Siiri Sippo, Henrik Nikkilä, Eetu Satosalmi

Aqueous two phase extraction

Introduction
Aqueous two-phase extraction has applications in biochemical processes involving protein
separation and purification. Aqueous two-phase extraction includes two different immiscible
polymers or polymer and salt systems for protein recovery. To minimize costs, the protein
must be recovered in highly purified form. In biotechnology industry one of the major
challenges is large-scale purification on desired protein from fermentation broth which
contains wide range of biomolecules. Aqueous two-phase system downstream process
offers a solution for this challenge. In this group work we are introducing background of the
process, theory to understand basis of the process and importance part which introduces
biotechnology processes and applications which utilizes aqueous two-phase extraction.

1. Background
Since the late 19th century aqueous two phase extraction has been known. Nowadays,
aqueous two phase can be formed with a wide variety of natural or synthetic water-soluble
polymers. Aqueous two phase extraction is used for secure separation and purification of
biomolecules, such as proteins, and extraction of enzymes. (Stanbury et al., 2016) Aqueous
extraction has many advantages; it is biocompatible, it has high water content and low
interfacial surface tension between phases, the process has an ability to integrate and a
capability for intensification (Benavides et al., 2010; Goja et al., 2013). Also, the degree of
degradation for biomolecules is low (Saravanan et al., 2007).

2. Theory
The basic principle of extraction is the same as usual; the goal is to separate the solute
selectively from its carrier. Aqueous two phase system is formed by adding two
uncompetitive water-soluble polymers (such as polyethylene glycol, PEG) or salt and one
polymer into water above the critical concentration. These polymers react with the water and
two immiscible aqueous phases are formed. Then soluble material, like proteins, are added
to the system and it is distributed into two phases (Fig 1.). (Stanbury et al., 2016) The
distribution of extracted component results from the interactions with the surrounding phase,
such as Van der Waals, hydrophobic, hydrogen bond, and ionic interactions (Saravanan et
al., 2007).

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Siiri Sippo, Henrik Nikkilä, Eetu Satosalmi

Figure 1. PEG/salt aqueous two phase extraction principle (Divkar, 2015).

Stanbury et al. (2016) listed four different possible aqueous two phase systems.
The first of these is to use two different nonionic polymers, such as polyethylene glycol and
dextran, with water extraction. Another option is to use a polyelectrolyte, such as sodium
carboxymethyl cellulose with a nonionic polymer, again using water extraction in separation.
Third, two different polyelectrolytes may be used. The fourth option is to use a polymer and a
low molecular weight compound, such as propyl alcohol. (Stanbury et al., 2016)

The ease of extraction is descripted by the distribution coefficient K:

!"#$%#&'(&)"#  !"  !"#$%&  !"  !"#$%&#

𝐾= (1)
!"#$%#&'(&)"#  !"  !"#$%&  !"  !"##$%"&'

As equation 1 shows, the larger the value of K, the more stable the product which results in
better separation. K is also known as the partition coefficient. For better phase separation
centrifugal and magnetic separators can be used. The distribution of solute between two
phases can be influenced by temperature, pH, polymers and solutes molecular weight and
size and by salt concentration. (Stanbury et al., 2016)

Extraction can be performed by batch or as a continuous operation. Commonly a continuous

extraction process is more efficient and involves less processing time. (Goja et al., 2013)

However, there has been some difficulties in some industrial scale process using aqueous
two phase extraction. Especially difficulties with PEG and salt extraction have been
common, since the salts caused corrosion effects and precipitation of product. Furthermore,
a high amount of waste salt formed is not environmentally sustainable and effective salt
recycling is expensive. These are partially the reasons why still two polymer systems are
more used than polymer and salt systems. Although mentioned polymer/salt extraction
problems, it is still widely studied, because it is still less expensive than PEG/dextran
extraction.
Nonetheless, aqueous two phase extraction is not widely used in large scale, because of
high costs of the polymers (dextran) and difficulties in solvent recovery. For those reasons

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Siiri Sippo, Henrik Nikkilä, Eetu Satosalmi

interest for developing new polymers for aqueous two phase extraction grows increasingly.
(Goja et al., 2013)

3. Importance
Penicillin G and penicillin V have small use medically today, they are however the raw
materials used in producing all semisynthetic penicillins (Marynova et al., 2014). Penicillin G
is manufactured in multiple countries in a quantity of over 11 000 tonnes per year. Its large
usage can be attributed to its lack of toxicity and irritancy. (Hossain & Dean, 2008)
Traditionally penicillins are extracted from the fermentation broth with butyl acetate.
However, many other techniques have been developed, including aqueous two-phase
extraction. In aqueous two-phase system PEG is often used as polymer, while salts, such as
sodium sulphate and ammonium sulfate, are introduced to the system in order for partition to
take place. The process uses flotation by nitrogen bubbling, in order to get the PEG phase
on the surface of the water solution. This sort of process is carried out at neutral pH, which is
preferable to traditional extraction (pH=2), since penicillin quickly decays in low pH (half-life
of 15 min at pH=2, 20 ) (Standbury et al., 2016). Aqueous two-phase extraction with
polymers allows for penicillin extraction from the whole broth. (Marynova et al., 2014)

Another potential use for aqueous two-phase extraction would be extraction of carotenoids.
The interest in carotenoids has increased extensively due to their nutraceutical potential,
with their estimated market value being 1000 million US$ by the end of the decade.
Commercially carotenoids are produced with cyanobacteria. Traditionally their extraction
involves cell disruption, cell debris removal and organic-aqueous fractionation. This
however, involves large expenses and potential for environmental risks caused by the large
amount of organic solvents used. As an alternative, aqueous two-phase extraction could be
utilized to extract the product. In this sort of system, PEG-dextran could be used, in which
the cyanobacteria would grow (upper phase). Lutein would continuously be extracted from
this phase. If an additional cell-removal phase is added, such as centrifugation or
microfiltration, β-carotene can be removed constantly from the upper phase. (Chavez-
Santoscoy A. et al., 2010)

4. Conclusion
Aqueous two phase extraction is a suitable separation and purification technique for
bioproducts in biotechnological field. Aqueous two phase extraction (ATPE) downstream
processes are suitable for bioproducts by providing mild conditions that don’t harm
bioproducts. In biotechnology ATPE processes are preferred instead of solvent extraction,
because ATPE process provides lower surface tension. PEG/salt downstream processing
technologies(DSP) offer efficient liquid-liquid extraction for a large variety of bioproducts
including proteins and enzymes. However, using PEG/salt in the large scale may cause
environmental problems due to the great amount of chemicals used during phase forming
process and also high costs for effective recycling.

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Siiri Sippo, Henrik Nikkilä, Eetu Satosalmi

References
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