Separation and Purification of Lactic Acid a Study on Cross-flow Nano Filtration as the Final Down-stream Process Using Different Commercial Membranes

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Separation and purification of lactic acid: A study

on cross-flow nanofiltration as the final down-

stream process using different commercial

membranes

Document By: Bharadwaj

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Abstract

This work is focused on the purification of fermentation broths using different

commercial nanofiltration membranes in order to optimize the final downstream process

for separation of lactic acid produced by fermentation. The processes of lactic acid production include two key stages, fermentation and product recovery. Lactic acid was

produced in a 20-liter fermentor and the purification was achieved using a 0.01 m2

filtration unit. Pure sugar cane juice was fermented using Lactobacillus plantarum,

NCIM 2912, for production of lactic acid. The cells were then separated fromfermentation broth by microfiltration. The produced lactic acid was then purified from

fermentation broth by nanofiltration. The commercially available NF2, NF3, and NF20

polyamide composite nanofiltration membranes on polyester backing with polysulfonesubstrate were used. The studies were performed in a pilot plant equipped with three

cross-flow flat-sheet membrane modules arranged in parallel. The effects of

transmembrane pressure and cross-flow velocity on the permeate flux, and on theretention of unconverted sugars were investigated. Experiments were performed at five

levels of transmembrane pressure (5, 7, 9, 11 and 13 bars) where cross-flow velocity was

varied within the range of 1.77 m/s to 2.48 m/s. Higher transmembrane pressure and

cross-flow velocity yielded higher permeate flux. The pH value affected both rejectionand permeates flux. Rejection increased with pH while flux decreased with this variable.

Keywords: Nanofiltation, Lactic acid, Cross-flow module, Transmembrane pressure.

IntroductionIn the recent years, there has been a spurt in the demand for lactic acid due to itsapplication potentials in a wide range of fields like foods, pharmaceuticals, cosmetics and

in production of biodegradable and biocompatible polylactate [1, 2]. Though it can be

manufactured both in chemical synthesis process as well as in fermentation-based process, the latter predominates lactic acid manufacturing industry having the capability

of producing desirable L-lactic acid instead of a racemic mixture of L and D lactic acids.

But the major cost of lactic acid production is due to its downstream separation and

purification steps. For effective exploitation of huge application potential of lactic acid,

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efficient separation and purification at relatively low cost remains a challenge. The

traditional downstream separation and purification involves quite a series of steps like

filtration, acidification, neutralization, crystallization, carbon adsorption, evaporation, ionexchange etc. Such schemes not only involve harsh chemicals they are energy-intensive

also [3, 4]. This is where membrane-based nanofiltration steps in as an entirely

environmentally benign process [5]. Nanofiltration in downstream purification canreplace the multiple purification steps by a single step while yielding a monomer grade

lactic aid from a mixture of unconverted sugars and lactic acid. Separation of microbial

cells has to be done in a prior microfiltration step. Such a membrane-based process thatcan be operated at relatively low pressure ranges of 6 to 20 bars vis a vis requirement of

high transmembrane pressure (above 15-20 bars always) by reverse osmosis is gaining

importance in the recent years. Separation mechanism draws heavily on Donnan-steric

effects and understanding these effects is essential to successful modeling and scaling upof the process. To better understand the hydrodynamics of the system and the Donnan-

steric effects, the present study evaluated the effects of transmembrane pressure and

cross-flow velocity on the permeate flux, and on the retention of unconverted sugars

while comparing performances of three commercial nanofiltration membranes. TheSepro-made polyamide composite membranes (NF2, NF3, and NF20) on polyester

backing with polysulfone substrate were procured in flat sheet forms and tested on cross-flow module using fermentation broths directly from a membrane cell recycle bioreactor

(MCRB) in the laboratory. The studies were performed in a pilot plant equipped with

three cross-flow flat-sheet membrane modules arranged in parallel. The surface area of

each flat sheet membrane was 0.01 m2. Experiments were performed at five levels of transmembrane pressure (5, 7, 9, 11 and 13 bar) where cross-flow velocity was varied

within the range of 1.77 m/s to 2.48 m/s.

Experimental

Materials

Membrane

The microfiltration PVDF-MFB membrane was supplied by Sepro Membranes(Oceanside, CA 92056) as flat sheets. The important characteristics, as provided by the

supplier, are pore size of 0.13 µm and normalized water flux 1800 L /m2 hr bar at pH 7.5

and 250C. The nanofiltration NF2, NF3 and NF20 membranes were also supplied bysepro membranes (Oceanside, CA 92056) as flat sheets. All are made of polyamide, thin-

film composite membranes on polyester backing with a polysulfone substrate. Other

important characteristics for different membranes, as provided by the supplier, are 97%,

98%, and 98% rejection of MgSO4 and 40%, 50%, 35% rejection of NaCl respectively(for [MgSO4] and [NaCl] = 2 g/L and ∆P = 10.3 bar), and water flux of 135, 42, 42 L/m 2

hr respectively at specified pressure at 250C and pH 7.5. The same piece of membrane

was used throughout the experiments.

Fermentation broth preparation

Experiment was carried out using microfiltrate fermentation broth (MFB). Pure sugar cane juice (14% w/v), supplied by local farmer, contents 126 sucrose as disaccharides, 8



g/l glucose and 6 g/l fructose as monosaccharide were supplemented with the following

medium: peptone 5 g/l , yeast extract 10 g/l, polysorbate80 2 ml/l, Sodium acetate 1.5 g/l

,NH4HCO3 0.75 g/l , MgSO4.7H2O 0.2 g/l , MnSO4.4H2O 0.005 g/l , KH2PO4 1.5 g/l,K 2HPO4 1.5 g/l at 370C [2, 6]. All the chemicals except yeast extract (Hi-Media, Mumbai,

India) were supplied by Merck Limited (Mumbai, India). A selected strain of

Lactobacillus plantarum NCIM 2912 (National Chemical Laboratory, Pune,Maharashtra, India) was used for fermentation. The fermentation broth was then

microfiltrate using PVDF-MFB membrane.

Methods

Experimental method

Fig.1. is a schematic diagram of the cross-flow membrane module, fabricated bysupercritical solutions, Kolkata, India. Only the relevant module was used for

nanofiltration study has been shown here. Effective surface area of the flat sheet

membrane was 0.01 m2. The membrane was compacted before each experiment by

filtering high-purity water at 15 bars until it reached a constant permeability. The feedwas pumped through diaphragm pump (Hydra-cell pump, Minneapolis, MN 55403 USA)

from a 20 liters feed vessel, kept at 37 0C, the temperature of the fermentation broth, into

the cell and flowed tangentially to the membrane. A stainless steel control valve ismounted on the retentate outlet to control the transmembrane pressure which was

monitored through two digital manometers located on the inlet and outlet of the

membrane module. Retentate was recycled back to the feed tank. A volume of 10 ml of

permeate was collected for each pressure and timed to estimate the permeation flux. After each run the membranes were cleaned. Fully open the recirculation and permeate valves

and flush with tap water for 5 min. Circulate 5 l of 2% potassium metabyphophite for 10

min and again rinse with tap water for15 min. Finally rinse with distilled water for 5 min.

Fig. 1. Experimental set-up for nanofiltration

Analytical method

The concentrations of lactic acid and residual sucrose, glucose, fructose were determined by High Performance Liquid Chromatography (C18 column, Perkin Elmer, Series 200,

USA) equipped with UV- VIS detector and Refractive Index Detector (Perkin Elmer,



Series 200, USA). The column temperature was set to 300C, and the mobile phase was

70% acitonitrile (Sigma Aldrich) and 30% ultrapure water (Milli-Q) at a flow rate of 0.8

ml/min. An injection volume of the sample was 50 µl.

Results and discussion

Permeate collection and measurement were done after running the cross flow membrane

module (Fig.1.) with ultra pure water (Milli-Q) for first 30 minutes under a constant

pressure of 13 bars to allow for complete wetting and normal compaction of themembrane under normal nanofiltration pressure regime. The pure water flux and flux of

fermentation broth were then measured in our experimental cross flow membrane module

over a trans-membrane pressure range (TMP) of 5-13 bars and cross-flow velocitiesvarying from 1.77 to 2.48 m/s. The filtration area of the membrane was 0.01 m2. Pure

water flux was calculated as PWF (litre/m2hr) = Q (amount of water permeated through

membrane, liter) / [A (effective membrane cross sectional area, m2) × ∆T (sampling time,

hr)]. Permeate flux (PMF) of the lactic acid fermentation broth was calculated using the

same relation.

0

100

200

300

400

0 5 10 15

Transmembrane Pressure (bar)

W a t e r f l u x

/ B r o t h F l u x ( L / m 2 h r )

NF2/PW

NF3/PW

NF20/P

NF2/FBFNF3/FBF

NF20/FB

Fig. 2. Effect of trans-membrane pressure on pure water flux, PWF (___) and

fermentation broth flux, FBF (----) for different membranes at 37 0C and 2.48 m/s.

Flux study results as presented in Fig. 2 show that all the three membranes (NF2, NF3and NF20) show increase of pure water flux (PWF) as TMP increases from 5 bars to 13

bars. The NF2 exhibits the highest flux of 360 L/ m2 hr at 13 bar pressure and NF20

exhibits the lowest flux and the highest attained value being 135 L/ m2 hr at 13 bars. For

all selected membranes fermentation broth flux were lower than the pure water flux dueto osmotic pressure difference induced by the separation, higher viscosities of the

fermentation broth than water and reversible fouling of the membranes [7]. After certain



pressure (9 bar), no significant effect on permeate flux was observed due to cake

formation over the membrane. Compared to all the other membranes, although NF3

started from low microfiltrate broth permeate flux exhibits the highest microfiltrate broth permeate flux, 111 L/m2 hr at 13 bar, in contrast, NF20 showed the lowest broth permeate

flux (78 L/ m2 hr) at the same pressure. Broth fluxes of NF2 and NF3 membrane are

nearly same. These results help in setting the best membrane operating cross flowvelocity at around 2.48 m/s and 13 bar pressure for NF3 membrane. The flux obtained by

NF3, 111 L/m2 hr at 13 bar, is suitable for industrial application as this flux are higher

than those obtained in literature [7, 8,9].

0

20

40

60

80

100

120

B r o t h F l u x ( L / m 2 h

NF2 NF3 NF20

1.

2.

Fig. 3. Effect of cross-flow velocity on fermentation broth permeates flux at constant

transmembrane pressure of 13 bars for different membranes.

As shear rate or cross-flow rate plays an important role in reducing membrane fouling,optimization of cross flow is essential in minimizing membrane area requirement [7, 8].

Fig. 3 shows that flux increases with increase of cross flow velocities for all selected

membranes. This is because of greater convective force that minimizes cake formation

and concentration polarization effects. Though compared to all the other membranes, NF3 exhibits the highest broth permeate flux (111 L/ m2 hr) at 13 bars and 2.48 m/s cross-

flow velocity.

Lactic acid and sugar rejection was calculated as rejection (%) = [1- (concentration of component in permeate / concentration of component in the feed stream)] × 100. Fig. 4

shows lactic acid and sugar rejection as a function of permeate flux of microfiltratefermentation broth. For both component fermentation broth rejection increases withincreasing transmembrane pressure. The observed phenomena can be explained as the

pressure increases flux also increases resulting a dilution of the components in permeate,

though retentate concentration did not change significantly [8]. Lactic acid rejection for microfiltrate fermentation broth increased in NF3 membrane from 53% to 67% when flux

increased from 69 to 111 L/ m2 hr and transmembrane pressure increased from 5 bars to



13 bars. The increased rejection of selected membranes were observed in the order of

NF3>NF2>NF20.

40

50

60

70

80

90

100

0 5 10 15

L a c t i c a c i d / S u g a r r e j e c t i o n ( % )

NF2/LACR

NF3/LACR

NF20/LAC

NF2/SUGR

NF3/SUGR

NF20/SUG

Fig. 4. Rejection of lactic acid (____) and sugar (-----) of selected membranes of

microfiltrate fermentation broth as a function of transmembrane.

0

10

20

30

40

50

60

70

80

90

100

R e j e c t i o n ( %

2.7 (LA) 2.7 (SUG) 5.5 (LA) 5.5 (SUG)

pH

NF2

NF3

NF20

Fig. 5. Comparative lactic acid rejection of the selected membranes as a function of

pH at 13 bars pressure.

In, Fig 5, the effect of pH on lactic acid rejection is compared for the three membranesconsidered. The fermentation broth was acidified to pH 2.7, and the microfiltrate

fermentation broth pH was 5.5. The results shown were obtained at a constant

transmembrane pressure of 13 bar. It can be observed that rejection increases with pH for



three membranes. The membrane charge becomes more negative with increase in pH

resulting increase in electrostatic repulsion and higher rejection of lactic acid (negatively

charged).

Conclusions

When the microfiltrate fermentation broth was treated by NF the electrostatic effect is alimiting factor as at the acidic pH lactic acid rejection was low. Lactic acid transport

through three membranes was affected by pH. Permeate flux increased with pressure.

NF3 membrane showed 96% sugar removal and 67 % lactic acid rejection at 13 bar pressure attaining flux 111 L/ m2 hr. The successful integration of the membrane with a

cross-flow module in a continuous lactic acid production and purification system paves

the way for scale up of the hybrid system not only for production of lactic acid but for

other similar organic acids as well.

Acknowledgements The authors are thankful to the Department of Science and Technology, Government of

India (DST) for the grants under DST-FIST Program (SR/FST/ET1-204/2007) and GreenChemistry/Technology Program (SR/S5/GC-05/2008) with which the infrastructure for

the present research was developed and necessary research materials were procured.

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Separation and Purification of Lactic Acid a Study on Cross-flow Nano Filtration as the Final Down-stream Process Using Different Commercial Membranes