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).     

反相高效液相色谱法测定肠球菌FQ15发酵液中的有机酸

目的研究肠球菌FQ15发酵液中的有机酸在不同发酵时间的变化趋势。方法建立反相高效液相色谱法测定此株益生菌发酵液中有机酸的主要成分及其在发酵不同时间的变化趋势。结果丙酸、丙酮酸、乳酸、乙酸的浓度都是在菌体生长处于衰亡期时达到最大值,乳酸随发酵时间的延长浓度明显下降,而乙酸在发酵后期含量呈上升趋势。结论此方法重现性好,精密度高,为研究微生物合成中有机酸种类及变化趋势提供了一种可供参考的快捷分析手段。     

Recovery of succinic acid produced by fermentation of a metabolically engineered Mannheimia succiniciproducens strain

There have recently been much advances in the production of succinic acid, an important four-carbon dicarboxylic acid for many industrial applications, by fermentation of several natural and engineered bacterial strains. Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is able to produce succinic acid with high efficiency, but also produces acetic, formic and lactic acids just like other anaerobic succinic acid producers. We recently reported the development of an engineered M. succiniciproducens LPK7 strain which produces succinic acid as a major fermentation product while producing much reduced by-products. Having an improved succinic acid producer developed, it is equally important to develop a cost-effective downstream process for the recovery of succinic acid. In this paper, we report the development of a simpler and more efficient method for the recovery of succinic acid. For the recovery of succinic acid from the fermentation broth of LPK7 strain, a simple process composed of a single reactive extraction, vacuum distillation, and crystallization yielded highly purified succinic acid (greater than 99.5% purity, wt%) with a high yield of 67.05wt%. When the same recovery process or even multiple reactive extraction steps were applied to the fermentation broth of MBEL55E, lower purity and yield of succinic acid were obtained. These results suggest that succinic acid can be purified in a cost-effective manner by using the fermentation broth of engineered LPK7 strain, showing the importance of integrating the strain development, fermentation and downstream process for optimizing the whole processes for succinic acid production.     

盐析萃取生物基化学品的研究进展

廉价生物质的生物炼制研究主要集中在菌种和发酵方面,对下游分离研究较少。廉价生物质资源的利用导致发酵液中引入更多杂质,成分较单糖发酵更复杂,致使生物基化学品的下游分离过程成为其工业化生产亟需解决的关键问题。文中介绍了一种基于两相分配差异分离亲水性生物基化学品的盐析萃取技术及其在生物基化学品分离方面的应用,重点阐述了短链醇和盐对双水相形成的影响,并对1,3-丙二醇、2,3-丁二醇、乙偶姻、乳酸等的盐析萃取研究进展进行了总结和展望。盐析萃取技术可有效地回收发酵液中的小分子亲水性产品,同时除去大多数的杂质 (细胞和蛋白质等),在生物基化学品的分离过程中将是一种有前景的分离技术。     

Application of bipolar electrodialysis on recovery of free lactic acid after simultaneous saccharification and fermentation of cassava starch

The efficiency of bipolar electrodialysis (BED) for the recovery of lactic acid from fermentation broth was evaluated. Three systems of BED (bipolar-anion, bipolar-cation and bipolar-anion-cation) at fixed voltage (20 V) were compared using a model solution of ammonium lactate (100 g l(-1)). Results showed that bipolar-anion (BED-anion) was the most beneficial in terms of lactate flux, current efficiency, energy consumption and recovery ratio. Consequently, BED-anion was used to purify lactic acid from fermentation broth which had been pre-treated with mono-polar electrodialysis (MED). The final lactic acid concentration and lactate flux obtained were 144 g l(-1) and 393 g m(-2) h(-1), respectively. Using the two-step process (MED and BED-anion) the concentration of fermentation broth was increased by 33% and the total energy consumption was 2.76 kW h kg(-1).     

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.     

Lactate production in an integrated process configuration: reducing cell adsorption by shielding of adsorbent

The problem of binding of microbial cells to an adsorbent matrix during in situ recovery of bioproducts from a fermentation broth has been addressed by shielding the adsorbent with a thin layer of a non-ionic polymer. Extractive bioconversion of lactic acid by integrating ion-exchange adsorption with the fermentation stage was studied. The effect of coating of the ion-exchanger with agarose on product recovery and cell adsorption was evaluated. Extractive fermentation with both uncoated and coated resin resulted in an increase in reactor productivity as compared to the normal fermentation. The free cell density in the system with agarose-coated beads was similar to that in control fermentation, but was significantly lower in the system with the uncoated ion-exchanger. Electron microscopic scanning of the bead surface after passage of the fermentation broth showed cells attached to the native adsorbent but not to the coated one.     

Recovery of lactic acid from fermentation broth by the two-stage process of nanofiltration and water-splitting electrodialysis

A two-stage process of nanofiltration and water-splitting electrodialysis was investigated for lactic acid recovery from fermentation broth. In this process, sodium lactate is isolated from fermentation broth in the first stage of nanofiltration by using an NTR-729HF membrane, and then is converted to lactic acid in the second stage by water-splitting electrodialysis. To determine the optimal operating conditions for nanofiltration, the effects of pressure, lactate concentration, pH and known added impurities were studied. Lactate rejection was less than 5%, magnesium rejection approximated 45%, and calcium rejection was at 40%. In subsequent water-splitting electrodialysis, both the sodium lactate conversion to lactic acid and sodium hydroxide recovery, were about 95%, with a power requirement of 0.9∼1.0 kWh per kg of lactate.     

Effect of operating variables on back-extraction characteristics of succinic acid from organic phase

Tri-n-octylamine (TOA) is an effective extractant for the recovery of succinic acid from fermentation broth. The recovery of succinic acid from organic phase depends on the operating variables such as temperature, pH, volume of aqueous phase, and use of displacer. In thermal recovery of succinic acid, 34% of succinic acid was recovered at 90°C. More than 90% of succinic acid was back-extracted by pH-swing. Efficiency of back-extraction was increased 23% as increasing volume of aqueous phase. Use of oleic acid as a displacer increased efficiency of back-extraction to 76%. Aqueous phase volume and concentration of oleic acid were controlled simultaneously to increase the efficiency of back-extraction and percent recovery of succinic acid reached to 90.     

In situ separation of lactic acid from fermentation broth using ion exchange resins

Lactic acid fermentation is an end product inhibited reaction. In situ separation of lactic acid from fermentation broth using ion exchange resins was investigated and compared with conventional fermentation system. Amberlite resin (IRA-400, Cl⁻) was used to separate lactic acid from fermentation broth and pH was controlled online with an automatic pH controller. The effect of process variables on lactic acid production by Lactobacillus casei in whey permeate was studied. The maximum productivity was obtained at pH = 6.1, T = 37 °C and impeller speed = 200 rpm. The maximum concentration of lactic acid at optimum condition was found to be 37.4 g/L after 38 h of fermentation using in situ separation system. The productivity of in situ separation system was five times increased in comparison with conventional system.     

New reactive extraction systems for separation of bio-succinic acid

Biotechnologically produced succinic acid has the potential to displace maleic acid and its uses and to become an important feedstock for the chemical industry. In addition to optimized production strains and fermentation processes, an efficient separation of succinic acid from the aqueous fermentation broth is indispensable to compete with the current petrochemical production processes. In this context, high molecular weight amines are known to be effective extractants for organic acids. For this reason, as a first step of isolation and purification, the reactive extraction of succinic acid was studied by mixing aqueous succinic acid solutions with 448 different amine–solvent mixtures as extraction agents (mixer-settler studies). The extraction agents consist either of one amine and one solvent (208 reactive extraction systems) or two amines and two solvents (240 reactive extraction systems). Maximum extraction yields of succinic acid from an aqueous solution with 423 mM succinic acid at pH 2.0 were obtained with more than 95% yield with trihexylamine solved in 1-octanol or with dihexylamine and diisooctylamine solved in 1-octanol and 1-hexanol. Applying these optimized reactive extraction systems with Escherichia coli fermentation broth resulted in extraction yields of 78–85% due to the increased ionic strength of the fermentation supernatant and the co-extraction of other organic acids (e.g., lactic acid and acetic acid), which represent typical fermentation byproducts.     

Downstream separation and purification of bio-based alpha-ketoglutaric acid from post-fermentation broth using a multi-stage membrane process

In this study, a multi-stage membrane process, assisted by vacuum evaporation and crystallization, for recovery of bio-based alpha-ketoglutaric acid from the actual post-fermentation broth was designed and investigated. In the first part of this study, pre-treatment of crude fermentation broth (centrifugation-ultrafiltration-nanofiltration) was carried out to remove biomass, proteins, sugars, part of inorganic ions and color compounds. The commercial ceramic UF membrane (15 kDa) and nanofiltration ceramic membrane (200 Da or 450 Da) were applied. Electrodialysis with a bipolar membrane was proposed for separation of ionic compounds and simultaneous electro-acidification to the acid form. During bipolar membrane electrodialysis carried out under acidic conditions, it was possible to remove close to 90 % of alpha-ketoglutaric acid. Moreover, the migration of other acids present in the fermentation broth (lactic and acetic) was significantly limited. The calculated specific energy consumption was low and equal to 0.6 kW h/kg. The final purification using crystallization assisted vacuum evaporation allowed obtaining alpha-ketoglutaric acid in solid form. Analysis of the final product showed that the proposed method of alpha-ketoglutaric acid recovery gives the acid of high purity equal to 94.8 %. Furthermore, the presented results have practical relevance and may in future be the basis for the development of separation technologies of alpha-ketoglutaric acid from the fermentation broth on industrial scale.     

Extraction of penicillin-G by aqueous two-phase partition

Summary Pure potassium penicillin-G solution and penicillin-G fermentation broth were extracted using the aqueous two-phase systems of poly-ethylene glycol and salts. The partition coefficients of penicillin-G were above 10 in both pure solution and broth systems. The partition coefficients of phenyl acetic acid were about 0.25. Cell mass and solid residue in the broth system were partly concentrated around the interfacial region and partly settled to the bottom when under 1000 × g centrifugal force. Accordingly, this technique is a promising alternative for the recovery of penicillins.     

Emulsion liquid membrane extraction of lactic acid from aqueous solutions and fermentation broth

Studies on the batch extraction of lactic acid using an emulsion liquid membrane system are reported. The membrane phase consists of the tertiary amine carrier Alamine 336 and the surfactant Span 80 dissolved in n-heptane/paraffin and aqueous solutions of sodium carbonate in the internal phase. The effects of internal phase reagent, extraction temperature, and initial external phase pH on the extraction efficiency and the emulsion swelling are examined. A statistical factorial experiment on extraction from clarified lactic acid fermentation broth was carried out to obtain knowledge of the performance of the extraction system from a broth. The extraction efficiency from the fermentation broth is found to be lower as compared to aqueous solutions of pure lactic acid. The effect of pH and the presence of other ionic species on selectivity are discussed. (c) 1993 John Wiley & Sons, Inc.     

Selection and optimization of an aqueous two‐phase system for the recovery of 1,3‐propandiol from fermentation broth

In this study a suitable alcohol/salt aqueous two‐phase (ATP) system was selected for the recovery of 1,3‐propandiol (1,3‐PD) from fermentation broth. From the different alcohol/salt systems studied the ethanol and dipotassium hydrogen phosphate ATP system appeared to be favorable. To examine the potential of this ATP system the partition coefficient of 1,3‐PD in synthetic solutions was first optimized with the response surface methodology. The parameters studied were concentrations of ethanol (21.99–38.81% w/w), dipotassium hydrogen phosphate (14.99–31.81% w/w) and 1,3‐PD (6.36–73.64 g/L). The optimum conditions were found to be 35.39% w/w for ethanol, 28.40% w/w for dipotassium hydrogen phosphate and 73.6 g/L for 1,3‐PD. Under these conditions the maximum partition coefficient of 1,3‐PD and the extraction yield were determined as 23.14 and 97.82%, respectively. The optimum extraction conditions were then used to guide the recovery of 1,3‐PD from a real fermentation broth. The partition coefficient and extraction yield of 1,3‐PD reached 20.28–97.20% in this case, respectively. A favorable partition of the organic acids lactate, acetate and butyrate in the bottom phase was also achieved. We have also studied the removal of cells and macromolecules from the broth. Removal ratio of cells and proteins were 96.47 and 93.05%, respectively. Thus, the ethanol/dipotassium hydrogen phosphate ATP system appears to be an interesting alternative or can be used as one useful step in the downstream processing of 1,3‐PD from fermentation broth.     

Recovery of potassium clavulanate from fermentation broth by ion exchange chromatography and desalting electrodialysis

Ion exchange chromatography (IEC) and desalting electrodialysis (DSED) processes were developed for the recovery and purification of potassium clavulanate (KCA) from fermentation broth. A strong anion exchanger, Amberlite IRA 400 resin, a potassium acetate solution as equilibrium buffer, and a potassium chloride (KCl) solution as elution buffer were used for the recovery of KCA in IEC. In order to determine optimal operating conditions, the effects of various operating parameters such as equilibrium buffer pH and concentration, elution buffer concentration, gradient length, and volumetric flow rate on KCA recovery and by-product removal were investigated using a simulated fermentation broth. In the subsequent step of DSED, employing cation (Neocepta CMS, Tokuyama, Japan) and anion (Neocepta ACS, Tokuyama, Japan) exchange membranes were carried out to remove KCl that existed in a large amount in the ion exchanged solution. The effects of operation voltage and feed composition on the performance of DSED were investigated. Based on the operating conditions determined above, IEC and DSED were applied in sequence to an ultrafiltered fermentation broth. Almost complete removal of KCl was possible with no significant loss of KCA, although the KCA recovery was slightly lower than that with the simulated fermentation broth. Based on this observation, it was concluded that IEC and DESD could be an effective process combination for the recovery of KCA from fermentation broth.     

Integrated separation process for isolation and purification of biosuccinic acid

Biotechnologically produced succinic acid has the potential to displace maleic acid and its uses. Therefore, it is of high interest for the chemical, pharmaceutical, and food industry.In addition to optimized production strains and fermentation processes, an efficient separation of succinic acid from the aqueous fermentation broth is indispensable to compete with the current petrochemical production of succinic acid. Isolation and purification of succinic acid from an Escherichia coli fermentation broth were studied with two amine-based reactive extraction systems: (i) trihexylamine in 1-octanol and (ii) diisooctylamine and dihexylamine in a mixture of 1-octanol and 1-hexanol. Back extraction of succinic acid from the organic phase was carried out using an aqueous trimethylamine solution. The trimethylammonium succinate generated after back extraction was split with an evaporation-based crystallization.The focus was on process integration, for example, reuse of the applied amines for extraction and back extraction. It was shown that the maximum trimethylamine concentration for back extraction should not exceed the stoichiometric amount (2 mol trimethylamine/mol the succinic acid in the organic phase) to ensure maximal extraction yields with the reused organic phase in subsequent extractions. Moreover, mixer-settler extraction and back extraction of succinic acid were scaled up from the milliliter- to the liter-scale making use of liquid–liquid centrifuges. The overall yield was 83.5% of the succinic acid from thefermentation supernatant. The final purity of the succinic acid crystals was 99.5%. Organic phase and amines can easily be recycled and reused.     

高效液相色谱法测定必特螺旋霉素发酵液中有机酸

必特螺旋霉素是运用基因工程技术获得的工程茵产生的一组以异戊酰螺旋霉素为主要成分的多组分新型抗生素,其前体为乙酸、丙酸、丁酸和异戊酸等有机酸。本研究利用高效液相色谱法以0．01mol／L磷酸缓冲液（pH2．3）和甲醇为流动相,分别在反相C8（α-酮戊二酸、乙酸、柠檬酸、琥珀酸、丙酸）和C18（丁酸、异戊酸）柱上定量测定了必特螺旋霉素前体酸和三羧酸循环相关有机酸。所建立的测定方法的相对标准偏差为0．10％～0．42％,回收率为93．19％～102．08％。     

Kinetic study of electrodialysis of acetic acid and phosphate in fermentation broth

Ion fluxes of acetic acid and phosphate in the electrodialysis of a model fermentation broth were examined using a solution of acetic acid and potassium dihydrogenphosphate. As the molar fraction of the phosphate was increased the ion flux of the acetic acid decreased, which was as predicted theoretically. As the influent pH was increased the flux also increased notably, reached a maximum, and then decreased slightly. The maximum flux was obtained in a pH range from 5.2 (near pK_a) to 6.6. The pH at the exit of the electrodialyzer became markedly alkaline when the influent pH was in the range of 6 to 7 in the absence of the phosphate. The pH range shifted to the alkaline side and the degree of alkalinity reduced as the molar fraction of phosphate increased. This may be ascribed to the buffering effect of the phosphate. It is concluded that the optimum pH for the electrodialysis of an organic acid fermentation broth is around (pKa + 1), where a relatively high ion flux with high current efficiency and a small pH increase at the exit of the electrodialyzer are established.     

植物乳杆菌DY6主要抑菌代谢物的分析和鉴定

【背景】被广泛应用于食品和饲料等多个行业的乳酸菌已成为制作生物防腐剂的研究热点。【目的】探究抑菌性能良好的植物乳杆菌DY6的抑菌物质,为其进一步应用提供参考依据。【方法】对植物乳杆菌发酵液中抑菌物质的理化特性进行研究,采用GC-MS分析发酵上清液代谢物,通过多元统计学分析方法推测主要抑菌物质,抑菌物质通过半制备进行初步分离后用GC-MS鉴定。【结果】植物乳杆菌DY6对金黄色葡萄球菌、大肠杆菌、沙门氏菌都有较强的抑制作用。采用不同发酵时间的发酵液作为研究对象,测定发酵上清液的抑菌能力,发酵0-4 h上清液无抑菌能力,发酵至8 h抑菌能力逐步上升,发酵24-48 h发酵上清液抑菌能力趋于稳定,在48 h时抑菌能力最佳,抑菌直径为15.28mm。通过多元统计学分析乳酸菌发酵液差异标志物,发现主要差异物为有机酸(如乳酸、乙酸、丙酸等)和脂肪酸(如辛酸、癸酸等)。经过半制备液相分离发酵上清液得到的抑菌组分,主要有有机酸(如乳酸、乙酸、3-苯基乳酸、苯丙酸等)和脂肪酸(如癸酸、辛酸、壬酸等),另外还有少量的醛类和醇类物质。【结论】确定了植物乳杆菌DY6的抑菌物质主要为有机酸和脂肪酸,为其进一步防腐应用提供了理论基础。