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Campus Monterrey

School of Engineering and Sciences

Scale-up of a competitive and low-cost medium for prodigiosin production in a S. marcescens culture using biphasic systems as an

integrated in-situ recovery process

A thesis presented by

Ulises Andrés Salas Villalobos

Submitted to the

School of Engineering and Sciences

in partial fulfillment of the requirements for the degree of

Master of Science In Biotechnology

Monterrey Nuevo León, June 15th, 2020


patience, and encouragement thought this adventure.

A special dedication to:

God and my family, that always accompany and support me in every step I give. Thanks to them and their guidance, I’m the person who I currently am.

To my girlfriend, that supports and understand me across all the path to reach this goal.


path to reach this goal in my life. I’m especially grateful:

To God, for all the opportunities that he has put in my life and also the strength given to me to affront them.

To my family, especially my parents and sister, who always take care of my well-being and stand by my side no matter the distance.

To my advisors, Dr. Alex Aguilar Jimenez for his continuous support and guidance, and Dra.

Josefina Castillo Reyna, to believe in me and push me to start the path to this goal.

To my girlfriend, who encourage and heard me in all the moments I needed.

And to all the professors that share their knowledge with me and the friends that I found across this time, that in one or another way, help me to reach this goal.

I wish to express my gratitude for the financial support of the Bioprocess Research Group of Tecnológico de Monterrey and for providing me with all the necessary facilities for the research.

Also, I am grateful to Dr. José González Valdez for accept me in the group.

I am grateful to CONACyT for the MSc fellowship that helps me to focus on my research.


Product inhibition is an undesirable effect during fermentation that causes not only low yields but also a reduction in bioproduction feasibility of certain molecules. Prodigiosin is a secondary metabolite with an intense red produced by different strains of Serratia marcescens. Several in- vitro activities such as anticancer, antibacterial, immunosuppressive, anti-malaria and bio- colorant properties, make it particularly attractive to the pharmaceutical, food and textile industries.

However, production of this natural alkaloid can be dramatically affected by media components and its antimicrobial activity causes end-product inhibition, additional to the complexity of typical production media making downstream processing complex and thus increasing the final cost of the purified product. In this study an optimization of a low-cost media using different sources and concentrations of carbon and nitrogen were performed. Extractive fermentation was evaluated as an integrated recovery strategy by coupling the upstream stage with different volumes of mineral oil as an extractive phase and we addressed the feasibility of scaling-up such extractive fermentation using batch and continuous processes modes. Peanut and defatted soybean meal in a 40 g/L - 5.25 g/Lratio, at 25 °C, achieved the best results, obtaining 584 mg/L of prodigiosin.

The coupling of the medium with mineral oil at a 5% v/v presented a 1.12-fold in the prodigiosin content, which represents a change from 674 mg/L to 756 mg/L; and a 2-fold in cell growth. At scale-up tests, prodigiosin direct extraction produced in batch mode resulted in a prodigiosin concentration of 1282 mg/L. While continuous fermentation reached a concentration of 493 mg/L in steady state, which represents a 1.5-fold in the production rate compared to batch. A mathematical model, using Logistic and Luedeking-Piret equation was used to describe the microbial growth and product to correlate biomass and pigment production, obtaining correlation coefficients above 0.9747. The obtained results provide an opportunity to enhance prodigiosin production at the industrial level by alleviating end-product inhibition phenomenon using a two- phase system for in-situ primary recovery and putting in perspective with an economical approach.

Also, this proposed strategy opens the possibility to transfer this experimental approach, for the recovery of several other hydrophobic molecules.

Keywords: Serratia marcescens, prodigiosn, medium optimization, extractive fermentation, scale-up.


L Liter

DSP Downstream processing

mL Milliliter

μL Microliter

mg/L Milligrams per liter

mg/mL Milligrams per milliliter

μg/mL Micrograms per milliliter

M Molarity

PEG Polyethylene Glycol

g/L Grams per liter

ATPS Aqueous two-phase system

μm Micrometer

nm nanometer

min Minute

h-1 Hour at inverse

h Hour

°C Celsius degrees

x g Artificial gravity

rpm Revolutions per minute

Wt Weight

v/v Volume per volume

w/v Weight per volume

psi Pound per square inch

vvm Volume of air per volume of medium per minute

F Flow of feed and outlet in continuous reactor

V Volume of work in the bioreactor

D Dilution rate

σ Standard deviation of peaks in chromatogram

μ Specific growth rate

μ max Maximum specific growth rate

Rt Time of residence

X Actual biomass

Xo Biomass at early exponential phase

Xmax Maximum biomass

T Time at X biomass

Rx Biomass formation rate

Rp Product formation rate

α Product growth associated coefficient

𝞫 Product non-growth associated coefficient

𝑃𝑜 Power consumption


Np Power Number

Pg Ungassed power consumption

Q Volumetric mass flow

Cu2+ Divalent copper ion

Cu1+ Monovalent copper ion

Fe Iron

Cu Copper

Mn Manganese

MnSO4∙H2O Manganese sulfate monohydrated

MgSO4∙7 H2O Magnesium sulfate heptahydrated FeSO4∙7 H2O Iron sulfate heptahydrated

CuSO4 Copper sulfate anhydrous

HCL Hydrochloric acid

NaOH Sodium hydroxide

UV-Vis Ultraviolet/Visible wavelengths

ISPR In situ product recovery

P53 Tumor suppressor p53

ATCC American type culture collection

USA United states of America

NJ New Jersey

DEQ Desarrollo de especialidades químicas

USD or dlls United states dollar

e.g. For example,

RSM Response Surface Methodology

ATP Adenosine triphosphate

MA Massachusetts

AOT Sodium bis(2-ethylhexyl) sulfosuccinate

HTS High throughput screening

CFU/mL Colony forming units per milliliter

N Sample size

Ln Natural logarithm


List of Figures

Figure 1.1. Comparison of traditional vs integrated DSP. ... 6 Figure 1.2. Process diagram for selection of ISPR technique. ... 8 Figure 1.3. Process diagram for selection of ISPR technique. ... 8 Figure 1.4. Main driving forces involved in membrane recovery processes. A) Pressure gradient and B) concentration gradient (diffusion), usually more involved in the hybrid processes like perstraction. ...13 Figure 1.5. Standard structures of (A) a submerged membrane system and B) an external loop process. ...14 Figure 3.1. Scheme of the system used for continuous reactor fermentations. ...36 Figure 4.1. Effect of different carbon sources in prodigiosin production rate. Peanut 2% (■) and 1.5% m/v (▲); sunflower 2% (♦) and 1.5% m/v (x); coconut 2% m/v (●) and peanut 2% without peptone (▬). Batch fermentation at 28ºC, 250 rpm for 58 h. ...45 Figure 4.2. Effect of nitrogen source and optimization of carbon-nitrogen ratio. ...46 Figure 4.3. Effect of 25 °C (▲), 28°C (▬), 30 °C (■) and temperature shifting (●) on prodigiosin yields. ...48 Figure 4.4. Effect of mineral oil in production of prodigiosin and growth of S. marcescens in 96 h fermentations. ...49 Figure 5.1. Prodigiosin production (▬) and growth kinetics (▬) in a 1.5 L batch bioreactor. ...63 Figure 5.2. Prodigiosin production (▬) and biomass (▬) kinetics profile in a 1.5 L continuous reactor. ...65 Figure 5.3. S. marcescens growth (●) and prodigiosin kinetic profile (■) obtained in batch fitted into a two stages mathematical model...67


Table 1.1 Benefits obtained in different fermentation processes by the implementation of ISPR techniques. ... 7 Table 3.1. Design obtained for the Response Surface Methodology using non-coded variables.

...33 Table 4.1. Concentration and type of soybean flour in prodigiosin production, evaluated after 40 hours fermentation. ...47 Table 5.1. Comparison of prodigiosin production rates obtained in batch and continuous fermentations at steady state. ...66 Table 5.2. Comparison between the parameters obtained from the experimental data and the proposed by the model to describe biomass and prodigiosin behavior. ...68 Table 5.3. Estimation of the electrical power consumption required for the fermentations using a 3L Applikon bioreactor. ...69 Table 5.4. Estimated costs comparison between batch and continuous reactor operating at steady state. ...70




List of Figures………viii

List of Tables………...ix

1. A Review - Recovery of microbial metabolites: Integrated downstream processing as a strategy to deal with inhibitory products………..2

Abstract: ...3

Introduction ... 4

Development of integrated DSP. ... 6

ISPR techniques at DSP ... 7

Liquid-liquid systems ... 8

Solid-liquid systems ...12

Gas-liquid system ...17

Conclusion and future trends ...17

References ...18

2. Problem Statement………...27

2.1 Problem Statement and Context ...28

2.2 Hypothesis ...29

2.3 Objectives ...29

3. Materials and Methods……….31

3.1 Materials ...32

3.2 Microorganism and culture conditions ...32

3.3 Carbon source screening ...32

3.4 Nitrogen source screening ...33

3.5 Sources ratio optimization using Response Surface Methodology. ...33

3.6 Effect temperature and defatted soybean meal. ...34

3.7 Extractive fermentation composition screening ...34


3.10 Determination of prodigiosin and cell growth. ...36

3.11 Model proposal ...37

3.12 Economic considerations ...37

3.13 Statistical Analysis ...38

4. A simple two-step strategy to increase prodigiosin production rates in Serratia marcescens: an optimal low-cost media coupled with in-situ product recovery.………39

Abstract ...40

Introduction ...41

Materials and methods ...42

Materials ...42

Microorganism and culture conditions ...42

Carbon source screening ...43

Nitrogen source screening ...43

Sources ratio optimization using Response Surface. ...43

Effect temperature and defatted soybean meal. ...44

Extractive fermentation composition screening ...44

Determination of prodigiosin and cell growth ...44

Statistical analysis ...45

Results ...45

Effect of carbon and nitrogen sources ...45

Effect of concentration and type of soybean flour in prodigiosin production. ...47

Effect of temperature and temperature shifting in prodigiosin production. ...47

Effect of mineral oil and its concentration in prodigiosin production and cell viability. ...48

Discussion ...49

Conclusions ...52

References ...53


Introduction ...58

Methodology ...59

Materials ...59

Microorganism and culture conditions ...59

Inoculum preparation ...60

Scale-up in batch and continuous bioreactors ...60

Determination of prodigiosin and cell growth ...61

Model proposal ...61

Economic considerations ...62

Statistical Analysis ...62

Results and discussion ...62

Batch Scale-up fermentation ...62

Continuous scale-up fermentations ...64

Model proposal ...67

Fermentation economic considerations ...69

Conclusions ...70

References ...71

6. General conclusions and future work.………...75

6.1 General conclusions ...76

6.2 Future work ...76


Curriculum Vitae………90



1. A Review - Recovery of microbial metabolites: Integrated

downstream processing as a strategy to deal with inhibitory



A Review - Recovery of microbial metabolites: Integrated downstream processing as a strategy to deal with inhibitory products

Ulises Andres Salas-Villalobos; Rigel Valentín Gómez-Acata; Josefina Castillo-Reyna; Oscar Aguilar*

Tecnologico de Monterrey, Escuela de Ingeniería y Ciencias, Av. Eugenio Garza Sada 2501 Sur 64849, Monterrey, Nuevo León, México

*Corresponding author: alex.aguilar@tec.mx


Product inhibition is an undesirable effect during fermentation that causes not only low yields but also a reduction in bioproduction feasibility of certain molecules. Common downstream processing techniques have been explored to attenuate this behavior. These approaches represent the most expensive part of a low-yield process. In-situ downstream techniques come into view as an alternative for both points: fermentation product recovery and downstream operations reduction. Distinct in-situ techniques have been reported. Their characteristics determine the suitability to improve or worsen the bioproduct recovery. In this work, different metabolite recovery in-situ techniques were reviewed, standing out their fundamental characteristics and appropriateness for a specific metabolite.

Key Words:

Recovery; metabolites; downstream; inhibitory products; titers


Introduction ... 4

Development of integrated DSP. ... 6

ISPR techniques at DSP ... 7

Liquid-liquid systems ... 8

Organic solvents. ... 8

ATPS ... 10


Solid-liquid systems ... 12

Membrane bioreactors ... 12

In-situ adsorbents ... 16

Gas-liquid system ... 17

Gas stripping ... 17

Conclusion and future trends ... 17

References ... 17


Metabolites are defined as chemical compounds isolated from diverse living beings, that can be classified into primary or secondary, depending on which metabolism they are derived from (Bérdy, 2005). Examples of primary metabolites are amino acids, nucleotides, and some fermentation ending products e.g. organic and fatty acids, which are essential for the optimal growth of microorganisms. Secondary metabolites are organic compounds usually associated to the growth stationary phase and adaptive response of microorganisms, e.g. antimicrobial agents, enzyme inhibitors, pigments, and other compounds that are not essential for proper cell growth (Singh et al., 2017). Some of these secondary metabolites have important bioactive functions such as antioxidants, antimicrobials, antifungal, anticancer, insecticidal, herbicidal, anti- inflammatory, or anti-coagulants (Singh et al., 2017; Rastogi & Sinha, 2009). Due to these characteristics, food, pharmacological and agricultural industries, have shown an interest in providing capital for the research of these metabolites and design strategies for their production (Rastogi & Sinha, 2009).

Metabolite production through a controlled bioprocess confers desired characteristics, e.g.

process control, implementation, and maintenance of optimized conditions and no dependence on climate and geographical conditions, which may be reflected in higher yields (Kallscheuer et al., 2019; Chattopadhyay et al., 2002). Production or upstream stage is followed by the downstream processing (DSP), that consists in a sequence of steps that are necessary to accomplish the extraction and desired purity of the final product from the culture broth and regularly represents the main cost of production strategy (Balasundaram et al., 2009).


Bioprocessing economic features and demand have led to increasing yields and efficiency of processes through fermentation upstream stage enhancement, e.g., culture condition optimization, use of genetic engineering, bioprospection of new organisms, which together allow cultures to boost their yield and productivity. (D’Souza et al., 2013). Still, limitations as the product inhibition effect may appear due to the presence of certain metabolites under a concentration at which the microorganisms are not usually exposed. (Meyniall, Dorotyn & Soucaille, 2007).

Advances in the downstream process are not obtained at the same rate compared with those at the upstream, and this delay directly affects the cost and efficiency of the production process.

Furthermore, the linear scale-up of traditional downstream processing has encountered their limit, making difficult to fulfill product demand in terms of cost, space, and process time (D’Souza et al., 2013). To face these problems, new technologies have emerged, offering downstream process integration and leading to a decrease in unit operations, time and costs while maintaining a high product recovery rate and purity degree. (D’Souza et al., 2013). Downstream processing integration using in-situ recovery techniques can help to achieve higher yields during fermentation by lowering the inhibition caused by certain end-products. (Chávez & Aguilar, 2016). This strategy can be also useful in scenarios where molecules are unstable (Carstensen, Apel & Wessling, 2012).

Product inhibition effect is characterized by metabolite cytotoxicity in producer cells above certain concentrations (Maiorella, Harvey & Wilke, 1983) e.g. Saccharomyces cerevisiae for ethanol production (Jönsson, Alriksson & Nilvebrant, 2013); Serratia marcescens for prodigiosin obtaining (Chávez & Aguilar, 2016); Pseudomonas putida for phenolic compounds generation (Heerema et al., 2011) and different Clostridium strains for butanol biosynthesis (Li et al., 2016). Or by targeting enzymes from specific metabolic pathways that are necessary for the biosynthesis of the product, as is the case of (i) cellulose degradation to glucose by cellulase Cel7A, which can be repressed by cellobiose (Atreya, Strobel & Clark, 2016) and (ii) amines production by Transaminase- catalyzed synthesis, where the product (1-methyl-3-phenylpropylamin) and coproduct (pyruvate) inhibit the enzyme (Börner et al., 2015). Product inhibition is related to a decrease in product yield, avoiding exploiting the true potential of the bioprocess and thus becoming a significant problem in process efficiency.

The objective of this work is to highlight some downstream processing strategies which were designed to deal with high concentrations of microbial inhibitory products, such strategies can enhance bioproduct yields and reduce the cost of the process.


Development of integrated DSP.

Downstream processing represents the bottleneck of bioproduct obtention (Chávez & Aguilar, 2016). The degree of purity of the final product will depend on the industry to which it is intended, for example, the pharmaceutical and food industries generally require a high degree of purity in their products and to achieve the desired level the downstream process may justify 80% of production cost (D'Souza et al., 2013).

Traditional DSP has many steps that increase resources, time, and even space consumption.

Harvest, clarification, capture, and concentration are the initial basic steps for downstream processing; and in the case of intracellular compounds, even a disruption step must be added (Balasundaram et al., 2009). Integrated downstream processing has the qualities of a feasible solution to optimize primary recovery at downstream, using techniques that make possible the reduction of steps from the beginning of the process (Figure 1.1) (D’Souza et al., 2013).

Figure 1.1. Comparison of traditional vs integrated DSP.

Implementation of in-situ recovery techniques at the downstream is called in-situ product removal (ISPR) and consists in the integration of the primary recovery step to the fermentation, causing the direct removal of the product from the reactor, where both, production and extraction/separation of the metabolite, are going to occur at the same time (Carstensen, Apel &

Wessling, 2012).

Change in traditional primary recovery methods for this kind of techniques, give advantages to the process, for instance: (i) higher yields, avoiding the product interaction or interference with compounds in culture media or microorganisms and thus helping to maintain maximum


expression levels; and (ii) less number of DSP steps, saving product losses and diminishing the final cost (Freeman et al., 1993).

Table 1.1Benefits obtained in different fermentation processes by the implementation of ISPR techniques.

Technique Target

Molecule Recovery

System Benefits Ref


Solvent Styrene n-dodecane

Minimize styrene toxicity.

• Improve compound recovery.

Lee et al., 2019

ATPS Alkaline

phosphatase PEG 4000 and Dextran T500

Accomplish a 1.4-fold in the

enzyme content. Pandey &

Banik, 2011 Adsorbent Prodigiosin X-5 resin

Simplified the separation process.

• Caused a 1.7-fold in the recovery yield.

Wang et al., 2004 Membrane

bioreactors Lactic Acid External Hollow- fiber membrane

Increment cell density and

volumetric productivity. Kwon et al., 2001 Perstraction Butanol

PDMS membrane and

ionic liquids as extractant

• Overcome inhibition.

Improve the extraction percentage.

Merlet et al., 2017


Acetone, Butanol and


Gas striping coupled to pervaporation

Reduce inhibition.

• Increase the recovery titers.

Decrease energy

requirements compare to similar methods.

Cai et al., 2016

ISPR techniques at DSP

ISPR advantages earlier mentioned have led to diverse studies to focus on the development of new techniques in which separation principles are differential solubility, magnetic forces, mechanical separation, etc. Each strategy must be selected depending on the target bioproduct characteristics or main contaminants (Figure 1.2).


Figure 1.2. Process diagram for selection of ISPR technique.

A representation of the principal integrated in-situ techniques that are discussed across this review is presented in Figure 1.3. These strategies can be used either at continuous or batch operation modes.

Figure 1.3. Process diagram for selection of ISPR technique.

Liquid-liquid systems Organic solvents.

Biphasic systems composed of aqueous-organic solvents were the first and most studied ISPR strategies based on two liquid immiscible phases. The basis of this technique is to add to the fermentation a solvent with a high recovery rate and selectivity towards the product (Freeman et al., 1993). It has been reported as an efficient recovery strategy for several inhibitory compounds


such as lactic acid (Ataei & Vasheghani, 2008), 2-Phenylethanol (Mei et al., 2009), and monoterpenes (Brennan et al., 2012). In a comprehensive review, Dafoe & Daugulis (2014), mentioned new and interesting insights of this type of extraction, discussing points like the improvement of the recovery rates by analyzing the interactions when using multiple extractants or taking advantage of organic solvent extraction for biofuels using an extractant that can be part of the fuel mixture. However, shortcomings like toxicity over the culture, ecological impact, emulsion formation, and complex posterior phases separation, may reduce the preference of this method (Heerema et al., 2011).


Aqueous two-phase system (ATPS) is a liquid-liquid partition technique that combines hydrophilic solutes above critical concentrations in an aqueous media, forming solutions that create two separate immiscible phases. This technique has been used for the recovery of different bioproducts, for example, proteins, hormones, monoclonal antibodies, etc (Dafoe & Daugulis, 2014). One of the two phases will act as an extractive phase for the bioproduct due to a higher affinity, the transfer process from one phase to other can be described by the partitioning coefficient (which describes the fractionation behavior of biomolecules) and is governed by the molecular weight, hydrophobicity, electrochemical interactions, and affinity. ATPS can be classified into five main groups depending on the compound used to form the phases: (i) polymer- polymer systems, (ii) polymer-salt systems, (iii) ethanol, (iv) micelles and (v) ionic fluids. Each group has different characteristics, making them preferable for certain biomolecules (Rito &

Benavides, 2017).

Some advantages such as eco-friendly, low cost, easy to scaling-up, capable of continuous operation, short processing time, and high efficiency for recovery and purification of biomolecules in a primary stage, make ATPS a widely studied method in biotechnological processes.

Furthermore, because of water is the main component in both phases, it creates a suitable environment for biomolecules extraction, while other techniques (e.g. organic solvents extraction) could damage bioproducts (Iqbal et al., 2016). The main disadvantage of this technique may be the relatively complex nature of selecting the components for the extraction phase, considering the high amount of interactions that can affect the process (Dafoe & Daugulis, 2014) and the lack of studies related with industrial scaling up (Espitia-Saloma et al., 2018).

Additionally, ATPS has been used for extractive fermentation, overcoming low product yield caused by inhibition or toxicity of end products (Iqbal et al., 2016). For example, removal of


inhibitors by this method, just as enzyme inhibitors, can also help to improve yields in extractive fermentation (Molino et al., 2013). Some examples of bioproducts enhanced by in-situ extraction using ATPS are: lipase, clavulanic acid, B-carotene, butanol, and prodigiosin.

Polymer-Polymer ATPS

One of the most popular ATPS types requires poly(ethylene glycol) (PEG) and high molecular weight dextran polymers to form an extractive phase (Ooi et al., 2011). These compounds have advantages as: low required concentrations, moderate viscosity and they can be easily buffered (Schindler & Nothwang, 2006). Since low ionic strength between the polymers exists, the system is usually used for recovery of compounds sensitive to ionic environments, as well as those susceptible to osmotic shocks, like cells and organelles (Rito & Benavides, 2017).

Viscosity is one of the main factors that can cause problems in the process, Ooi et al. (2011) tested different PEGs over different concentrations in the extraction of a lipase produced by Burkholderia pseudomallei culture, they used a PEG/dextran ATPS recovery system and concluded that high concentrations and molecular weight of PEG can affect the cell growth by increasing the medium viscosity, thus, limiting the oxygen mass transfer. Dextran molecular weight also has been reported as limiting for microbial growth. This behavior evinces the complexity of determining the biocompatibility and the election of the components in ATPS processes (Dafoe & Daugulis, 2014).

Ooi et al. (2011) also reported that the bottom phase (formed principally by dextran), can be used as a filter, separating contaminant proteins and biomass, exploiting target product affinity towards the top phase. This bottom phase can be recycled or reused to take advantage of the captured biomass.

Polymer-Salt ATPS

ATPS using salt and polymeric solutions are the most common systems, in view of its lower cost compared with a polymer-polymer system and the different ranges of hydrophobicity between phases. They have been extensively used for bioseparation of proteins, enzymes, and polyketides (Esmanhoto & Kilikian, 2004).

The concentration of the phase-forming compounds, the molecular weight of the polymer, pH, and ionic strength are the main factors influencing partitioning (Esmanhoto & Kilikian, 2004).

Pandey & Banik (2011) studied polymer-polymer and polymer-salt ATPS in extractive


fermentation for alkaline phosphatase production by Bacillus licheniformis and concluded that the most suitable treatment was a polymer-polymer ATPS (using PEG 4000 and dextran T 500) as result of the enzyme got majorly entrapped in salt bottom phase stage at polymer-salt ATPS because due to the interactions between the anionic amino acids on the protein and metal ions from the salt, leading lower yields of recuperation.

Ooi et al. (2011) reported extensive inhibitory activity of ionic salts such as potassium phosphate and magnesium sulfate for the biosynthesis of lipases inhibiting its direct application as an in-situ extraction system. However, other studies have used these salts as successful components for in-situ bioproducts extraction (Karkaş & Önal, 2012; Esmanhoto & Kilikian, 2004; Wei, Zhu & Cao, 2002), making evident the complicated nature of selecting phase-forming components for the recovery of a specific metabolite as Dafoe & Daugulis (2014) described.

Alcohol-salt ATPS

In contrast to those ATPS using polymers, alcohol-salt ATPS is a cheaper option and avoid viscosity problems through short-chain organic solvents and salt solutions for the extraction, also they present high polarity, easiness for further downstream processing and the possibility of recycling the organic phase by evaporation (Ooi et al., 2009). The main disadvantage of this type of extraction is that some proteins and other compounds can be affected by the organic solvent, causing inactivation or denaturalization of the molecules (Rito & Benavides, 2017). Nevertheless, alcohol-salt ATPS has been successfully applied for the extraction of proteins such as lipases and plant biomolecules like anthraquinones using a 2-propanol/potassium phosphate/NaCl system and 1-propanol/ammonium sulfate system respectively (Ooi et al., 2009; Tan, Li & Xu, 2013).

Micellar ATPS

Micellar ATPS type, also known as cloud point system, uses surfactants (generally non-ionic) to generate the extractive phase. This process is triggered by an increase in temperature to overcome the threshold of cloud point temperature; this causes that micelles aggregate, forming two different water affinity zones, one hydrophobic and one hydrophilic, a property that can be applied for selective separation and extraction of bioproducts (Rito & Benavides, 2017). Micellar ATPS is very useful for the extraction of bioparticles that are sensitive to ionic environments and it has also been used for the extraction of membrane proteins, viruses, and nucleic acids (Iqbal et al., 2016).


Santos et al. (2011) reported micellar ATPS using Triton X-114 and sodium bis(2-ethylhexyl) sulfosuccinate (AOT) for recovery of clavulanic acid, a β-lactamase inhibitor produced by Streptomyces clavuligerus, and obtained favorable recovery yields, reduction in extraction steps and a target molecule stable in the micellar phase. Micellar ATPS also has been used for in-situ recovery of intracellular compounds, which avoids harvesting and cell lysis steps, necessary in traditional DSP for intracellular compounds, and may help to overcome product inhibition (Hu et al., 2012; Chávez y Aguilar, 2016).

Ionic liquid ATPS

Ionic liquid (IL) ATPS uses salts that are liquid at room temperature and are composed of a cation and an anion (Dafoe & Daugulis, 2014). Other characteristics of these salts include: low vapor pressure, high thermal stability, and low viscosity (Rito & Benavides, 2017). IL is an alternative process to replace organic solvent extraction methods by not dealing with solvent toxicity, volatility, and flammability (Cull et al., 2000).

Common IL ATPS uses hydrophobic compounds for phase formation to extract hydrophobic or charged molecules (Dreyer, Salim & Kragl, 2009). However, targets that present a hydrophilic behavior, like proteins, are difficult to recover. To overcome these limitations, hydrophilic ionic liquids combined with kosmotropic salts or polymers have been studied, and good recovery rates exploiting the electrostatic force as the main driving force have been obtained. Selectivity can be also be improved by modifying pH (Oppermann, Stein & Kragl, 2011). Penicillin (Jiang et al., 2009), erythromycin (Cull et al., 2000), amoxicillin (Soto et al., 2005), and lactic acid (Matsumoto et al., 2004) are some examples of compounds recovered by ionic liquid two-phase systems.

Solid-liquid systems Membrane bioreactors

Membranes have been widely used in biotechnology, mainly as fermentation end product separation method. Compared with other primary recovery techniques, membrane separation presents some advantages e.g. high surface area per unit of volume, which allows better contact between the phases (culture broth and membrane); they are usually performed at low temperatures and pressures, and do not require the addition of chemicals to the culture, presenting a low rate of interference with the media compounds, such as degradation or denaturalization of bioproducts (Charcosset, 2006).


This technique uses membranes made off ceramics, fibers, steel, or polymers that are low reactive materials, avoiding unwanted interactions between the membrane and the media components. They present small pore size (usually up to 0.2 m) that allows the entrance of media with the compounds of interest but not the biomass (maintaining it in the culture broth) (Figure 1.4), thus separating the product from the broth and causing an accumulation of cells while the product inhibition is alleviated (Carstensen, Apel & Wessling, 2012).

Two driving forces are involved in the separation of the molecules in membrane bioreactors (Figure 1.4). (i) The pressure gradient, that is the one that governs the process; and (ii) the concentration gradient (diffusion), which participates to a lesser extent (Carstensen, Apel &

Wessling, 2012).

A) B)

Figure 1.4. Main driving forces involved in membrane recovery processes. A) Pressure gradient and B) concentration gradient (diffusion), usually more involved in the hybrid processes (like perstraction).

According to Heerema (2011), two types of configurations can be used with membrane systems, the first consists of a submerged membrane inside the reactor, directly in contact with the culture broth. While the second known as "external loop membrane systems" consists of a membrane placed outside the reactor which is fed with culture media using a recirculation system (see Figure 1.3).


A) B)

Figure 1.5. Standard structures of (A) a submerged membrane system and B) an external loop process.

Carstensen, Apel & Wessling (2012) discussed three main filtration modes for membrane processes. Membrane bioreactors mostly use a cross-flow filtration (a flux passes in a horizontal way to the membrane) at external loop systems, while submerged membranes are typically encased inside a filtration system at the interior of the bioreactor (Figure 1.5). Membranes bioreactors typically present fewer problems with cake layer formation compared with downstream traditional methods that use in-line filtration modes (flux entering vertically); either by the flux flow in external membranes (Baker, 2004) or by the presence of air bubbles in the submerged systems, that help to control the layer formation (Chang et al., 2002).

In their extensive review, Carstensen, Apel & Wessling (2012), highlighted the main external systems disadvantages; considerations as problematic sterilization, high energy consumption, reaction volumes, more space required, and oxygen limitations in recirculation have to be considered. However, they present advantages at fouling control and biomass layer formation on the membrane surfaces (Carstensen, Kasperidus, & Wessling, 2013; Germain & Stephenson, 2005). Also, the commercial availability of external units is major than submerged membranes (Stark & Von Stockar, 2003).

Membrane filtration can be combined with other recovery strategies for the development of hybrid processes like perstraction (membrane extraction), in which solvent extraction is added. Heerema et al. (2011) reported the use of a submerged membrane for phenols compounds recovery, by coupling a solvent extraction using circulation of 1-octanol at the inner phase of the membrane unit. The compounds are transferred by affinity to the solvent, which is physically separated culture broth, minimizing the interactions between the two phases and avoiding emulsion formation, as well as the losses of culture broth in the solvent phase (Figure 1.4-B). This process


can be improved by changing the solvent for one with a higher partition coefficient (Heerema et al., 2011). Solvent extraction also facilitates the recovery of the compound by evaporation of the solvent. In addition to perstraction, other methods like pervaporation (a mix between membrane permeation and vacuum evaporation) and vapor permeation (a strategy derived from pervaporation), have been studied as hybrid techniques for the recovery of butanol (Jiménez &

Wang, 2017) and ethanol (Sun et al., 2017), respectively.

In-situ adsorbents

The use of solid adsorbents that may be added directly to the fermentation is probably the less restrictive and more commonly used type of ISPR for its simple implementation, being the most studied at in-situ product recovery researches (Dafoe & Daugulis, 2014). Adsorbents can be also placed as external units (adsorption columns) in which the media is circulated; nonetheless, this configuration can lead to lower recovery efficiencies compare to the in-situ (submerged) process due to a decrease in the interactions between the adsorbent resin and the culture broth (Wang et al., 2004).

Ion-exchange resins and natural adsorbents, e.g. activated charcoal, were the first type of adsorbent materials reported. However, drawbacks as non-selective adsorption and resin fouling with cells and polar compounds, have limited the use of ion-exchange resins by cause of a decrease in the binding capacity (Phillips et al., 2013). Non-ionic resins appeared as a more selective solution and are currently the most widely used. These adsorbents, such as zeolites and polymeric resins, bind to the target molecules by hydrophobic interactions and their selectivity can be modified by characteristics like the pore size of the adsorbent (Dafoe & Daugulis, 2014) and the resin chemistry (Phillips et al., 2013).

Recently, exchange-resins are receiving attention for the recovery of compounds that are charged at the fermentation pH, like organic acids (Xue et al., 2010; Mirata, Heerd & Shrader, 2009). Ataei

& Vasheghani (2008), used this approach for the in-situ recovery of lactic acid, and determine that pH, temperature, and agitation rates affect the recovery yields of the absorbents. They optimized the parameters previously mentioned and reported a 4.3-fold in the lactic acid production yield by overcoming product inhibition.

Wang et al. (2004) noted that the concentration of the adsorbent play an important role in the efficiency of the process and must be also optimized for enhancing product recovery (in ionic and non-ionic resins); Mei, Min & Lü (2009) reported an improvement in yields of phenylalanine


biotransformation into 2-phenylethanol, a process affected by product inhibition effect, by adding 2 g of a non-ionic resin to the culture media. The amount of adsorbent added has to be optimized based on the degree of product inhibition and the adsorption capacity of the resin in a case by case analysis.

Also, for a single process, many adsorbents have to be tested to select the most suitable resin, considering selectivity and adsorption capacity. This may represent a time-consuming and exhausting work. For this reason, high throughput screening (HTS) techniques offer a fast and economical way, mainly by miniaturization and automation for the screening process (Cramer &

Holstein, 2011). Nevertheless, even when the selection process has been sped up by HTS, adsorbents still have some important issues, for example, the recovery of the adsorbed molecules, a process that usually needs highly concentrated salt solutions or heat, generates wastes and increase the require energy (Dafoe & Daugulis, 2014).

In the last years, nanotechnology has been applied to the adsorbents field, showing an improvement in the efficiencies. Several investigations have studied the use of nanostructured adsorbents for the in-situ recovery of products like toxic organic/volatiles compounds in microalgae culture (Rocha et al., 2016), excessive phosphate in water bodies (Lǚ et al., 2013) and noxious heavy metals in wastewater effluents (Burakov et al., 2018), reporting increments in the selectivity. These materials can be also combined with other techniques as membrane reactors. Hazrati, Jahanbakhshi & Rostamizadeh (2018), studied the use of zeolite nano- adsorbents in a membrane bioreactor and determine a reduction in membrane fouling and transmembrane pressure. Similar results were reported by Mohamadi, Hazrati & Shayengan (2019) applying zeolites and activated carbon in membrane systems.

In addition to adsorbent materials, there are also absorbents or “soft” polymers, which have a different sorption mechanism ruled by the diffusion of the molecule into the polymer. These materials avoid problems related to competition for binding sites as observed at the surface of adsorbents (Dafoe & Daugulis, 2014). Considering the advantages of these "soft polymers", their simple implementation and effectivity, more research has been done in this field for product removal and the understanding of the process, as well as criteria for better polymer selection (Poleo & Daugulis, 2013).


Gas-liquid system Gas stripping

Gas stripping is a recovery technique applicable only for the capture and separation of volatile products. The method consists of gas sparged into the medium (that can be carbon dioxide, nitrogen, air, etc) to capture the volatile compounds and separating them from the media for further condensation and separation of the components. This is a very simple technique that can be easily implemented to continuous operation mode without harm to the culture (Pyrgakis et al., 2016).

This technique has been widely studied for bio-fuels extraction (Silva et al., 2015; Schläfe et al., 2017; Sonego et al., 2017; Thani, Laopaiboon & Laopaiboon, 2017; Xue et al., 2013) and demonstrated to be a feasible method to accomplish an acetone-butanol-ethanol (ABE) extractive fermentation (Li et al., 2016).

Gas temperature and pressure can be modified to enhance the recovery rate, and the gas type can variate to reduce the cost of the process. Many studies have addressed gas stripping modeling, making easier the behavior and yield prediction of the process under certain parameters using mathematical models (Silva et al., 2015; Pyrgakis et al., 2016).

Gas stripping has demonstrated to be a flexible technique that can be coupled to other primary recovery methods, developing different strategies for extraction and separation of volatile compounds. The first recovery technique is used to separate and concentrate the compound of interest, increasing the yields of capture by the subsequent gas stripping technique.

Pervaporation (Cai et al., 2016), adsorbents (Pyrgakis et al., 2016) and liquid-liquid partition (Lu et al., 2016) have been tested as suitable primary extraction techniques. Oleyl alcohol (O.A) has been studied as an extractive phase for butanol during fed-batch fermentation with Clostridium actetobutylicum coupled with nitrogen gas stripping at the O.A phase, and demonstrate to be an efficient hybrid ISPR technique, allowing good recovery yields and enhancing fermentation productivity.

Conclusion and future trends

In-situ downstream processing emerges as an opportunity to increase the fermentation yields by alleviating product inhibition that can be caused by cytotoxic compounds or catabolic regulation;

and by the recovery of target metabolites that may be metabolized by the cells, leading to low


yields. A great range of extractive techniques has been studied, making possible to obtain a variety of compounds.

Each extraction technique has its own drawbacks and limitations, a recent trend turns to combine different extraction techniques in hybrid modes to take advantage of the strong points of each of the recovery steps, but in exchange, the complexity of the procedure is increased. Gas stripping is probably one of the most used techniques to be combined with different strategies and develop hybrid processes, this because of its simplicity, facility for compound separation, and reduced interaction with the media. However, as a major limitation, it only can be used for volatile compounds extraction. Also, adsorption, as traditional as it may be, it has been taking fresh air with the use of newly developed ceramic materials and other kinds of nanostructured adsorbents, that present high affinity and selectivity for the target molecule.

ISPR strategies involving the addition of new components to a fermentation media may present undesired effects over the process that should be considered. In an extractive fermentation, the components of the recovery phase will be in contact with the media during the fermentation, and if they are not carefully chosen and pre-tested, the fermentation yields can be negatively affected due to the interaction of these components with the media and the microorganism. Mathematical models can be used as a powerful tool to improve the actual process to select and optimize ISPR, offering a fast and feasible way to describe/predict the behavior of the process.


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2. Problem Statement


2.1 Problem Statement and Context

During the last decades, the search and production of natural pigments of industrial interest has increased considerably, especially in the pharmaceutical and food area, due to its diverse bioactive properties and lower toxicity index, with particular attention to compounds from microbial and vegetable origins (Venil et al., 2013). Prodigiosin is an alkaloid produced as a secondary metabolite in various microorganisms, being the gram-negative bacteria Serratia marcescens probably the most common and abundanet producer (Wei & Chen, 2005). It is an intense red pigment with plenty of reported bioactivities, such as, immunosuppressive, antimicrobial and anticancer (Naik et al., 2012). Most of the studies that address the biological production of this pigment, have focused on developing high-yield culture media and culture conditions optimization, aiming to increase the pigment production rate (Darshan and Manonmani, 2015). However, the high cost of some components of the proposed culture media, such as glycerol, diminishes the interest of an industrial scaling up (Casullo et al., 2010; Naik et al., 2012). On the other hand, the total production yields for prodigiosin bioprocesses have been reported to be affected by end- product microbial inhibition, when prodigiosin reaches a particular concentration in the fermentation media (Bae et al., 2001; Chavez & Aguilar, 2016). These factors, combined to the typically high cost of a traditional downstream processing required for primary extraction and purification of this metabolite, represent important drawbacks for the industrial implementation of a bioprocess by limiting the economic feasibility and thus affecting the final cost of the pigment and their potential use for the bioactivities previously mentioned.

A highly (>95%) pure prodigiosin extract is also highly expensive, around 700 USD for 1 mg. And due to this fact, the therapeutic uses of this compound has been very limited, even when in vitro studies have shown the ability of prodigiosin to stimulate apoptosis in cancerous cells (without attacking the normal cells) by having a role in activation of p-53 and other pathways (Sigma- Aldridch, 2018). Also, as previously mentioned, antimicrobial (E. coli, P. vulgaris, C. albicans, P.

notamun…), antiviral (antimalarial), immunosuppressive, food colorant properties, photo- protective ability and cell cycle inhibitor activity have been scientifically proved (Darshan &

Manonmani, 2015). Considering the potential of this compound, is necessary to develop cost- effective strategies that allow prodigiosin production to become more economically viable and thus, increase the therapeutic use of this alkaloid.

Previous research has focused on trying to solve these drawbacks, addressing them separately.

In one hand, different low-cost media culture using sources rich in fatty acids have been proposed.


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