢𝑐𝑡 𝑚𝑎𝑠𝑠 𝑟𝑒𝑐𝑜𝑣𝑒𝑟𝑒𝑑

𝑦𝑖𝑒𝑙𝑑 =
𝑏𝑖𝑜𝑝𝑟𝑜𝑑𝑢𝑐𝑡 𝑚𝑎𝑠𝑠 𝑖𝑛 𝑓𝑒𝑒𝑑
Recovery yields of the final product can range from about 20% to 60–70% of the initial
molecule present in the feed stream. Some clarification of raw fermentation or cell-culture
feed streams prior is usually required to analyze their bioproduct content, which makes
accurate assessment of recovery yields difficult. It is particularly important to preserve
biological activity during the bioseparation steps by maintaining the structure or assembly of
the bioproduct.
Table 1.2 classifies common bioseparation operations according to their type, purpose, and
illustrative species removed.

Table 1.2: Synthesis of Bioseparation Sequences

4
Eg. Penicillin Block-flow diagram

Figure 1.2: Block-flow diagram for Penicillin KV process

5
Example 1. Cumene Manufacture (Due 30 September 2016 @ 1600)
A more complex example is the manufacture of cumene (isopropyl benzene) by the
alkylation of benzene with propylene, taken from the 1997 National Student Design
Competition of the AIChE. Cumene is widely used to make acetone and phenol. The fresh
feeds are as follows, where the benzene feed is nearly pure, but a refinery cut of a propylene-
propane mixture is used rather than a more expensive feed of nearly pure propylene.

The main reaction, conducted with a catalyst, is:

Propylene + Benzene → Isopropylbenzene (Cumene)
A number of undesirable side reactions involving the main reactants also occur, including:
Propylene + Benzene → n-Propylbenzene
Cumene + Propylene → m-Diisopropylbenzene (m-DIPB)
Cumene + Propylene → p-Diisopropylbenzene (m-DIPB)
Other reactions that produce alkylation heavies
All of the impurities in the propylene and benzene fresh feed streams, including the large
amount of propane in the propylene feed, are essentially inert, with the exception of 1-
Butene, which enters into the following undesirable side reactions:
1-Butene + Benzene → t-Butylbenzene (t-BB)
1-Butene + Benzene → 1-isopropyl,4-methyl Benzene (p-Cymene)

6
Potential products and byproducts include cumene, propane, DIPBs, t-BB, p-cymene, inert
light hydrocarbons, inert aromatic compounds, and water. A main objective of the process is
to maximize the production of cumene and minimize the amounts of byproduct and waste
streams. The cumene product must meet the following specifications:

The propane byproduct is used as either fuel gas or LPG. Thus, it can contain water and light
hydrocarbons. However, the aromatic content cannot exceed 0.01 wt%.
Experimental alkylation data show that the two reactions above that produce DIPBs can
result in a serious loss (> 10%) of potential cumene product. To reduce this loss, two
remedies are applied, the first of which is related to Heuristic 2 in Table 1.3:
(1) the use of a large excess of benzene in the combined feed to the alkylation reactor, for
example, a 4.0 molar ratio of benzene to propylene to reduce the DIPB formation reactions,
and (2) the addition of a trans-alkylation reactor where the DIPBs are reacted with benzene to
produce cumene according to the reaction:
DIPB + Benzene → 2 Cumene
Other reactions that produce trans-alkylation heavies

7
Table 1.3: Heuristics of Process synthesis

8
9
10
11
12