Effective lactic acid production by two-stage extractive fermentation

For effective microbial lactic acid production using Lactobacillus delbrueckii, two-stage extractive fermentation was examined. Extractants were screened from the viewpoints of a high distribution coefficient for lactic acid and less toxicity toward the microorganism. Even if the extractant showed some toxicity toward the microorganism, it was found that a reduction of toxicity was possible by back-extraction using oleyl alcohol. As a result, 40% Alamine 336 diluted with oleyl alcohol, and oleyl alcohol, were selected as the extractant and the back-extractant, respectively. After two-stage extraction by these extractants, the growth rate was improved by the removal of lactic acid. This method was then applied to continuous extractive fermentation using a jar-fermentor. During 4-h extraction, lactic acid accumulation in the broth was significantly suppressed, while the cell growth and glucose consumption rates were also found not to be reduced. After 24 h, the cell concentration attained an OD₆₆₀ of 14, which corresponded to a level 1.25 times higher than that of the control culture without extraction. Total lactic acid productivity was 1.4 times level compared with the control culture.     

Analysis of phase composition in aqueous two-phase systems using a two-column chromatographic method: Application to lactic acid production by extractive fermentation

A new chromatographic system for the simultaneous analysis of polyethylene glycol, dextran, sugars, and low-molecular-weight fatty acids was developed. The system is based on a gel exclusion column which allows a first separation between high- and low-molecular-weight compounds, and a cationic exchange column used to further separate the low-molecular-weight compounds. Two applications of the system were demonstrated: (i) after optimizing eluent conditions the gel exclusion column was used to determine the influence of lactic acid, phosphate buffer, and lactic acid bacteria on the ethylene oxide propylene oxide-dextran T40 phase diagram by HPLC; (ii) the ion exchange column was coupled in series with the gel exclusion column and the concentration of polyethylene glycol, dextran, glucose, lactate, acetate, and formate was determined in samples from the fermentative production of lactic acid in a polyethylene glycol 8000-dextran T40 aqueous two-phase system. The fermentation was operated without pH control in a repeated extractive batch mode, where the cell-free top phase was replaced four times, whereas the cell-containing bottom phase was reused repeatedly. The yield was 1.1 mol of lactic acid formed per mole of glucose added and the productivity was 4.7 mM.h(-1). The polymeric composition of the fermentation system was monitored during the five repeated extractive batches, and it showed a progressive depletion in polyethylene glycol and a progressive enrichment in dextran. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 303-311, 1997.     

Extractive lactic acid fermentation in poly (ethyleneimine)-based aqueous two-phase system

The potential of an aqueous two-phase system composed of a polycation, poly(ethyleneimine) (PEI), and an uncharged polymer, (hydroxyethyl) cellulose (HEC), for extractive lactic acid fermentation was tested. Batch fermentation with 20 g/L glucose in two-phase medium using Lactococcus lactis without external pH control resulted in 3-4 times higher amount of lactate and biomass produced as compared to that in a conventional one-phase medium. Lactic acid was preferentially partitioned to the PEI-rich bottom phase. However, the cells which favored the HEC-rich top phase in a fresh two-phase medium were partitioned to a significant extent to the bottom phase after fermentation. Addition of phosphate buffer or pH adjustment to 6.5 after fermentation caused fewer cells to move to the bottom phase. With external pH control, fermentation in normal and two-phase medium showed no marked differences in glucose consumption and lactic acid yield, except that about 1.3 times higher cell density was obtained in the two-phase broth, especially at initial glucose concentrations of 50-100 g/L. Use of higher concentration of phosphate during batch fermentation in the two-phase medium with 50 g/L sugar provided a 15% higher yield of lactic acid, but the growth rate of cells was nearly half of the normal, thus affecting the productivity. Continuous fermentation with twice the normal phosphate concentration resulted in higher cell density, product yield, and productivity in two-phase medium than in monophasic medium. (c) 1996 John Wiley & Sons, Inc.     

Technological aspects of extractive fermentation using aqueous two-phase systems

World Journal of Microbiology and Biotechnology -     

Sugar-cane pressmud as a novel and inexpensive substrate for production of lactic acid in a solid-state fermentation system

Lactobacillus casei subsp. casei CFTRI 2022 produced a higher concentration of lactic acid (5.27 g/100 g dry sugar-cane pressmud) in a solid-state fermentation (SSF) system as compared to L. helveticus CFTRI 2026 and Streptococcus thermophilus CFTRI 2034. The lactic acid production by L. casei subsp. casei CFTRI 2022 was found to be significantly influenced by the initial moisture content, initial pH and initial sugar concentration of the medium. Studies on four inert materials to reduce the initial sugar concentration in the medium showed the high potential of microcrystalline cellulose whereas the use of diatomaceous earth, acid-washed river sand and washed pith bagasse posed problems. The data indicate the potential of lactic acid production from sugar-cane pressmud in an SSF system.     

Enhanced sensitivity in PCR detection of Listeria monocytogenes in soft cheese through use of an aqueous two-phase system as a sample preparation method.

A sample treatment method based on an aqueous two-phase system containing polyethylene glycol and dextran was developed for enhancing sensitivity in the detection of Listeria monocytogenes in soft cheese with PCR. The results suggest that the improved detection sensitivity following partitioning of the cheese homogenate in an aqueous two-phase system may be due to partitioning of the PCR inhibitors to the polyethylene glycol phase.     

Aqueous two-phase: the system of choice for extractive fermentation

Extractive fermentation in aqueous two-phase systems is a meaningful approach to overcome low product yield in a conventional fermentation process, and by proper design of the two-phase system it is possible to obtain the product in a cell-free stream. The characteristics of an aqueous two-phase system, various polymers used for forming an aqueous two-phase system, the physicochemical parameters of the aqueous two-phase system, partitioning of biomolecules and cell mass and the effect of individual phase forming polymers on cell growth and product formation are reviewed in this article. The various extractive fermentation processes are also summarised here. At the end, the economic viability and scope of aqueous two-phase fermentation are briefly discussed in relation to the wider application of this topic. Received: 16 June 1999 / Received revision: 29 December 1999 / Accepted: 4 January 2000     

Improving the lactic acid production of Actinobacillus succinogenes by using a novel fermentation and separation integration system

This work describes the development of a novel integrated system for lactic acid production by Actinobacillus succinogenes. Fermentation and separation were integrated with the use of a microfiltration (MF) membrane, and lactic acid was recovered by resin adsorption following MF. The fermentation broth containing residual sugar and nutrients was then recycled back into the fermenter after lactic acid adsorption. This novel approach overcame the problem of product inhibition and extended the cell growth period from 41 h to 120 h. Production of lactic acid was improved by 23% to 183.4 g L⁻¹. The overall yield and productivity for glucose were 0.97 g g⁻¹ and 1.53 g L⁻¹ h⁻¹, respectively. These experimental results indicate that the integrated system could benefit continuous production of lactic acid at high levels.     

Analysis of mass transfer for immobilized cells in an extractive lactic acid fermentation

The use of immobilization in extractive lactic acid fermentation by Lactobacillus delbrueckii is preferred. In this article, the mathematical simulations to examine the influences of substrate and product transport were performed to assess the overall performance. The simulations showed that transport of the substrate in k-carrageenan beads was not a rate limiting factor. However, the model observed significant buildup of inhibitory product in large beads. The model was validated through comparisons with the experimental results. Finally, the model was used to predict the performance of the extractive fermentation under different operating strategies.     

Application of metabolically engineered Saccharomyces cerevisiae to extractive lactic acid fermentation

In this study, Saccharomyces cerevisiae OC-2T T165R, metabolically engineered to produce optically pure L(+)-lactic acid, was used to develop a high performance extractive fermentation process. Since the transgenic yeast could produce lactic acid efficiently even at lower than pH 3.5, high extractive efficiency was achieved when tri-n-decylamine (TDA), a tertiary amine, was used as the extractant. Separation of microorganisms by means of a hollow fiber module could not only improve the total amount of lactic acid produced but also increase the lactic acid concentration in the solvent. Moreover, pH had a significant effect on extractive fermentation. The highest rate of recovery of lactic acid could be obtained on pH-uncontrolled fermentation (pH 2.5); however, the lowest amount of lactic acid was produced. Taking into account the trade-off between the fermentation and extraction efficiencies, the optimum pH value was considered to be 3.5, with which the largest amount of lactic acid was produced and the highest lactic acid concentration in the solvent was obtained. The results show promise for the use of the transgenic yeast for extractive fermentation.     

Partitioning of pristinamycins in aqueous two-phase systems: A first step toward the development of antibiotic production by extractive fermentation

The partitioning of pristinamycins was studied in dextran and polyethylene glycol (PEG) aqueous two-phases systems. Pristinamycins partitioned preferentially into the PEG-rich top phase. The partition coefficient was independent of molar mass of PEG and dextran and of antibiotic concentration, but, increased exponentially with the tieline length of the system. Partition of pristinamycins was greatly improved when fatty acids esters of PEG were mixed with PEG. In such mixtures, the partition of coefficient increased up to a value of 24, dependent on the carbon chain length of fatty acids and the modified PEG concentrations. Moreover, in such system, the two groups of pristinamycins, I and II, were extracted in accordance with their hydrophobicity. Recovery of pristinanamycins produced by Streptomyces pritinaespiralis in a fermentation broth was achieved with a dextran/PEG system. Cells were confined into the bottom phase and pristinamycins partitioned in the top phase. However, due to binding of the pristinamycins to the cells, the partition coefficient was slightly lower than of pure antibiotics solutions. (c) 1994 John Wiley & Sons, Inc.     

Steroid bioconversion in a novel aqueous two-phase system

A novel aqueous two-phase system, based on polyethyleneglycol (PEG) and monosodium glutamate, was tested for the 1-dehydrogenation of hydrocortisone-based substrates. This system led to higher substrate solubilities and biocatalyst/steroid separation levels when compared with alternative systems. The addition of short-chain monohydric alcohols resulted in higher solubilities and more favourable partition coefficients for the tested substrates. Bioconversion activities in PEG/glutamate systems with 2,5% (v/v) methanol were comparable to those measured in monophasic buffer-methanol medium.     

Extractive fermentation of clavulanic acid by Streptomyces DAUFPE 3060 using aqueous two-phase system

The influence of four variables, specifically PEG molar mass (400, 1,000, and 8,000 g/mol), concentrations of PEG and phosphate salts (15, 20, and 25% for both), and agitation intensity (110, 150, and 200 rpm), on clavulanic acid (CA) extraction by extractive fermentation with PEG/phosphate salts aqueous two-phase system was investigated in shaken flasks using a 2(4-1) -fractional factorial design. After selection of the two most significant variables (agitation intensity and PEG molar mass), an optimization study conducted according to a 2(2) -central composite design revealed that 25% PEG 8,000 g/mol and phosphate salts at 240 rpm (run 6) were the best conditions for the extractive fermentation, leading to the best results in terms of partition coefficient (k = 8.2), yield of CA in the PEG-rich phase (η(T) = 93%) and productivity (P = 5.3 mg/Lh). As a first attempt to make a scale-up of these results, the effectiveness of the extractive fermentation was then checked in a bench-scale bioreactor under conditions as close as possible to the optimum ones determined in flasks. The highest CA concentration obtained in the PEG-rich phase (691 mg/L) was 30% higher than in flasks, thus demonstrating the potential of such a new process, integrating the production and extraction steps, as a promising, low-cost tool to obtain high yields of this and similar products.     

Isolation and characterization of a novel lactic acid bacterium for the production of lactic acid

We isolated a novel lactic acid bacterium from a Korean traditional fermented food, soybean paste. The newly isolated strain, dubbed RKY2, grew well on glucose, sucrose, galactose, and fructose, but it could not utilize xylose, starch, or glycerol. When the partially amplified 16S rDNA sequence (772 bp) of the strain RKY2 was compared with 10 reference strains, it was found to be most similar toLactobacillus pentosus JCM 1588^T, with 99.74% similarity. Therefore, the strain RKY2 was renamedLactobacillus sp. RKY2, which has been deposited in the Korean Collection for Type Cultures as KCTC 10353BP.Lactobacillus sp. RKY2 was found to be a homofermentative lactic acid bacterium, because its end-product from glucose metabolism was found to be mainly lactic acid. It could produce more than 90 g/L of lactic acid from MRS medium supplemented with 100 g/L of glucose, with 5.2 g L⁻¹ h⁻¹ of productivity and 0.95 g/g of lactic acid yield.     

Effects of extractive fermentation on butyric acid production byClostridium acetobutylicum

Summary The addition of an oleyl alcohol extractant to a batch fermentation of glucose byClostridium acetobutylicum resulted in a concentration profile that was distinctly different from the non-extractive control fermentation. The concentration of butyric acid increased and subsequently decreased in the control fermentation. The concentration of butyric acid increased but did not subsequently decrease in the oleyl alcohol extractive fermentation. The production of butyric acid was found to have been prolonged into the solventogenic phase in the oleyl alcohol extractive fermentation. Butyric acid was continually replenished from glucose while it was being converted to butanol. Supplementation of exogenous acetic and butyric acids, the metabolic uncoupler carbonyl cyanide 3-chlorophenylhydrazone, or decanol to the oleyl alcohol extractive fermentation helped to reinstate the normal butyric acid concentration profile. These findings are discussed with respect to the effects of these additives on the pH ofC. acetobutylicum and its importance with regard to the production of butyric acid.     

pH-Uncontrolled lactic acid fermentation with activated carbon as an adsorbent

In this paper, we presented a novel process involving activated carbon (AC) as an adsorbent for lactic acid fermentation, separation and oligomerization. It was found that pH has a significant effect on the adsorption of lactic acid on AC. The use of AC for in situ removal of lactic acid successfully decreased the inhibitory effect of lactic acid, resulting in significant increases in both productivity and yield. Acetone was used to desorb lactic acid and it was confirmed that the acetone treatment did not decrease the optical purity of the lactic acid, i.e., the optical purity was as high as 99.5% after desorption. Due to the presence of little materials influencing lactic acid oligomerization, oligomers with an optical purity of above 96% and a weight-average molecular weight (M(w)) of 2400 were obtained in the oligomerization process.     

Sugarcane-pressmud as a novel substrate for production of citric acid by solid-state fermentation

Sugarcane-pressmud, a by-product of cane-sugar manufacture, was used as a substrate for production of citric acid by Aspergillus niger CFTRI 30, in a solid-state fermentation system. Of the 170 g of sugar supplied, 131 g were consumed, with a 79% yield of citric acid over 120 h. Potassium ferrocyanide improved the conversion to about 88% and lowered the fermentation time by 24 h. Enrichment with sugar and NH₄NO₃ was essential to improve productivity. About 174 g citric acid/kg dry sugarcane-pressmud were produced after 120 h in ferrocyanid-treated medium which initially contained 12.5% (w/w) effective sugar and 0.1% (w/w) NH₄NO₃. About 3% (w/w) of the original sugar present in the sugarcane-pressmud was non-utilizable. This is the first report on the potential of sugarcane-pressmud for citric acid production.V.S. Shankaranand and B.K. Lonsane are with the Fermentation Technology and Bioengineering Discipline, Central Food Technological Research Institute, Mysore-570 013, India     

Novel polymer-polymer conjugates for recovery of lactic acid by aqueous two-phase extraction

A new family of polymer conjugates is proposed to overcome constraints in the applicability of aqueous two-phase systems for the recovery of lactic acid. Polyethylene glycol-polyethylenimine (PEI) conjugates and ethylene oxide propylene oxide-PEI (EOPO-PEI) conjugates were synthesized. Aqueous two-phase systems were generated when the conjugates were mixed with fractionated dextran or crude hydrolyzed starch. With 2% phosphate buffer in the systems, phase diagrams with critical points of 3.9% EOPO-PEI-3.8% dextran (DEX) and 3.5% EOPO-PEI-7.9% crude starch were obtained. The phase separation temperature of 10% EOPO-PEI solutions titrated with lactic acid to pH 6 was 35 degrees C at 5% phosphate, and increased linearly to 63 degrees C at 2% phosphate. Lactic acid partitioned to the top conjugate-rich phase of the new aqueous two-phase systems. In particular, the lactic acid partition coefficient was 2.1 in 10% EOPO-PEI-8% DEX systems containing 2% phosphate. In the same systems, the partitioning of the lactic acid bacterium, Lactococcus lactis subsp. lactis, was 0.45. The partitioning of propionic, succinic, and citric acids was also determined in the new aqueous two-phase systems.     

Extractive lactic acid fermentation with tri-n-decylamine as the extractant

In this study, the possibility of tri-n-decylamine (TDA) was investigated as the extractant in extractive lactic acid fermentation with a recombinant yeast being used. Extractive fermentation with TDA did not provide high l-lactic acid production relative to fermentation without extraction. Through determination of trace components of the TDA, it was found that the TDA contained a high concentration of 1-decylaldehyde, which was confirmed to be toxic to yeast and to have an inhibitory effect on cell growth even at low concentration (40 ppm). When 1-decylaldehyde in TDA was reduced from 700 ppm to 33 ppm, the productivity and total concentration of lactic acid increased by 1.8 times and 2.5 times, respectively.     

Purification of acetohydroxy acid synthase by separation in an aqueous two-phase system

Extraction in a polyethylene glycol (PEG)–phosphate aqueous two-phase system was considered as a primary step in purification of the acetohydroxy acid synthase III large catalytic subunit from an E. coli extract. Extraction optimization was achieved by varying the system parameters. Two systems with the following weight compositions were chosen for purification: PEG-2000 (16%)–phosphate (6%) and PEG-4000 (14%)–phosphate (5.5%)–KCl (8%), both at pH 7.0 and 1 mg total protein per 1 g system. Significant purification was achieved by a single extraction step with 70% recovery of the enzyme. After an additional ion-exchange chromatography step, pure enzyme was obtained in a 50% overall yield.