1836 Review Received: 5 July 2017 Revised: 22 August 2017 Accepted article published: 11 September 2017 Published online in Wiley Online Library: 25 October 2017 (wileyonlinelibrary.com) DOI 10.1002/jctb.5434 Novel aspects and future trends in the use of aqueous two-phase systems as a bioengineering tool José González-Valdez, a† Karla Mayolo-Deloisa a† and Marco Rito-Palomares a,b* Abstract Traditionally, aqueous two-phase systems (ATPS) have been used as a liquid–liquid extraction technique for the primary recovery and purification of biological samples. The enormous potential of their usage comes with great economical and technical advantages mainly due to the mild physicochemical environment. Nowadays, the use of ATPS as a bioengineering technique is approaching an era where new possibilities are being explored to maximize their use and implementation in the development of novel practical applications and tools. In this context, ‘intelligent’ polymers are being used as phase forming chemicals in ATPS in route to process integration. Extractive fermentation in ATPS is being re-evaluated with the aim of effectively growing microorganisms while recovering their fermentation products in different phases. ATPS are also being used as a tool for refolding of proteins. There are also several innovative efforts being made towards implementing this bioengineering tool as a continuous process moving away from traditional batch operations. In general, the possibilities of implementing ATPS in different bioprocessing aspects are growing considerably and gaining importance. This review aims to present the novel trends in the use and development of ATPS strategies as complete bioengineering tools and to provide a full perspective of their possibilities in the near future. © 2017 Society of Chemical Industry Keywords: aqueous two-phase systems; ATPS-based tools; bioprocessing; phase-forming chemicals; process integration NOTATION ATPS aqueous two-phase systems K P partition coefficients TLL tie-line length V R volume ratio PEG poly (ethylene glycol) EOPO ethylene oxide/propylene oxide PPO-Ph poly (propylene oxide)-phenyl group UCON copolymer of ethylene oxide and propylene oxide CI2 chymotrypsin inhibitor 2 BSA bovine serum albumin INTRODUCTION Aqueous two phase-systems (ATPS) are biphasic systems consti- tuted of two hydrophilic compounds (usually two polymers or one polymer and one salt) mixed above critical conditions. Since two immiscible coexisting aqueous phases are formed, they have been traditionally used as a liquid–liquid extraction technique where solutes partition to either of the phases according to the differences in their partition coefficients (K P ). 1 In this manner, by selecting the appropriate system design parameters, the selec- tive fractionation of the solute(s) of interest to a phase different from the rest of the contaminants is promoted. As seen in Fig. 1, in recent years, compounds such as low molecular weight alcohols, surfactants, and ionic liquids have also been used in the forma- tion of ATPS. 2,3 The main advantages of this methodology include a very fast to reach partition equilibrium, low cost, low toxicity and recyclability of the raw materials, scale-up potential, easiness of process integration, capability of continuous application leading to a reduction of process time and costs at increasing yields, 4 and a high biocompatibility with the molecules being separated. 2 The physicochemical events involved in molecule partition are not fully understood yet. The preference of a solute to migrate to a particular phase depends on several factors such as molecular weight, surface area, net electrochemical charge, isoelectric point and content of hydrophobic moieties or residues in its surface. 5 Along with this, system design parameters of ATPS play an impor- tant role during the partition of a molecule towards a particu- lar phase. Therefore, to obtain high purification factors and yields it is necessary to perform an appropriate selection of the type ∗ Correspondence to: M Rito-Palomares, Escuela de Medicina y Ciencias de la Salud, Tecnológico de Monterrey, Av. Morones Prieto 3000 Pte, Col. Los Doctores, Monterrey, NL 64710, México. E-mail: mrito@itesm.mx † The authors contributed equally to this work. a Tecnologico de Monterrey, Escuela de Ingeniería y Ciencias, Monterrey, NL, México b Tecnologico de Monterrey, Escuela de Medicina y Ciencias de la Salud, Monter- rey, NL, México J Chem Technol Biotechnol 2018; 93: 1836–1844 www.soci.org © 2017 Society of Chemical Industry