In Situ Product Recovery Research Articles

In situ product removal

Abstract During current years, the industrial biotechnology area has grown at giant steps, supported by the necessity of a sustainable supply chain and the inevitable depletion of petrochemical feedstocks. From this accelerated growth, the need for the development of more efficient bioprocesses in term of productivity and cost has emerged. A substantial number of bioprocesses have their potential hindered by product inhibition, a phenomenon that appears due to microbial metabolites produced in concentrations that become toxic even for the producing microorganism. In situ product recovery (ISPR) appears as a strategy to overcome such problems by primary recovery stage to the upstream, thus continuously extracting a desired or undesired target molecule from the fermentation broth as soon as it is produced. In this chapter, we will review the inherent advantages of implementing this technology in the production process, not only in terms of productivity, but also in equipment. A revision across the main the ISPR technologies can be found, explaining their main mechanisms and configurations, the appropriate scenarios to use each one and the main factors that must be considered that affect process efficiency. The chapter will be divided into three parts according to the types of ISPR that are reviewed, liquid–liquid, solid–liquid and gas–liquid techniques. Some recent trends and further perspectives for each method are also mentioned leaving space for further analysis of these technologies.

Increased paclitaxel recovery from Taxus baccata vascular stem cells using novel in situ product recovery approaches

In this study, several approaches were tested to optimise the production and recovery of the widely used anticancer drug Taxol® (paclitaxel) from culturable vascular stem cells (VSCs) of Taxus baccata, which is currently used as a successful cell line for paclitaxel production. An in situ product recovery (ISPR) technique was employed, which involved combining three commercial macro-porous resin beads (HP-20, XAD7HP and HP-2MG) with batch and semi-continuous cultivations of the T. baccata VSCs after adding methyl jasmonate (Me-JA) as an elicitor. The optimal resin combination resulted in 234 ± 23 mg of paclitaxel per kg of fresh-weight cells, indicating a 13-fold improved yield compared to the control (with no resins) in batch cultivation. This resin treatment was further studied to evaluate the resins’ removal capacity of reactive oxygen species (ROS), which can cause poor cell growth or reduce product synthesis. It was observed that the ISPR cultivations had fourfold less intracellular ROS concentration than that of the control; thus, a reduced ROS concentration established by the resin contributed to increased paclitaxel yield, contrary to previous studies. These paclitaxel yields are the highest reported to date using VSCs, and this scalable production method could be applied for a diverse range of similar compounds utilising plant cell culture.Graphical

Co-culture of Kluyveromyces marxianus and Meyerozyma guilliermondii with In Situ Product Recovery of 2-Phenylethanol.

Production of 2-phenylethanol (2-PE) via Kluyveromyces marxianus is well-established. However, co-culture with other microbes in combination with in situ product recovery (ISPR) yields improved selectivity and volumetric productivity. Fermentation ofK. marxianus (MUCL 53775) with direct inclusion of absorptive polymer Hytrel3548 achieved ISPR, but accumulation of the byproduct phenylethyl acetate (PEA) was strongly favored. Co-culture of K. marxianus (MUCL 53775) with Meyerozyma guilliermondii (MUCL 28072) with ISPR limited PEA production, thereby improving the 2-PE selectivity from 13 to 90%, compared to a pure culture of K. marxianus (MUCL 53775) under similar conditions. This improved the volumetric productivity by 85% compared to 2-PE ISPR with a pure culture of K. marxianus. This is the first report of co-culture in a two-phase fermentation for 2-PE bioproduction and demonstrates that interactions between co-culture and ISPR techniques can modulate bioproduction between 2-PE and byproduct PEA, and this technique will be explored for other strain combinations and for other high-value molecules of interest.

Development of In Situ Product Recovery (ISPR) System Using Amberlite IRA67 for Enhanced Biosynthesis of Hyaluronic Acid by Streptococcus zooepidemicus

High broth viscosity due to the accumulation of hyaluronic acid (HA) causes a limited yield of HA. It is a major problem of HA production using Streptococcus zooepidemicus. Extractive fermentation via in situ product recovery (ISPR) was utilized to enhance the HA production. Resins from Amberlite: IRA400 Cl; IRA900 Cl; IRA410 Cl; IRA402 Cl; and IRA67 were tested for the HA adsorption. IRA67 showed high adsorption capacity on HA. The study of the adsorption via a 2 L stirred tank bioreactor of S. zooepidemicus fermentation was investigated to elucidate the adsorption of HA onto IRA67 in dispersed and integrated internal column systems. The application of a dispersed IRA67 improved the HA production compared to the fermentation without resin addition by 1.37-fold. The HA production was further improved by 1.36-fold with an internal column (3.928 g/L) over that obtained with dispersed IRA67. The cultivation with an internal column shows the highest reduction of viscosity value after the addition of IRA67 resin: from 58.8 to 23.7 (mPa·s), suggesting the most effective ISPR of HA. The improved biosynthesis of HA indicated that an extractive fermentation by ISPR adsorption is effective and may streamline the HA purification.

Systematic metabolic engineering of Escherichia coli for the enhanced production of cinnamaldehyde

Cinnamaldehyde (CAD) derived from cinnamon bark has received much attention for its potential as a nematicide and food additive. Previously, we have succeeded in developing an Escherichia coli strain (YHP05) capable of synthesizing cinnamaldehyde; however, the production titer (75 mg/L) was not sufficient for commercialization. Herein, to develop an economical and sustainable production bioprocess, we further engineered the YHP05 strain for non-auxotrophic, antibiotic-free, inducer-free hyperproduction of CAD using systematic metabolic engineering. First, the conversion of trans-cinnamic acid (t-CA) to CAD was improved by the co-expression of carboxylic acid reductase and phosphopantetheinyl transferase (PPTase) genes. Second, to prevent the spontaneous conversion of CAD to cinnamyl alcohol, 10 endogenous reductase and dehydrogenase genes were deleted. Third, all expression cassettes were integrated into the chromosomal DNA using an auto-inducible system for antibiotic- and inducer-free production. Subsequently, to facilitate CAD production, available pools of cofactors (NADPH, CoA, and ATP) were increased, and acetate pathways were deleted. With the final antibiotic-, plasmid-, and inducer-free strain (H-11MPmR), fed-batch cultivations combined with in situ product recovery (ISPR) were performed, and the production titer of CAD as high as 3.8 g/L could be achieved with 49.1 mg/L/h productivity, which is the highest CAD titer ever reported.

In Situ Product Recovery of Bio-Based Industrial Platform Chemicals: A Guideline to Solvent Selection

In situ product recovery (ISPR), in the form of an extractive fermentation process, can increase productivity and product titers in the sustainable production of platform chemicals. To establish a guideline for the development of industrially relevant production processes for such bio-based compounds, a wide screening was performed, mapping the potential of an extensive range of solvents and solvent mixtures. Besides solvent biocompatibility with Saccharomyces cerevisiae, distribution coefficients of three organic acids (protocatechuic acid, adipic acid and para-aminobenzoic acid) and four fragrance compounds (2-phenylethanol, geraniol, trans-cinnamaldehyde and β-ionone) were determined. While for highly hydrophobic fragrance compounds, multiple pure solvents were identified that were able to extract more than 98%, reactive extraction mixtures were proven effective for more challenging compounds including organic acids and hydrophilic alcohols. For example, a reactive mixture consisting of 12.5% of the extractant CYTOP 503 in canola oil was found to be biocompatible and showed superior extraction efficiency for the challenging compounds as compared to any biocompatible single solvent. This mapping of biocompatible solvents and solvent mixtures for the extraction of various classes of industrial platform chemicals can be a tremendous step forward in the development of extractive fermentations.

Using poly(vinyldodecylimidazolium bromide) for the in-situ product recovery of n-butanol.

The mitigation of end-product inhibition during the biosynthesis of n-butanol is demonstrated for an in-situ product recovery (ISPR) system employing a poly(ionic liquid) (PIL) absorbent. The thermodynamic affinity of poly(vinyldodecylimidazolium bromide) [P(VC12 ImBr)] for n-butanol, acetone and ethanol versus water was measured at conditions experienced in a typical acetone-ethanol-butanol (ABE) fermentation. In addition to providing a high n-butanol partition coefficient (PC = 6.5) and selectivity (αBuOH/water = 46), P(VC12 ImBr) is shown to be biocompatible with Saccharomyces cerevisiae and Clostridium acetobutylicum. Furthermore, the diffusivity of n-butanol in a hydrated PIL provides absorption rates that support ISPR applications. Using a 5 wt% PIL phase fraction relative to the aqueous phase mass, P(VC12 ImBr) improved the volumetric productivity of a batch ABE ISPR process by 31% relative to a control fermentation. The concentration of n-butanol in the P(VC12 ImBr) phase was sufficient to increase the alcohol concentration from 1.5 wt% in the fermentation medium to 25 wt% in the saturated PIL, thereby facilitating downstream n-butanol recovery.

Enhanced production of styrene by engineered Escherichia coli and in situ product recovery (ISPR) with an organic solvent

BackgroundStyrene is a large-volume commodity petrochemical, which has been used in a wide range of polymer industry as the main building block for the construction of various functional polymers. Despite many efforts to produce styrene in microbial hosts, the production titers are still low and are not enough to meet the commercial production of styrene.ResultsPreviously, we developed a high l-phenylalanine producer (E. coli YHP05), and it was used as a main host for de novo synthesis of styrene. First, we introduced the co-expression system of phenylalanine-ammonia lyase (PAL) and ferulic acid decarboxylase (FDC) genes for the synthesis of styrene from l-phenylalanine. Then, to minimize cell toxicity and enhance the recovery of styrene, in situ product recovery (ISPR) with n-dodecane was employed, and culture medium with supplementation of complex sources was also optimized. As a result, 1.7 ± 0.1 g/L of styrene was produced in the flask cultures. Finally, fed-batch cultivations were performed in lab-scale bioreactor, and to minimize the loss of volatile styrene during the cultivation, three consecutive bottles containing n-dodecane were connected to the air outlet of bioreactor for gas-stripping. To conclude, the total titer of styrene was as high as 5.3 ± 0.2 g/L, which could be obtained at 60 h.ConclusionWe successfully engineered E. coli strain for the de novo production of styrene in both flask and fed-batch cultivation, and could achieve the highest titer for styrene in bacterial hosts reported till date. We believe that our efforts in strain engineering and ISPR strategy with organic solvent will provide a new insight for economic and industrial production of styrene in a biological platform.

Separation of Protocatechuic Acid: Emphasizing on Intensifying Approaches

Many recent studies focus on polyphenols, which are categorized into stilbenes, lignans, flavonoids and phenolic acids which are known to possess numerous biological actions. Protocatechuic acid, 3, 4 Dihydroxy, benzoic acid, and a polyphenol found abundantly in more than 500 plant species, known for its pharmacological effects in invivo and invitro studies as well as for its applications in polymer and plastic industries. The requirement for the development of a profitable and competitive method for the separation of carboxylic acids from the fermenter and aqueous streams is met by the process intensification technique- reactive extraction. Further, in Situ Product Recovery (ISPR) that is, integration of fermentation and separation unit is a cost-effective approach for the production of carboxylic acids. The reactive separation of protocatechuic acid using natural and conventional diluents in aminic and phosphorous extractants has been explored. The results of the study will be worthwhile for intensifying the recovery of Protocatechuic from the aqueous waste stream and fermentation broth and for the application of the process intensification at a marketable level, a systematic fundamental understanding at laboratory scale is vital by use of batch experiments.

In situ product recovery of bio-based ethyl esters via hybrid extraction-distillation

In situ product recovery (ISPR) of bio-based chemicals from microbial cultivation is a means to improve titer, rate, and yield because it alleviates end product inhibition.

Covalent immobilization of halohydrin dehalogenase for efficient synthesis of epichlorohydrin in an integrated bioreactor.

Halohydrin dehalogenase (HHDH)-mediated dehalogenation of 1,3-dichloro-2-propanol (1,3-DCP) is a key step in the chemoenzymatic synthesis of epichlorohydrin (ECH) from glycerol. In this study, a covalent immobilization strategy was employed to enhance the stability of Agrobacterium tumefaciens HHDH using epoxy resin ES-103B as a carrier. Under optimal conditions, the activity recovery of ES-103B-immobilized HHDH (HHDH@ES-103B) was 62.4% and the specific activity was 1604 U/g. The HHDH@ES-103B exhibited excellent thermostability, with a half-life of 68.6 days at 40°C, which is 8.0-times higher than that of the free HHDH. A semicontinuous biotransformation of 1,3-DCP to ECH was performed using HHDH@ES-103B as biocatalyst in a recirculating packed bed reactor (RPBR), resulting in an ECH yield of 94.2%, with an average productivity of 5.2 g/L/h. The RPBR system exhibited a high operational stability and even after 50 cycles of reaction, it retained > 90% of the initial conversion. Furthermore, an integrated bioprocess based on in situ product recovery (ISPR) was developed in RPBR to overcome product inhibition. The integrated bioreactor equipped with an external macroporous adsorption resin HZD-9 column led to another 1.6-fold increase in ECH productivity to 8.46 g/L/h. This improved stability and reusability of HHDH@ES-103B demonstrated its potential for the biotransformation of 1,3-DCP to ECH. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:784-792, 2018.

Hybrid in situ product recovery technique applied to (A)IBE fermentation

Abstract Fermentative production of alcohols is a promising alternative to petroleum-based fuels industry. However, the development of a competitive biological process implies the achievement of higher production titer and enhanced productivity. In the case of butanol production by solventogenic strains, In Situ Product Recovery (ISPR) techniques help to overcome the inhibition threshold of the microorganism and to reduce the overall cost of downstream process. In this work, gas stripping (GS) was preliminary studied in an abiotic system and high flow rates (vvm – vessel volume minute- >2 min−1) were needed to achieve higher butanol stripping rate than its production rate. Combined liquid extraction (LE) with pulse Gas Stripping (GS) were applied to batch fermentations resulting in a synergic effect giving a higher glucose consumption rate and a lower aqueous butanol concentration. Moreover, in this hybrid system, biphasic bioreactor acted as a “liquid–liquid equilibrium step” and both phases were stripped concomitantly.

A comparison of the energy use of in situ product recovery techniques for the Acetone Butanol Ethanol fermentation

The productivity of the Acetone Butanol Ethanol (ABE) fermentation can be significantly increased by application of various in situ product recovery (ISPR) techniques. There are numerous technically viable processes, but it is not clear which is the most economically viable in practice. There is little available information about the energy requirements and economics of ISPR for the ABE fermentation. This work compares various ISPR techniques based on UniSim process simulations of the ABE fermentation. The simulations provide information on the process energy and separation efficiency, which is fed into an economic assessment. Perstraction was the only technique to reduce the energy demand below that of a batch process, by approximately 5%. Perstraction also had the highest profit increase over a batch process, by 175%. However, perstraction is an immature technology, so would need significant development before being integrated to an industrial process.

Bioreactors and in situ product recovery techniques for acetone-butanol-ethanol fermentation.

The microbial fermentation process is one of the sustainable and environment-friendly ways to produce 1-butanol and other bio-based chemicals. The success of the fermentation process greatly relies on the choice of bioreactors and the separation methods. In this review, the history and the performance of bioreactors for the acetone-butanol-ethanol (ABE) fermentation is discussed. The subject is then focused on in situ product recovery (ISPR) techniques, particularly for the integrated extraction-gas stripping. The usefulness of this promising hybrid ISPR device is acknowledged by its incorporation with batch, fed-batch and continuous processes to improve the performance of ABE fermentation.

Debottlenecking product inhibition in 1,3-propanediol fermentation by In-Situ Product Recovery

The present work describes the application of liquid–liquid extraction as an In-Situ product recovery (ISPR) technique to overcome the problem of product inhibition in 1,3-PD fermentation. As a part of initial screening experiments, six solvents were subjected to phase separation and biocompatibility tests to find the best extractant for in-situ removal of 1,3-PD from the bioreactor. These included tributylphosphate, ethyl acetate, butyl acetate, oleyl alcohol, oleic acid and hexanol. Of these, ethyl acetate was found to be the most suitable solvent for 1,3-PD extraction. Use of the selected extractant in continuous integrated fermentation–extraction was established by batch and fed-batch extractive fermentations which demonstrated a significantly improved 1,3-PD production of 35g/L and 74.5g/L, respectively. A steady state 1,3-PD concentration of 58g/L was obtained in continuous extractive system. Continuous cultivation with in-situ cell retention and in-situ 1,3-PD removal demonstrated a 5-fold enhancement in 1,3-PD productivity over non-extractive batch.

A membrane stirrer for product recovery and substrate feeding.

During fermentation processes, in situ product recovery (ISPR) using submerged membranes allows a continuous operation mode with effective product removal. Continuous recovery reduces product inhibition and organisms in the reactor are not exposed to changing reaction conditions. For an effective in situ product removal, submerged membrane systems should have a sufficient large membrane area and an anti-fouling concept integrated in a compact device for the limited space in a lab-scale bioreactor. We present a new membrane stirrer with integrated filtration membranes on the impeller blades as well as an integrated gassing concept in an all-in-one device. The stirrer is fabricated by rapid prototyping and is equipped with a commercial micromesh membrane. Filtration performance is tested using a yeast cell suspension with different stirring speeds and aeration fluxes. We reduce membrane fouling by backflushing through the membrane with the product stream.

The use of high pressure CO2‐facilitated pH swings to enhance in situ product recovery of butyric acid in a two‐phase partitioning bioreactor

Through the use of high partial pressures of CO2 (pCO2 ) to facilitate temporary pH reductions in two-phase partitioning bioreactors (TPPBs), improved pH dependent partitioning of butyric acid was observed which achieved in situ product recovery (ISPR), alleviating end-product inhibition (EPI) during the production of butyric acid by Clostridium tyrobutyricum (ATCC 25755). Through high pressure pCO2 studies, media buffering effects were shown to be substantially overcome at 60 bar pCO2 , resulting in effective extraction of the organic acid by the absorptive polymer Pebax® 2533, yielding a distribution coefficient (D) of 2.4 ± 0.1 after 1 h of contact at this pressure. Importantly, it was also found that C. tyrobutyricum cultures were able to withstand 60 bar pCO2 for 1 h with no decrease in growth ability when returned to atmospheric pressure in batch reactors after several extraction cycles. A fed-batch reactor with cyclic high pCO2 polymer extraction recovered 92 g of butyric acid to produce a total of 213 g compared to 121 g generated in a control reactor. This recovery reduced EPI in the TPPB, resulting in both higher productivity (0.65 vs. 0.33 g L(-1) h(-1) ) and yield (0.54 vs. 0.40). Fortuitously, it was also found that repeated high pCO2 -facilitated polymer extractions of butyric acid during batch growth of C. tyrobutyricum lessened the need for pH control, and reduced base requirements by approximately 50%. Thus, high pCO2 -mediated absorptive polymer extraction presents a novel method for improving process performance in butyric acid fermentation, and this technique could be applied to the bioproduction of other organic acids as well.

Demonstration of in situ product recovery of butyric acid via CO2‐facilitated pH swings and medium development in two‐phase partitioning bioreactors

Production of organic acids in solid-liquid two-phase partitioning bioreactors (TPPBs) is challenging, and highly pH-dependent, as cell growth occurs near neutral pH, while acid sorption occurs only at low pH conditions. CO2 sparging was used to achieve acidic pH swings, facilitating undissociated organic acid uptake without generating osmotic stress inherent in traditional acid/base pH control. A modified cultivation medium was formulated to permit greater pH reduction by CO2 sparging (pH 4.8) compared to typical media (pH 5.3), while still possessing adequate nutrients for extensive cell growth. In situ product recovery (ISPR) of butyric acid (pKa = 4.8) produced by Clostridium tyrobutyricum was achieved through intermittent CO2 sparging while recycling reactor contents through a column packed with absorptive polymer Hytrel® 3078. This polymer was selected on the basis of its composition as a polyether copolymer, and the use of solubility parameters for predicting solute polymer affinity, and was found to have a partition coefficient for butyric acid of 3. Total polymeric extraction of 3.2 g butyric acid with no CO2 mediated pH swings was increased to 4.5 g via CO2 -facilitated pH shifting, despite the buffering capacity of butyric acid, which resists pH shifting. This work shows that CO2 -mediated pH swings have an observable positive effect on organic acid extraction, with improvements well over 150% under optimal conditions in early stage fermentation compared to CO2 -free controls, and this technique can be applied other organic acid fermentations to achieve or improve ISPR.

In-situ Product Recovery as a Strategy to Increase Product Yield and Mitigate Product Toxicity

Product inhibition is often the cause limiting the maximum product concentration attainable in fermentation. This study showed the product yield of p-cresol could be improved by in-situ product recovery (ISPR). Escherichia coli transformed with the hpd BCA operon from Clostridium difficile was shown in this study to express phydroxyphenylacetate decarboxylase which converted p-hydroxyphenylacetate into p-cresol under anaerobic fermentation. Toxicity of p-cresol found at a concentration as low as 5 mM in a broth spiked with p-cresol was shown to have limited the maximum product concentration at 1 ± 0.1 mM after 30 hours of batch fermentation. Product yield was however shown to increase by 51% when activated carbon was used to remove p-cresol in-situ production. The activated carbon concentrated p-cresol on the solid adsorbent which was subsequently separated by sedimentation and p-cresol recovered by ultrasonic-assisted solvent extraction. Desorption of p-cresol from the spent activated carbon allowed the adsorbent to be regenerated for further product recovery. The ISPR strategy reported here was shown to improve the yield of a toxic product, was sustainable, and when adapted to a continuous process would increase productivity.

A Novel Integrated Bioprocess for Efficient Production of (R)-(−)-Mandelic Acid with Immobilized Alcaligenes faecalis ZJUTB10

An integrated bioprocess for the enantioselective hydrolysis of mandelonitrile to (R)-(−)-mandelic acid (R-MA) with immobilized Alcaligenes faecalis ZJUTB10 cells was constructed. Production of A. faecalis ZJUTB10 nitrilase in a pilot-scale fermenter (700 L) with high activity was achieved after optimizing cultivation conditions. A. faecalis ZJUTB10 cells were then immobilized in Ca-alginate. Efficient reusability of the biocatalyst up to 9 batches was obtained by immobilization, and treatment with polyethyleneimine (PEI) and glutaraldehyde (GA) further extended the longevity to 19 batches. The immobilized cells showed maximum activity at 40 °C and pH 8.0. A method for in situ product recovery (ISPR) based on an external extraction loop was established to overcome product inhibition. Anion-exchange column containing resin HZ202 was coupled to the packed bed bioreactor and enabled product recovery by continuously recirculating reaction mixture through the ISPR unit. This integrated bioprocess led to a high...