Aqueous two-phase extraction of 2,3-butanediol from fermentation broths using an ethanol/phosphate system

Separation of 2,3-butanediol from the complex fermentation broths is a difficult task and becomes a bottleneck in industrial production. Aqueous two-phase systems composed of hydrophilic solvents and inorganic salts could be used to extract 2,3-butanediol from fermentation broths. Aqueous two-phase extraction of 2,3-butanediol from fermentation broths was studied by ethanol and dipotassium hydrogen phosphate system. The influences of phase composition on partition of 2,3-butanediol, removal of cells and biomacromolecules were investigated. The partition coefficient and recovery of 2,3-butanediol reached up to 28.34 and 98.13%, respectively, and the selective coefficient of 2,3-butanediol to glucose was 615.87 when the system was composed of 24% (w/w) ethanol and 25% (w/w) dipotassium hydrogen phosphate. Simultaneously, cells and proteins could be removed from the fermentation broths and the removal ratio reached 99.63 and 85.9%, respectively. This process is convenient and economic, furthermore, the operation is easy to scale-up, that is, this method provides a new possibility for the separation and refining of 2,3-butanediol.     

Continuous countercurrent salting-out extraction of 1,3-propanediol from fermentation broth in a packed column

The possibility of continuous extraction of 1,3-propanediol in a experimental packed column was investigated using a salting-out extraction system of dipotassium phosphate/ethanol. Mass transfer of 1,3-propanediol takes place from the dispersed phase (salt-rich solution) to the continuous phase (ethanol). The influences of flow rate of dispersed phase and size of packing material on partition coefficient and recovery of 1,3-propanediol were investigated and the results were compared with those obtained in spray column and test tube. Furthermore, the influences of various system compositions on hold up of dispersed phase, mass transfer coefficient, and system stability were also studied in the column packed by stainless steel Dixon 3 × 3 mm. It was found that the packed column showed a good extraction efficiency and stability. Besides, 1,3-propanediol recovery of 90.30% was obtained during a 11 h continuous operation when the real fermentation broth was used. At the same time, 94.4% of phosphate could be recovered when 0.2 volume of anhydrous ethanol was added into the raffinate phase at pH 4.0.     

乙醇-磷酸氢二钾双水相体系萃取L-精氨酸

采用乙醇-磷酸氢二钾(K2HPO4)双水相体系萃取L-精氨酸。实验考察了乙醇浓度、K2HPO4浓度、pH、萃取温度对萃取分离L-精氨酸的影响。结果表明,L-精氨酸在该双水相体系的分配系数K随体系乙醇浓度、K2HPO4浓度的增大、萃取温度的升高而增大,随着体系pH的增大而减小;L-精氨酸在该双水相体系的萃取率随体系乙醇浓度和pH的增大而减小,随着体系K2HPO4浓度增大、萃取温度的升高而增大。     

Highly pure 1,3‐propanediol: Separation and purification from crude glycerol‐based fermentation

A new separation and purification process was developed for recovering 1,3‐propanediol (1,3‐PD) from crude glycerol‐based fermentation broth with high purity. The downstream process integrated chitosan flocculation, activated carbon decolorization, fixed bed cation exchange resin adsorption, and vacuum distillation. Breakthrough curves were measured considering the effect of sample concentration, flow rate, temperature, and resin stack height. Yoon–Nelson model was proposed to fit the fixed bed adsorption. The characteristic column parameters were calculated. Optimal condition for adsorption was 1,3‐PD, 30.0 g/L; flow rate, 1.00 mL/min; stacking height, 30.0 cm; and temperature, 298 K. Ethanol‐water (75%, 1 mL/min) was used as eluent to separate 1,3‐PD and glycerol with 95.3% 1,3‐PD elution rate. After vacuum distillation, the overall purity and yield of 1,3‐PD were 99.2% and 80.8% in the purification process, respectively. This is a simple and efficient downstream strategy for 1,3‐PD purification.     

Enhanced hydrogen and 1,3‐propanediol production from glycerol by fermentation using mixed cultures

The conversion of glycerol into high value products, such as hydrogen gas and 1,3‐propanediol (PD), was examined using anaerobic fermentation with heat‐treated mixed cultures. Glycerol fermentation produced 0.28 mol‐H₂/mol‐glycerol (72 mL‐H₂/g‐COD) and 0.69 mol‐PD/mol‐glycerol. Glucose fermentation using the same mixed cultures produced more hydrogen gas (1.06 mol‐H₂/mol‐glucose) but no PD. Changing the source of inoculum affected gas production likely due to prior acclimation of bacteria to this type of substrate. Fermentation of the glycerol produced from biodiesel fuel production (70% glycerol content) produced 0.31 mol‐H₂/mol‐glycerol (43 mL H₂/g‐COD) and 0.59 mol‐PD/mol‐glycerol. These are the highest yields yet reported for both hydrogen and 1,3‐propanediol production from pure glycerol and the glycerol byproduct from biodiesel fuel production by fermentation using mixed cultures. These results demonstrate that production of biodiesel can be combined with production of hydrogen and 1,3‐propanediol for maximum utilization of resources and minimization of waste. Biotechnol. Bioeng. 2009; 104: 1098–1106. © 2009 Wiley Periodicals, Inc.     

Simultaneous solid–liquid separation and primary purification of clavulanic acid from fermentation broth of Streptomyces clavuligerus using salting‐out extraction system

Clavulanic acid (CA) is usually used together with other β‐lactam antibiotics as combination drugs to inhibit bacterial β‐lactamases, which is mainly produced from the fermentation of microorganism such as Streptomyces clavuligerus. Recently, it is still a challenge for downstream processing of low concentration and unstable CA from fermentation broth with high solid content, high viscosity, and small cell size. In this study, an integrated process was developed for simultaneous solid–liquid separation and primary purification of CA from real fermentation broth of S. clavuligerus using salting‐out extraction system (SOES). First, different SOESs were investigated, and a suitable SOES composed of ethanol/phosphate was chosen and further optimized using the pretreated fermentation broth. Then, the optimal system composed of 20% ethanol/15% K₂HPO₄ and 10% KH₂PO₄ w/w was used to direct separation of CA from untreated fermentation broth. The result showed that the partition coefficient (K) and recovery yield (Y) of CA from untreated fermentation broth were 29.13 and 96.8%, respectively. Simultaneously, the removal rates of the cells and proteins were 99.8% and 63.3%, respectively. Compared with the traditional method of membrane filtration or liquid–liquid extraction system, this developed SOES showed the advantages of simple operation, shorter operation time, lower process cost and higher recovery yield of CA. These results demonstrated that the developed SOES could be used as an attractive alternative for the downstream processing of CA from real fermentation broth.     

Aqueous two-phase extraction of 2,3-butanediol from fermentation broths using an ethanol/ammonium sulfate system

Separation of 2,3-butanediol from the fermentation broth is a difficult task that has become a bottleneck in industrial production. Aqueous two-phase systems composed of hydrophilic solvents and inorganic salts could be used to extract 2,3-butanediol from fermentation broth. The ethanol/ammonium sulfate system was investigated in detail, including phase diagram, effect of phase composition on partition, removal of cells and biomacromolecules from the broths and recycling of ammonium sulfate. The highest partition coefficient (7.10) and recovery of 2,3-butanediol (91.7%) were obtained by a system composed of 32% (w/w) ethanol and 16% (w/w) ammonium sulfate. The maximum selective coefficient of 2,3-butanediol to glucose was 30.74 in the experimental range. In addition, cells and proteins could be simultaneously removed from the fermentation broth. The removal ratio of cells and proteins reached 99.7% and 91.2%, respectively. The recovery of ammonium sulfate in the bottom phase reached 97.14% when two volumes of methanol were added to the salt-rich phase.     

Metabolism in 1,3‐propanediol fed‐batch fermentation by a D‐lactate deficient mutant of Klebsiella pneumoniae

Klebsiella pneumoniae HR526, a new isolated 1,3‐propanediol (1,3‐PD) producer, exhibited great productivity. However, the accumulation of lactate in the late‐exponential phase remained an obstacle of 1,3‐PD industrial scale production. Hereby, mutants lacking D ‐lactate pathway were constructed by knocking out the ldhA gene encoding fermentative D ‐lactate dehydrogenase (LDH) of HR526. The mutant K. pneumoniae LDH526 with the lowest LDH activity was studied in aerobic fed‐batch fermentation. In experiments using pure glycerol as feedstock, the 1,3‐PD concentrations, conversion, and productivity increased from 95.39 g L^?1, 0.48 and 1.98 g L^?1 h^?1 to 102. 06 g L^?1, 0.52 mol mol^?1 and 2.13 g L^?1 h^?1, respectively. The diol (1,3‐PD and 2,3‐butanediol) conversion increased from 0.55 mol mol^?1 to a maximum of 0.65 mol mol^?1. Lactate would not accumulate until 1,3‐PD exceeded 84 g L^?1, and the final lactate concentration decreased dramatically from more than 40 g L^?1 to <3 g L^?1. Enzymic measurements showed LDH activity decreased by 89–98% during fed‐batch fermentation, and other related enzyme activities were not affected. NADH/NAD⁺ enhanced more than 50% in the late‐exponential phase as the D ‐lactate pathway was cut off, which might be the main reason for the change of final metabolites concentrations. The ability to utilize crude glycerol from biodiesel process and great genetic stability demonstrated that K. pnemoniae LDH526 was valuable for 1,3‐PD industrial production. Biotechnol. Bioeng. 2009; 104: 965–972. © 2009 Wiley Periodicals, Inc.     

Potential application of aqueous two‐phase systems and three‐phase partitioning for the recovery of superoxide dismutase from a clarified homogenate of Kluyveromyces marxianus

Superoxide dismutase (SOD; EC 1.15.1.1) is an antioxidant enzyme that represents the primary cellular defense against superoxide radicals and has interesting applications in the medical and cosmetic industries. In the present work, the partition behavior of SOD in aqueous two‐phase systems (ATPS) (using a standard solution and a complex extract from Kluyveromyces marxianus as sample) was characterized on different types of ATPS (polymer–polymer, polymer–salt, alcohol–salt, and ionic liquid (IL)–salt). The systems composed of PEG 3350‐potassium phosphate, 45% TLL, 0.5 M NaCl (315 U/mg, 87% recovery, and 15.1‐fold purification) and t‐butanol‐20% ammonium sulfate (205.8 U/mg, 80% recovery and 9.8‐fold purification), coupled with a subsequent 100 kDa ultrafiltration stage, allowed the design of a prototype process for the recovery and partial purification of the product of interest. The findings reported herein demonstrate the potential of PEG‐salt ATPS for the potential recovery of SOD. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1326–1334, 2014     

Separation and determination of tetracycline hydrochloride in real water samples using binary small molecule alcohol‐salt aqueous two‐phase system coupled with high‐performance liquid chromatography

An environmental and sensitive sample pretreatment method was established and combined with high‐performance liquid chromatography (HPLC) for the analysis of tetracycline hydrochloride (TC) in feed water and lake water. One element small molecule alcohol‐salt aqueous two‐phase system (ATPS) cannot effectively adjust the polarity of the system, but binary small molecule alcohol‐salt ATPS can adjust the polarity and improve the extraction efficiency of antibiotics. In this work, a binary ATPS based on ethanol +2‐propanol + (NH₄)₂SO₄ system was formed and applied to the separation and purification of TC in real water samples. The influence factors on partition behaviors of TC were discussed, including the types and the concentration of phase salts, the volume ratio of alcohol, the pH value, extraction temperature, and the standing time. The response surface methodology was used to determine the best experimental conditions for multi‐factor experiments. Under this optimal condition, the extraction efficiency of TC reached 95.7%. This new method is considered to have significant application in the divorce of antibiotics.     

Separation of bio‐based chemicals from fermentation broths by salting‐out extraction

The possibility of creating a biorefinery using inexpensive biomass has attracted a great deal of attention, which is mainly focused on the improvement of strains and fermentation, whereas few resources have been spent on downstream processing. Bio‐based chemical downstream processing can become a bottleneck in industrial production because so many impurities are introduced into the fermentation broth. This review introduces a technique referred to as salting‐out extraction, which is based on the partition difference between chemicals in two phases consisting of salts and polymers or hydrophilic solvents, hydrophobic solvents, and amphipathic chemicals. The effects of solvents and salts on the formation of two phases were discussed, as was the use of this method to recover bio‐based chemicals. This review focused on the separation of hydrophilic chemicals (1,3‐propanediol, 2,3‐butanediol, acetoin, and lactic acid) from fermentation broths. Diols could be recovered at a high yield from fermentation broths without pretreatment especially with a hydrophilic solvent‐based system, whereas the recovery of organic acids was slightly lower. Most of the impurities (cells and proteins) were removed during the same step. Extractive fermentations were also used for polymer‐based aqueous two‐phase systems.     

A novel downstream process for highly pure 1,3‐propanediol from an efficient fed‐batch fermentation of raw glycerol by Clostridium pasteurianum

An efficient downstream process without prior desalination was developed for recovering 1,3‐propanediol (1,3‐PDO) with high purity and yield from broth of a highly productive fed‐batch fermentation of raw glycerol by Clostridium pasteurianum. After removal of biomass and proteins by ultrafiltration, and concentration by water evaporation, 1,3‐PDO was directly recovered from the broth by vacuum distillation with continuous addition and regeneration of glycerol as a supporting agent. Inorganic salts in the fermentation broth were crystallized but well suspended by a continuous flow of glycerol during the distillation process, which prevented salt precipitation and decline of heat transfer. On the other hand, ammonium salt of organic acids were liberated as ammonia gas and free organic acids under vacuum heating. The latter ones formed four types of 1,3‐PDO esters of acetic acid and butyric acid, which resulted in yield losses and low purity of 1,3‐PDO (< 80%). In order to improve the efficiency of final 1,3‐PDO rectification, we examined alkaline hydrolysis to eliminate the ester impurities. By the use of 20% (w/w) water and 2% (w/w) sodium hydroxide, > 99% reduction of 1,3‐PDO esters was achieved. This step conveniently provided free 1,3‐PDO and the sodium salt of organic acids from the corresponding esters, which increased the 1,3‐PDO yield by 7% and prevented a renewed formation of esters. After a single stage distillation from the hydrolyzed broth and a followed active carbon treatment, 1,3‐PDO with a purity of 99.63% and an overall recovery yield of 76% was obtained. No wastewater with high‐salt content was produced during the whole downstream process. The results demonstrated that the monitoring and complete elimination of 1,3‐PDO esters are crucial for the efficient separation of highly pure 1,3‐PDO with acceptable yield from fermentation broth of raw glycerol.     

AQUEOUS TWO-PHASE EXTRACTION FOR THE PURIFICATION OF ALKALINE AGARASES FROM CULTURE EXTRACTS OF Pseudomonas aeruginosa AG LSL-11

The agarases were purified for the first time an using aqueous two-phase system (ATPS) consisting of polyethylene glycol (PEG) and phosphate salt. The three extracellular, alkaline agarases produced by Pseudomonas aeruginosa AG LSL-11 were efficiently extracted into the top PEG-rich layer. The influencing factors on the partition of agarases—molecular weight of the PEG, system pH, system temperature, and NaCl concentration—were investigated. All the factors were found to have a significant effect on the partition of agarases except NaCl. The optimal ATPS parameters for the partitioning and purification of agarases were found to be 12% PEG 600 and 11.9% (w/w) phosphate salt at pH 8.0 and 4°C. All three agarases were concentrated in the top PEG phase with 6.19-fold purity and 71.21% recovery. The ATPS was found to be more convenient and economical than the conventional ion-exchange chromatography (IEC) method for extraction of three agarases and could be significantly employed for the purification of agarases from fermentation broth.     

Metabolic engineering of Clostridium acetobutylicum for the enhanced production of isopropanol‐butanol‐ethanol fuel mixture

Butanol is considered as a superior biofuel, which is conventionally produced by clostridial acetone‐butanol‐ethanol (ABE) fermentation. Among ABE, only butanol and ethanol can be used as fuel alternatives. Coproduction of acetone thus causes lower yield of fuel alcohols. Thus, this study aimed at developing an improved Clostridium acetobutylicum strain possessing enhanced fuel alcohol production capability. For this, we previously developed a hyper ABE producing BKM19 strain was further engineered to convert acetone into isopropanol. The BKM19 strain was transformed with the plasmid pIPA100 containing the sadh (primary/secondary alcohol dehydrogenase) and hydG (putative electron transfer protein) genes from the Clostridium beijerinckii NRRL B593 cloned under the control of the thiolase promoter. The resulting BKM19 (pIPA100) strain produced 27.9 g/l isopropanol‐butanol‐ethanol (IBE) as a fuel alcohols with negligible amount of acetone (0.4 g/l) from 97.8 g/l glucose in lab‐scale (2 l) batch fermentation. Thus, this metabolically engineered strain was able to produce 99% of total solvent produced as fuel alcohols. The scalability and stability of BKM19 (pIPA100) were evaluated at 200 l pilot‐scale fermentation, which showed that the fuel alcohol yield could be improved to 0.37 g/g as compared to 0.29 g/g obtained at lab‐scale fermentation, while attaining a similar titer. To the best of our knowledge, this is the highest titer of IBE achieved and the first report on the large scale fermentation of C. acetobutylicum for IBE production. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1083–1088, 2013     

Aqueous two-phase extraction for the purification of alkaline agarases from culture extracts of Pseudomonas aeruginosa AG LSL-11

The agarases were purified for the first time an using aqueous two-phase system (ATPS) consisting of polyethylene glycol (PEG) and phosphate salt. The three extracellular, alkaline agarases produced by Pseudomonas aeruginosa AG LSL-11 were efficiently extracted into the top PEG-rich layer. The influencing factors on the partition of agarases--molecular weight of the PEG, system pH, system temperature, and NaCl concentration--were investigated. All the factors were found to have a significant effect on the partition of agarases except NaCl. The optimal ATPS parameters for the partitioning and purification of agarases were found to be 12% PEG 600 and 11.9% (w/w) phosphate salt at pH 8.0 and 4°C. All three agarases were concentrated in the top PEG phase with 6.19-fold purity and 71.21% recovery. The ATPS was found to be more convenient and economical than the conventional ion-exchange chromatography (IEC) method for extraction of three agarases and could be significantly employed for the purification of agarases from fermentation broth.     

Extractive disruption process integration using ultrasonication and an aqueous two‐phase system for protein recovery from Chlorella sorokiniana

Microalgae emerge as the most promising protein sources for aquaculture industry. However, the commercial proteins production at low cost remains a challenge. The process of harnessing microalgal proteins involves several steps such as cell disruption, isolation and extraction. The discrete processes are generally complicated, time‐consuming and costly. To date, the notion of integrating microalgal cell disruption and proteins recovery process into one step is yet to explore. Hence, this study aimed to investigate the feasibility of applying methanol/potassium ATPS in the integrated process for proteins recovery from Chlorella sorokiniana. Parameters such as salt types, salt concentrations, methanol concentrations, NaCl addition were optimized. The possibility of upscaling and the effectiveness of recycling the phase components were also studied. The results showed that ATPS formed by 30% (w/w) K₃PO₄ and 20% (w/w) methanol with 3% (w/w) NaCl addition was optimum for proteins recovery. In this system, the partition coefficient and yield were 7.28 and 84.23%, respectively. There were no significant differences in the partition coefficient and yield when the integrated process was upscaled to 100‐fold. The recovered phase components can still be recycled effectively at fifth cycle. In conclusions, this method is simple, rapid, environmental friendly and could be implemented at large scale.     

Simultaneous clostridial fermentation,lipase‐catalyzed esterification,and ester extraction to enrich diesel with butyl butyrate

The recovery of 1‐butanol from fermentation broth is energy‐intensive since typical concentrations in fermentation broth are below 20 g L^?1. To prevent butanol inhibition and high downstream processing costs, we aimed at producing butyl esters instead of 1‐butanol. It is shown that it is possible to perform simultaneously clostridial fermentation, esterification of the formed butanol to butyl butyrate, and extraction of this ester by hexadecane. The very high partition coefficient of butyl butyrate pulls the esterification towards the product side even at fermentation pH and relatively low butanol concentrations. The hexadecane extractant is a model diesel compound and is nontoxic to the cells. If butyl butyrate enriched diesel can directly be used as car fuel, no product recovery is required. A proof‐of‐principle experiment for the one‐pot bio‐ester production from glucose led to 5 g L^?1 butyl butyrate in the hexadecane phase. The principle may be extended to a wide range of esters, especially to longer chain ones. Biotechnol. Bioeng. 2013; 110: 137–142. © 2012 Wiley Periodicals, Inc.     

Salting-out extraction and crystallization of succinic acid from fermentation broths

In this study, salting-out extraction (SOE) and crystallization were combined to recover succinic acid from fermentation broths. Of the different SOE systems investigated, the system consisting of organic solvents and acidic salts appeared to be more favorable. A system using acetone and ammonium sulfate was investigated to determine the effect of phase composition and pH. The highest partition coefficient (8.64) and yield of succinic acid (90.05%) were obtained by a system composed of 30% (w/w) acetone and 20% (w/w) ammonium sulfate at a pH of 3.0. Additionally, 99.03% of cells, 90.82% of soluble proteins, and 94.89% of glucose could be simultaneously removed from the fermentation broths. Interestingly, nearly 40% of the pigment was removed using the single-step salting-out extraction process. The analysis of the effect of pH on salting-out extraction indicates that a pH lower than the pK of succinic acid is beneficial for the recovery of succinic acid in an SOE system. Crystallization was performed for the purification of succinic acid at 4 °C and pH 2.0. By combining salting-out extraction with crystallization, an identical total yield (65%) and a higher purity (97%) of succinic acid were obtained using a synthetic fermentation broth compared with the actual fermentation broth (65% and 91%, respectively).     

Extraction of natural red colorants from the fermented broth of Penicillium purpurogenum using aqueous two‐phase polymer systems

Safety concerns related to the increasing and widespread application of synthetic coloring agents have increased the demand for natural colorants. Fungi have been employed in the production of novel and safer colorants. In order to obtain the colorants from fermented broth, suitable extraction systems must be developed. Aqueous two‐phase polymer systems (ATPPS) offer a favorable chemical environment and provide a promising alternative for extracting and solubilizing these molecules. The aim of this study was to investigate the partitioning of red colorants from the fermented broth of Penicillium purpurogenum using an ATPPS composed of poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA). Red colorants partitioned preferentially to the top (PEG‐rich phase). In systems composed of PEG 6,000 g/mol/NaPA 8,000 g/mol, optimum colorant partition coefficient (K_C) was obtained in the presence of NaCl 0.1 M (K_C = 10.30) while the PEG 10,000 g/mol/NaPA 8,000 g/mol system in the presence of Na₂SO₄ 0.5 M showed the highest K_C (14.78). For both polymers, the mass balance (%MB) and yield in the PEG phase (%η_TOP) were close to 100 and 79%, respectively. The protein selectivity in all conditions evaluated ranged from 2.0–3.0, which shows a suitable separation of the red colorants and proteins present in the fermented broth. The results suggest that the partitioning of the red colorants is dependent on both the PEG molecular size and salt type. Furthermore, the results obtained support the potential application of ATPPS as the first step of a purification process to recover colorants from fermented broth of microorganisms. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1295–1304, 2015