Batch Electrodialysis of Lactic Acid Obtained from Lab Fermentation

Abstract The aim of this work was to develop the method of lactic acid (LA) separation from fermented whey. CMI-7000 Cation Exchange Membrane and AMI-7001 Anion Exchange Membrane were employed in electrodialysis process. Experiments showed that the selected membranes separated organic acids effectively (including LA) from other organic ingredients present in medium. Selecting an appropriate volume of a receiving chamber could lead to LA concentration. Moreover, membrane fouling during separation was investigated. This phenomenon is negligible which is the main advantage of this process. As it was shown during batch processes, with the voltage increase, the rate of electrodialysis increases as well. It prompts to a reduction of residence time in electrodialyzer during a continuous separation.


INTRODUCTION
Lactic acid (LA) is one of the most common organic acids that is used widely in biotechnological and chemical technologies. For instance, in the food industry it is widely used as an acidulant, a preservative and also as an emulgator 1 . In 2016 the yearly world production reached 1.077 · 10 6 tons and an annual growth rate 14.2% is expected 2 .
Benefi cial properties of LA for skin condition have been applied in cosmetics production. It is common used as an acidity regulator 3 in cosmetics formulation, but could be also used as active ingredients too. W. P. Smith 4 treated patients' skin with 12% w/v lactic acid and this resulted in increased epidermal fi rmness and reduction of wrinkles amounts. LA after polymerization as polylactide is a great alternative to botox 5 .
A large application of LA comes with its consumption for the synthesis of the polyester PLA (polylactic acid or polylactide) -a biodegradable polymer. It is extensively used in tissue engineering and in drug delivery systems 6 . The global production capacity for PLA increases rapidly every year and the capacity of 800.000 tons should be established in 2020 7 (http://news.bio-based.eu). Hence, the lactic acid requirement is estimated at an annual growth of 5-8% per year 8, 9 .
Not only PLA production consumes LA. This organic acid may be converted into a wide range of useful products or semi -products such as acrylic acid, propylene glycol, 2,3-pentanedione, acetaldehyde, pyruvic acid 1 .
From the economical point of view and because of its enormous world consumption, new sources (especially natural -derived from food industry) used in lactic acid production are sought. It is quite obvious, that a fermentation process is preferable than a chemical synthesis 10 . The fermentation is performed by lactic acid bacteria -LAB 11 that are able to convert simple carbohydrates such as glucose, galactose and lactose (derived from whey) to LA. LA production from whey is certainly an eco-friendly process and gives surprising fermentable capability 12 .
A product of microbiological fermentation is contaminated by unreacted substrate, residual of nutrient solution or -what is the most problematic -propagated bacterial cells. In effect, a purifi cation of this stream is complicated and diffi cult. Membrane reactor is increasingly popular process solution to produce organic acids 13, 14 . Its application leads to cells and heterogenic pollution separation. Much more complicated are the further steps. In fermentation broth there are huge amounts of impurities such as unreacted sugars, residue of proteins, or other organic acids 12 . The most selectively method to separate these compounds is an ion exchange chromatography 14 . Gonzales et al. 16 used two types of ion exchangers -Lewatit S2568H and Lewatit S3428 and obtained a fi nal purity of lactic acid higher than 99%. There is huge amount of anion and cation exchange chromatography applied: Reillex 425 17 , IRA-420 18 or MWA-1 and AG3-X4 19 . The ion exchange chromatography was an effective process in terms of the product quality, but it gave the product recovery in an unfavorable quantity approach.
LA can be also extracted by n-butanol. The advantage of this process is high selectivity of lactic, however factors such as pH, mixing time, initial concentration of lactic acid, and volume ratio between the organic and the aqueous phase affect the extraction yield 20 . An integrated process including fi ltration, activated carbon treatment and evaporation should be also mentioned 20 . This method gives 85% concentration of LA. Another integrated process of lactic acid recovery and purifi cation process was esterifi cation with distillation. Purifi ed LA in extraction process with n-butanol was mixed with methanol (in molar ratio 1:1.5) and small amount of sulfuric acid. The mixture was directed to fractionating column. LA was a bottom fraction 22 . However, described process requires a lot of energy, therefore is not profi table.
This work tries to outline the promising fi eld of LA purifi cation by electrodialysis (ED). This approach is intensively developing nowadays. Membrane processes are cheaper, more effi cient than other purifying processes (e.g. chromatography or extraction) and works usually in continuous system, what make them valuable from industrial point of view. These are advantages and ED as a type of membrane process is characterized by the same.
ED membranes are mechanically and chemically stable in different electrolyte solutions 23 , so in effect they may be used for a long time, especially when it comes to continues system processes. Additionally, regeneration is quite easy and cheap (using NaOH, HCl or NaCl) Many membrane systems have been already described but this membrane technique is constantly studied. There are many various types of this process: desalting electrodialysis with membranes stacked alternately or water splitting electrodialysis with bipolar membrane, one stage system or multi -stage system etc. 24, 25 This type of separation process is widely used to the separation and purifi cation of organic acids 26 , besides lactic acid, also citric acid 27 . Habova et al. 28 received a fi nal concentration of LA (derived from a fermentation broth) 151 g L -1 . Boyaval et al. 29 coupled electrodialysis unit with continuous fermentation and achieved 85 g L −1 concentration in outlet stream. Kim and Moon 25 employed one-stage ED process, which lead to a high volumetric productivity -72 g L −1 h −1 , that in comparison to a multi -stage separation leads to a decrease in capital and operating costs. All of described above processes refer to continuous process.
ED is very specifi c type of membrane process, because as an one is a based on electric potential difference. Authors rarely investigate the problem of transport through the membrane, which is undoubtedly very important from a process design point of view. They had pretested results (often very satisfactory) of separation made on industry or laboratory equipment, but do not id not mention about mechanism.
In presented work, one -step batch electrodialysis process was tested. An investigation a process using a batch system could provide some information about mechanism of separation, what is a quite diffi cult in continues system case. CMI-7000 Cation Exchange Membrane and AMI-7001 Anion Exchange Membrane from Membranes International, USA, (which have not employed to LA separation yet) were tested. These membranes should have a great potential in LA purifi cation 30 . Separated fermented broth derived from membrane bioreactor with Lactobacillus rhamnosus while post-production whey was the carbon and energy source. The main task was to separate LA from another small organic compounds like lactose or peptides occurring in broth 31 .
Concentration of LA in outlet stream (simultaneously in electrodialyzed solution) was approximately 7.2 g · L -1 (was not constant because came from microbiological process). It contains small amount of unreacted lactose (1.4 g · L -1 ), proteins (0.5 g · L -1 ) and other (in insig-nifi cant concentration) organic acids -especially citric and acetic acid. A presence of these acids generated a pH value about 4.0.
In order to estimate a concentration of lactic acid salt (during process outside acid there is formed its salt too) 1mL medium was mixed with 1M H 2 SO 4 (Poch, Poland, CAS: 7664-93-9) and then lactic acid concentration was measured with HPLC. The difference between based on HPLC obtained value, and concentration (measured in medium without sulfuric acid), is approximate salt content.

Equipment
A laboratory equipment to electrodialysis (our construction) had three chambers divided by ion-exchanging membranes (CMI-7000 Cation Exchange Membranes and AMI-7001 Anion Exchange Membranes, Membranes International, USA). Parameters of these membranes A cathode was placed in the left chamber (chamber I, V = 30 mL) detached by the cation -exchanging membrane while in the right chamber (chamber III, V = 30 mL) an anode was placed. The electrodes were submerged in distilled water and connected with direct current source (MS Major Science MP -300V). A middle chamber (chamber II, V = 60 mL) was fi led with a post Figure 1. Scheme of an electrodialysis set -culture medium. The scheme of the system described is presented in Fig. 1.
During separation an electrical voltage, current, temperature and the concentration of lactic acid were measured.
Membranes were regenerated by 1% HCl (for the cation exchange membrane) and 1% NaOH (for the anion exchange membrane) poured into an adequately chamber. Additionally, ultrasounds for 5 min were applied.

Lactic acid analysis
The concentration of lactic acid was analyzed with HPLC (Waters, USA) under isocratic conditions using Synergii 4μ Hydro -RP 80 Å column (Phenomenex, USA).
Samples were fi rst fi ltered through a 0.22 μm syringe fi lter and then eluted with 20 mM K 2 HPO 4 /H 3 PO 4 at pH 2.9 and 30°C for 10 min. The fl ow rate was 0.042 L h -1 and peak absorbance was monitored at 214 nm.

RESULTS AND DISCUSSION
The separation of LA from a fermented broth was the main aim of this research. A processing stage of LA synthesis in bioreactor is a complex microbiological process, in which very various products can be formed. Moreover, LA salts (sodium or calcium lactate) can be found in fermented broth 33 . Electrodialysis process does not distinguish these ingredients; lactate anions derived from both acid and salts migrate to chamber III. In effect at the equal volume of all chambers we can obtain higher concentration of LA in chamber III than in chamber II in the initial stage.
The experiment for total amount of LA determination (extraction by 1M sulfuric acid) indicated that in our case almost 16% of LA molecules came from salts.

Composition of separated medium
Electrodialysis is a specifi c membrane technique where a driving force is an electric potential difference. Different ions with the same sign migrate throughout the membrane in the same direction. Thus, the presence of other acids (other than LA) can be observed in chamber III. They came from whey, which was used as a medium in bioreactor. L. rhamnosus is a homofermentative L-(+)lactic acid producer 34 ; due to its metabolism only LA is formed. Additionally, L. rhamnosus can used a citric acid as well as lactose 35 , as a carbon source. Therefore, the concentration of this acid decreases during fermentation process (Fig. 2).

Profi le of product concentration in chamber III
At the beginning, the middle (II) chamber was fi lled with the fermented broth, microfi ltrated previously. Average LA concentration in this solution was 7.2 g L -1 . Then the LA molecules, and also other organic acid molecules migrated to the right chamber using the appropriate voltage -from 25 to 100 [V].
At each voltage the concentration profi les in chamber III were similar, but the process rate increased with a voltage increase (Fig. 3). After 5 hours of separation process, the concentration of LA was approximately 3.6 g L -1 (it estimated for 25% initial mass of LA). The faster this condition was achieved the higher voltage was applied. As expected, ions migrated after this time, but with lower intensity. During electrodialysis, the concentration of acids in the middle chamber decreased (Fig. 4).

Profi le of resistance and temperature in the system
Electrical resistance and temperature were monitored during the whole electrodialysis process. In each chamber temperature was the same. For the fi rst 20 minutes electrical resistance of the system decreased quite rapidly and at the same time temperature increased for each applied voltage (Figure 5A and B). At the beginning of the process the resistance of system is very high and the reason of this is a presence of distilled water in I and III chamber (of course, an introduction of some ionic solution could decrease this phenomenon, but it could lead to product contamination and that idea was abandoned), so in effect the signifi cant rise of temperature takes place. After 1.5 hour of the electrodialysis process, temperature was quite stable and LA concentration and resistance increased modestly. These two last parameters are mutually bonded with each other. The higher acids concentration in liquid in chamber III resulted their lower concentration in liquid in chamber II. It leads to the situation, in which there are less and less energy conveyors and the middle chamber becomes a source of resistance.
A relative change of temperature associated with a given change in resistance for semiconductor (mentioned scheme is certainly one of them) is described by equation: [1] where: R t -resistance in given time, R 0 -resistance at the begging, ΔT -the difference between T and T 0 , α -temperature coeffi cient.
Obtained results (profi le of the temperature - Fig. 6A) confi rmed this theory, because according to it at the beginning of process there is rapidly increase of temperature and after initial time (in this case approximately 1 hour) slowly decline is observed. A mentioned high temperature at the beginning of separation is caused by I and III chamber -there is very low concentration of ions -so these chambers became resistors. As the concentration increase the scheme does not put up such resistance anymore. A further increase of temperature is present because the response of this parameter is delayed.

Fouling on electrodialysis membranes
One of the disadvantages of any membrane separation is membrane fouling 36 . During electrodialysis not only acids can block the membrane surface. The postproduction fermented broth used as a feed in presented research contained unreacted lactose (approximately at 1.5 g L -1 ) and whey proteins (approximately at 0.2 g L -1 ).
In order to check how the membranes are blocked, the experiment was performed with and without cleaning procedure between successive periodic processes lasting 5 hours. As it is presented in Fig. 7 the process run with the same effi ciency on the regenerated membranes as on the membranes using previously by 5 hours. It looks like using this type of membranes, fouling could be  neglected. It is a signifi cant advantage of this type of membrane separation.

CONCLUSIONS
The aim of the presented work was to recognize the separation process of LA, obtained during whey fermentation. The broth was rich in proteins, peptides, lactose and organic acids. CMI-7000 Cation Exchange Membrane and AMI-7001 Anion Exchange Membrane were selected to the separation of organic acids by electrodialysis. The research was conducted in a batch system.
Taking the obtained data into consideration a few advantages can be indicated: 1) LA is effi ciently separated from other organic compounds present in broth. Particularly, the acids transportation while lactose and peptides cannot be transported caused this process to be more interesting than nanofi ltration 15.
2) It is possible to concentrate all molecules of LA in the receiving chamber, also the ones obtained from lactates. Final concentration strongly depends on the receiving chamber volume.
3) Membrane fouling does not infl uence strongly on the process.
One the most important disadvantage of the proposed process is the presence of other acids in the receiving chamber. Their concentration is tightly connected with a broth quality, that strongly depends on other acids concentration in whey and the fermenting culture properties. These acids can be separated using chromatographic techniques 37 .
During analysis voltage increase was observed and the rate of electrodialysis increased as well. It suggests a reduction of residence time in electrodialyzer during a continuous separation at the application of the higher values of voltage. The estimated mass stream at the fi rst signifi cant hours was variable, but ranged several mg · L -1 · cm -2 · s -1 . The infl uence of this parameter on ED separation should be carefully examine during the continuous processes, in which a stagnation phase will be omitted.