Extractive Fermentation Process Research Articles

Ethanol removal by stripping with CO2 reduced-size bubbles: Mechanical and thermodynamic entrainments

The efficiency of stripping processes for volatile compounds is governed by mechanical and thermodynamic entrainments, which play crucial roles in ethanol removal during the extractive fermentation process. These mechanisms depend on variables such as bubble diameter, ethanol concentration, liquid volume, pressure, and temperature. However, the quantification of each of them separately and their dependence on bubble diameter have never been addressed. The present study proposes a new methodology to evaluate both mechanical and thermodynamic entrainments, based on experimental data and thermodynamic equilibrium analysis of the bubbles in reactors with working volumes of 10.0 and 50 L, aiming to clarify the role that bubble diameter (microbubbles, fine and coarse bubbles) plays in these mechanisms. The results indicated that thermodynamic equilibrium was reached for any size of bubbles up to 5 mm in diameter, under a wide range of experimental conditions with specific gas flow rates from 0.22 to 10 vvm. Equally important, mechanical entrainment was found to enrich the gas phase in ethanol by a factor of 15, representing a higher concentration factor than for thermodynamic entrainment, which was inconceivable initially. This introduces a new and promising approach to intensify the stripping process of volatile compounds by means of mechanical entrainment.

Extractive fermentation for the production and partitioning of lipase and citric acid by Yarrowia lipolytica

Extractive fermentation is an efficient downstream process to segregate the desired product in a fermentative process. Here, an integrated extractive fermentation process with an aqueous two-phase system (ATPS) was applied to evaluate the lipase and citric acid production by Yarrowia lipolytica. For this purpose, the influences of polyethylene glycol (PEG) molecular weights, different salts and different concentrations as well as the pH of the potassium phosphate buffer were studied. Citric acid preferentially migrated to the salt-rich phase, whereas lipase mostly partitioned to the PEG-rich phase. The PEG2000 (30 wt%) + salt (7 wt%) system provided higher cell growth and citric acid production, whereas the PEG2000/Na2SO4 ATPS showed a better recovery yield (79.75%) for citric acid. The increase in PEG molecular weight reduced lipase production and the partition coefficient (KL). In the PEG 2000/KH2PO4-K2HPO4 (pH 7) system, a lipase production of 634.32 U L−1 and KL = 2.22 was obtained. Lipase production kinetics in an Erlenmeyer (10 mL) and a bioreactor (1.5 L) demonstrated that in the ATPS, the fermentation medium caused a delay in the growth curve and a peak in lipase production, of which the latter allowed for a reduction in the dissolved oxygen concentration, limiting cell metabolism.

Extractive Fermentation Processes: Modes of Operation and Application

AbstractBioprocesses can be hampered by product concentrations within a fermentation leading to product‐inhibition, and reductions in yield and productivity. This limitation impacts operational considerations, process economics, and viability, and, ultimately, whether a technology is industrially viable. One approach to circumventing these concerns is in situ extraction of products. In this way, the organism is no longer exposed to the inhibitory product, and fermentation can proceed, often at improved rates. Significant advances in this field have been made in recent years, however, they are for the most part siloed into sub‐fields (focusing on the product extracted, or the method of extraction). This review considers in situ extraction more holistically in terms of i) the separation methodology, ii) the mode of reactor operation, and iii) the physical reactor configuration. The summary hopes to give insight into promising areas for research and potentially spark ideas for novel in situ extractive processes.

Improving carotenoid production in recombinant yeast, Saccharomyces cerevisiae, using ultrasound-irradiated two-phase extractive fermentation.

Carotenoids are hydrophobic compounds that exhibit excellent bioactivity and can be produced by recombinant S. cerevisiae. Irradiating microorganisms with ultrasonic waves increase the productivity of various useful chemicals. Ultrasonic waves are also used to extract useful chemicals that accumulate in microbial cells. In this study, we aimed to improve the carotenoid production efficiency of a recombinant S. cerevisiae using an ultrasonic‐irradiation based two‐phase extractive fermentation process. When isopropyl myristate was used as the extraction solvent, a total of 264 mg/L of carotenoid was produced when batches were subjected to ultrasonic‐irradiation at 10 W, which was a 1.3‐fold increase when compared to the control. Transcriptome analysis suggested that one of the reasons for this improvement was an increase in the number of living cells. In fact, after 96 h of fermentation, the number of living cells increased by 1.4‐fold upon irradiation with ultrasonic waves. Consequently, we succeeded in improving the carotenoid production in a recombinant S. cerevisiae strain using a ultrasonic‐irradiated two‐phase extractive fermentation and isopropyl myristate as the solvent. This fermentation strategy has the potential to be widely applied during the production of hydrophobic chemicals in recombinant yeast, and future research is expected to further develop this process.

Combustion of n-butyl acetate synthesized by a new and sustainable biological process and comparisons with an ultrapure commercial n-butyl acetate produced by conventional Fischer esterification

This paper reports a study of the combustion dynamics of n-butyl acetate (BA) using the configuration of a burning droplet. Two grades of BA were examined: one (SBA) synthesized by a new process described in the paper that uses a metabolically engineered solventogenic Clostridiumstrain through an extractive fermentation process using n-hexadecane as the extractant; and one commercially available as a high-purity (99.9%) ‘neat’ BA grade produced by conventional Fischer esterification (NBA). The initial droplet diameter was primarily 0.6 mm with some limited experiments carried out for 0.4 mm droplets to show the influence of convection. Experiments were performed in the standard atmosphere and ignition was by spark discharge. The results showed the presence of impurities in the SBA at mass concentrations totaling about 6% which included n-butanol, n-hexadecane, iso-propyl alcohol and ethyl acetate. Droplet burning rates and flame structures were not influenced by these impurities at this concentration level. In the presence of convection created by buoyancy, droplets burned faster with stretched flames and a luminosity revealing the presence of soot by incandescence at the flame tips. Reducing the initial droplet diameter to 0.4 mm eliminated the convective effect and resulted in near spherical flames. The results presented show that the new synthesis process is a sustainable alternative for BA production with burning characteristics identical to NBA in both convective and stagnant gas transport fields.

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.

Extractive Fermentation for Process integration and amplified pullulan production by A. pullulans in Aqueous Two Phase Systems

Extractive fermentation technique or in situ product recovery process is a novel technique to segregate the desired product simultaneously in a fermentation process. For economic and high yield production of pullulan, Extractive fermentation process was applied fermentation process of A. pullulans. Aqueous Two Phase system (ATPS) systems were designed with various molecular mass of PEG (400, 600, 4000 and 6000) and dextran or mono/bi-sodium phosphate salts. Systems with short Tie Line length (TLL) 6.7 and 7.5% w/w for PEG-Salt and PEG-dextran respectively were chosen. Volume ratio for all the systems was kept constant at 1.0 and pH 7.0 for PEG-dextran and PEG-NaH2PO4 was maintained, whereas pH 9.0 was kept for PEG-Na2HPO4. A. pullulans, was found to be viable with PEG-NaH2PO4 and PEG-dextran systems. The biomass partitioned in the PEG rich top phase and the exopolysaccharide pullulan shown affinity towards the bottom phase. A maximum yield (36.47 g/L) was found with PEG 4000-Dextran 500 system of extractive fermentation process. The proposed process aptly integrates upstream and downstream process for continuous production and recovery of pullulan from the biomass, thus reducing the time quotient of the whole process.

Organosolv pretreated beech wood as a substrate for acetone butanol ethanol extractive fermentation

Abstract Acetone-butanol-ethanol (ABE) fermentation has a high potential for industrial application in lignocellulose-based biorefinery. Nevertheless, strong end-product inhibition is a severe limitation of this process, which can be partially solved by in situ product recovery techniques, such as extractive fermentation. In this work, the hydrolyzed cellulose from organosolv pre-treated beech wood (Fagus sylvatica L.) was fermented by Clostridium beijerinckii CECT 508 and compared with synthetic P2 medium in conventional and extractive fermentation processes. The conventional control fermentation yielded 9.12 g l−1 of ABE with C. beijerinckii CECT 508, while ABE overproduction of 40% was observed with cellulose hydrolysate as a substrate. After extractive batch fermentation based on 2-butyl-1-octanol as extractant, the final ABE titer was doubled in the P2 control medium over the conventional batch, with respective increases of 50, 190, and 140% for butanol (B), acetone (A) and ethanol (E). In the case of ABE as a hydrolysate medium, the total production increased by 10% with liquid extraction, while mainly B was responsible for this improvement. The total productivity was nevertheless gradually decreased in extractive fermentation, with a significantly slower production rate and the microbial growth compared to conventional fermentation. Moreover, solvent production and sugar consumption stopped, although the aqueous B concentration was below the inhibition threshold. The mid-term toxicity of the extraction solvent 2-butyl-1-octanol seems responsible for these results and this issue should be considered in future research.

Novel biobutanol fermentation at a large extractant volume ratio using immobilized Clostridium saccharoperbutylacetonicum N1-4

Product inhibition by butanol and acetone is a known drawback in acetone-butanol-ethanol (ABE) fermentation. Extractive fermentation improves butanol production by several ABE-producing Clostridium spp., but only low volume ratios (<4) of extractant to broth (Ve/Vb) have been studied. Here, a novel extractive fermentation process was developedusing Clostridium saccharoperbutylacetonicum N1-4 and a large Ve/Vb ratio. A mixture of oleyl alcohol-tributyrin (1:1 (v/v)) yielded high distribution coefficients for both butanol (3.14) and acetone (0.660). Although a fed-batch culture using free cells and the oleyl alcohol-tributyrin mixture at a Ve/Vb ratio of 5 had a lag phase of >24h, it produced a higher concentration of total butanol (i.e., butanol produced in all the phases per broth volume used) of 24.2g/L-broth after 96h compared with 14.4g/L-broth at a Ve/Vb ratio of 1, resulting in a low butanol concentration in the aqueous phase. The use of cells immobilized with calcium alginate beads shortened the lag phase to <12h. Butanol production was achieved not only in a 3-phase mode (extractant, beads, and tryptone-yeast extract-acetate (TYA) medium) but also in a 2-phase mode (extractant and beads containing TYA medium, without an aqueous phase) at a Ve/Vb ratio of 5, resulting butanol concentrations of 30.9g/L-broth and 27.7g/L-broth, respectively. The 3-phases fed-batch extractive fermentation at a Ve/Vb ratio of 10 showed a better performance compared with published reports: a total butanol concentration of 64.6g/L-broth and a butanol yield to consumed sugar of 0.378C-mol/C-mol.

Production of butyric acid from acid hydrolysate of corn husk in fermentation by Clostridium tyrobutyricum: kinetics and process economic analysis

BackgroundButyric acid is an important chemical currently produced from petrochemical feedstocks. Its production from renewable, low-cost biomass in fermentation has attracted large attention in recent years. In this study, the feasibility of corn husk, an abundant agricultural residue, for butyric acid production by using Clostridium tyrobutyricum immobilized in a fibrous bed bioreactor (FBB) was evaluated.ResultsHydrolysis of corn husk (10% solid loading) with 0.4 M H2SO4 at 110 °C for 6 h resulted in a hydrolysate containing ~ 50 g/L total reducing sugars (glucose:xylose = 1.3:1.0). The hydrolysate was used for butyric acid fermentation by C. tyrobutyricum in a FBB, which gave 42.6 and 53.0% higher butyric acid production from glucose and xylose, respectively, compared to free-cell fermentations. Fermentation with glucose and xylose mixture (1:1) produced 50.37 ± 0.04 g L−1 butyric acid with a yield of 0.38 ± 0.02 g g−1 and productivity of 0.34 ± 0.03 g L−1 h−1. Batch fermentation with corn husk hydrolysate produced 21.80 g L−1 butyric acid with a yield of 0.39 g g−1, comparable to those from glucose. Repeated-batch fermentations consistently produced 20.75 ± 0.65 g L−1 butyric acid with an average yield of 0.39 ± 0.02 g g−1 in three consecutive batches. An extractive fermentation process can be used to produce, separate, and concentrate butyric acid to > 30% (w/v) sodium butyrate at an economically attractive cost for application as an animal feed supplement.ConclusionA high concentration of total reducing sugars at ~ 50% (w/w) yield was obtained from corn husk after acid hydrolysis. Stable butyric acid production from corn husk hydrolysate was achieved in repeated-batch fermentation with C. tyrobutyricum immobilized in a FBB, demonstrating that corn husk can be used as an economical substrate for butyric acid production.

Greener L-lactic acid production through in situ extractive fermentation by an acid-tolerant Lactobacillus strain.

Lactic acid (LA) fermentation requires a neutralizer for a physiologically acceptable range. However, a neutralizer generates a large amount of gypsum, an environmental pollutant. Furthermore, the downstream processing is complicated and expensive, comprising 50-70% of the total cost. We previously developed a Lactobacillus delbrueckii FM1, which can produce undissociated LA without neutralizer. Here, we improved FM1 by adaptive evolution at pH4.5, which generated Adp FM1 showing an ~ 1.80-fold increase in LA production compared to FM1. The LA production via fed-batch fermentation yielded 36.2g/L of LA, with a productivity of 0.500g/L/h. However, cell viability was reduced due to the acidic pH and/or end-product inhibition. Therefore, an in situ LA recovery process using an extractive solvent was employed to maintain cell viability. Adp FM1 produced 49.2g/L of LA via in situ LA-extractive fed-batch fermentation, which was ~ 1.4-fold higher than that without LA extraction. Adp FM1 provided a total LA productivity of 0.512g/L/h in 96h. Among the tested strains, Adp FM1 exhibited the highest H+-ATPase activity and a 415-fold increase in H+-ATPase gene expression compared to the parent strain. These results suggest that the in situ LA extractive fermentation process will ease downstream processing and prove to be a more economical and environmentally friendly option compared to the present fermentation. To our knowledge, this is the first report on the production of undissociated L-LA by Lactobacillus using an in situ recovery process, with high LA production levels and productivity.

In situ extractive ethanol fermentation in a drop column bioreactor

AbstractBACKGROUNDIn conventional ethanol fermentation processes, the low ethanol content in the wine is due to inhibition of its production by the yeast cells. Extractive fermentation is an alternative process that can be used to overcome product inhibition by removing the ethanol from the fermentation broth. The present study describes the modeling and experimental validation of ethanol production in extractive batch fermentation, with in situ ethanol extraction by oleic acid, in a non‐conventional drop column bioreactor (DCB) operated under industrial conditions.RESULTSA model was developed using the hybrid Andrews–Levenspiel equation and the ethanol distribution coefficient (KDE), which provided an excellent description of the extractive fermentation process with oleic acid. Furthermore, higher ethanol productivities were obtained in the extractive fermentations, with ethanol productivity of 11.27–12.98 kg m‐3 h‐1, 12.7–29.8% higher when compared with the conventional process without ethanol removal. This was especially evident for the best extractive fermentation, which finished around 2 h earlier than the conventional fermentation.CONCLUSIONThe DCB showed good performance for use in extractive fermentation with liquid–liquid extraction of the ethanol and this technique presented higher ethanol productivity compared with the conventional process. © 2017 Society of Chemical Industry

Perstraction of Intracellular Pigments through Submerged Fermentation of Talaromyces spp. in a Surfactant Rich Media: A Novel Approach for Enhanced Pigment Recovery.

A high percentage of the pigments produced by Talaromyces spp. remains inside the cell, which could lead to a high product concentration inhibition. To overcome this issue an extractive fermentation process, perstraction, was suggested, which involves the extraction of the intracellular products out of the cell by using a two-phase system during the fermentation. The present work studied the effect of various surfactants on secretion of intracellular pigments produced by Talaromyces spp. in submerged fermentation. Surfactants used were: non-ionic surfactants (Tween 80, Span 20 and Triton X-100) and a polyethylene glycerol polymer 8000, at different concentrations (5, 20, 35 g/L). The highest extracellular pigment yield (16 OD500nm) was reached using Triton X-100 (35 g/L), which was 44% higher than the control (no surfactant added). The effect of addition time of the selected surfactant was further studied. The highest extracellular pigment concentration (22 OD500nm) was achieved when the surfactant was added at 120 h of fermentation. Kinetics of extracellular and intracellular pigments were examined. Total pigment at the end of the fermentation using Triton X-100 was 27.7% higher than the control, confirming that the use of surfactants partially alleviated the product inhibition during the pigment production culture.

Change of Monascus pigment metabolism and secretion in different extractive fermentation process

Monascus pigments that were generally produced intracellularly from Monascus spp. are important natural colorants in food industry. In this study, change of pigment metabolism and secretion was investigated through fed-batch extractive fermentation and continuous extractive fermentation. The biomass, secreting rate of pigment and total pigment yield closely correlated with the activated time of extractive fermentation as well as the composition of feeding nutrients. Metal ions played a key role in both the cell growth and pigment metabolism. Nitrogen source was necessary for a high productivity of biomass but not for high pigment yield. Furthermore, fermentation period for the fed-batch extractive fermentation could be reduced by 18.75% with a nitrogen source free feeding medium. Through a 30-day continuous extractive fermentation, the average daily productivity for total pigments reached 74.9AUday-1 with an increase by 32.6 and 296.3% compared to that in a 6-day conventional batch fermentation and a 16-day fed-batch extractive fermentation, respectively. At the meantime, proportions of extracellular pigments increased gradually from 2.7 to 71.3%, and yellow pigments gradually became dominated in both intracellular and extracellular pigments in the end of continuous extractive fermentation. This findings showed that either fed-batch or continuous extractive fermentation acted as a promising method in the efficient production of Monascus pigments.

Liquid–liquid equilibria in the quinary aqueous two-phase system of poly(ethylene glycol) 6000 + sodium sulfate + water in the presence of glucose and ethanol: Experimental investigation and thermodynamic modeling

Extractive fermentation processes involving aqueous two-phase systems (ATPSs) are considered as viable means of overcoming the problems associated with product inhibition. Practical development of these processes requires accurate knowledge of the liquid–liquid equilibrium (LLE) of the ATPS forming components alongside the substrate and product of the fermentation process. In this work, the quinary aqueous two-phase LLE of poly(ethylene glycol) 6000+sodium sulfate+water in the presence of glucose and ethanol have been experimentally determined at 298.15K using spectrophotometric methods. The resulting LLE data were then satisfactorily correlated by the non-random two-liquid (NRTL) activity coefficient model based on mass fractions. In doing so, the binary energy interaction parameters of the NRTL activity coefficient model were obtained and reported. Calculated RMS deviations below 0.6% demonstrate that the original NRTL activity coefficient model can accurately correlate the LLE data of the quinary aqueous biphasic system of interest.

Liquid–Liquid Equilibrium in an Aqueous Two-Phase System of Polyethylene Glycol 6000, Sodium Sulfate, Water, Glucose, and Penicillin-G: Experimental and Thermodynamic Modeling

Aqueous two-phase systems can be applied to a penicillin fermentation process to extract the produced penicillin from the fermentation broth to achieve a higher production rate. Such systems contain a polymer and a salt or two polymers. In this study, the liquid–liquid equilibrium of the quinary system of polyethylene glycol 6000, sodium sulfate, glucose, penicillin G, and water at 298.15 K is investigated. The experimental partition coefficients of glucose and penicillin-G showed the high efficiency of the present extractive fermentation processes. Using the liquid–liquid equilibrium data, the parameters of the NRTL activity model were calculated for the quinary system. The average absolute relative deviation of 0.677 % between experimental and calculated mass fractions shows that the NRTL activity model has a good ability to predict the equilibrium concentrations of the present system. In addition, a simple and accurate turbidimetric method for the measurement of penicillin G is proposed.

Production of Ethanol as a Renewable Energy by Extractive Fermentation

One issue with batch fermentation is that product inhibition causes low yields and ethanol productivity. The objective of this study was to increase the yield and ethanol productivity via continuous fermentation in a packed bed bioreactor with both an integrated extraction process and recycling of the raffinate into the fermenter. Molasses was used as the feedstock, and the immobilized cells were supported by ĸ-carrageenan. This process used n-amyl alcohol, 1-octanol, and 1-dodecanol as solvents. The yield and ethanol productivity increased from 8.79% to 20.03% and 34.54 g/L·h to 118.16 g/L·h for experiments using n-amyl alcohol, 9.05% to 12.67% and 35.59 g/L·h to 74.71 g/L·h, for 1-dodecanol, 8.89% to 13.45% and 34.93 g/L·h to 84.62 g/L·h, for1-octanol by increasing recycle ratio from 0 to 0.5. Based on these results, n-amyl alcohol was the best solvent for the extractive fermentation process.

Analysis and Design of Extractive Fermentation Processes Using a Novel Short-Cut Method

One means of increasing the productivity of microbiological processes is to remove toxic inhibiting products as they are formed through extractive fermentation. The aim of this work was to develop a short-cut tool to allow the design and analysis of extractive fermentation processes. For this purpose, a rigorous model considering both the fermentation kinetics and the application of a thermodynamic phase equilibrium model was developed for batch and continuous regimes. The outcomes and regularities found during the analysis of extractive fermentation using the rigorous model were considered to develop the short-cut tool. The short-cut method was based on the principles of topological thermodynamics and was applied to several case studies in this article. This method enables preliminary information to be acquired and used during the subsequent rigorous simulation, enabling a decrease in the calculation efforts and in the number of experimental runs.

Computer-aided biocompatible solvent design for an integrated extractive fermentation–separation process

Computer-aided process/solvent design is introduced to find a feasible biocompatible solvent for an extractive fermentation and separation process. The designed biocompatible solvent serves as both the extractant for extractive fermentation and the entrainer for extractive distillation, to yield water-free ethanol. Several goals, such as maximizing production rate and extraction efficiency, and limiting solvent utilization, are simultaneously considered in the optimal solvent design problem. Thus, the design was formulated as a mixed-integer nonlinear optimization problem. A two-phase computational scheme was introduced to solve the problem. The mixed-integer hybrid differential evolution (MIHDE) algorithm was first applied to solve the problem in order to obtain a feasible solution. The feasible solution was then served as an initial starting point for the mixed-integer sequential quadratic programming (MISQP) solver to numerically confirm that the optimal design was achieved. We have compared the crisp and fuzzy approaches to the design problem. The fuzzy goal attainment approach is able to address goal trade-offs and yield an overall satisfactory grade for the problem.

Extractive fermentation in cloud point system for lipase production by Serratia marcescens ECU1010

Extractive microbial fermentation for production of lipase by Serratia marcescens ECU1010 has been carried out in cloud point system. The cloud point system is composed of mixture nonionic surfactants with a ratio of Triton X-114 to Triton X-45 4:1 in aqueous solution. The lipase prefers to partition into the surfactant rich phase (coacervate phase) whereas the cells and other hydrophilic proteins retain in the dilute phase of cloud point system. Thus, a concentration factor 4.2-fold and a purification factor 1.3-fold of the lipase have been achieved in the extractive fermentation process. This is the first report about extractive fermentation of proteins in cloud point system.