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Article
Production of L (+) glutamic acid in a fully membrane-integrated hybrid reactor
system: direct and continuous production under non-neutralizing conditions
Vikramachakravarthi D, Ramesh Kumar, and Parimal Pal
Ind. Eng. Chem. Res., Just Accepted Manuscript • Publication Date (Web): 17 Nov 2014
Downloaded from http://pubs.acs.org on November 18, 2014
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Page 1 of 23 Industrial & Engineering Chemistry Research
1
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3 1 Production of L (+) glutamic acid in a fully membrane-integrated hybrid reactor
4 2 system: direct and continuous production under non-neutralizing conditions
5
6 3 Vikramachakravarthi D.1, Ramesh Kumar1, Parimal Pal*
7
8 4 Environmental and Membrane Technology Laboratory, Department of Chemical Engineering,
9
10 5 National Institute of Technology Durgapur, India – 713209
11 6 ___________________________________________________________________________
12
13 7 ABSTRACT
14
15
16 8 Experimental investigations were carried out on continuous and direct production of L-
17
9 glutamic acid in a hybrid reactor system that integrated conventional fermentative production
18
19 10 step with downstream membrane-based separation and purification in flat sheet cross flow
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21 11 membrane modules. To ensure constant fermentor capacity, the system was operated at
22
12 steady state at different set flux values ranging from 76 L m-2 h-1 to 90 L m-2 h-1 at a dilution
23
24 13 rate of 0.15 h-1. Overcoming the substrate-product inhibitions of traditional batch production
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26 14 system, this new, compact, flexible and largely fouling-free design ensured steady and
27
15 continuous production of L-glutamic acid directly from a renewable carbon source at the rate
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29 16 of about 8.4 g L-1 h-1. Provisions of continuous product withdrawal, separation and recycling
30
31 17 of unconverted sugar and microbial cells ensured almost inhibition-free production under
32
18 high cell concentration. Well-screened nanofiltration membranes with high selectivity helped
33
34 19 achieve over 97% product purity while ensuring recovery and recycle of more than 95%
35
36 20 unconverted carbon source resulting in high yield of 0.95gg-1. The direct production scheme
37
21 involves no phase change and use of harsh chemicals. The study presents development of a
38
39 22 green process of glutamic acid production for sustainable business.
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41 23
42
24 Keywords
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44 25 Glutamic acid; Continuous process; Direct production; Membrane separation; Cross-flow
45 26 module; Green design
46 27 ___________________________________________________________________________
47
28 *Corresponding Author (P. Pal) email: parimalpal2000@yahoo.com/ppal.nitdgp@gmail.com
48
49 29 Tel.: +91 343 2755955 Mobile: +91 943446950 Fax: +91 343 2547375
50 30
51 31 1
Equal contribution
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ACS Paragon Plus Environment
Industrial & Engineering Chemistry Research Page 2 of 23
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3 1
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5 2
6 3 1. INTRODUCTION
7
8 4 In the recent years, fermentative production of L-glutamic acid (GA) or 2-aminopentanedioic
9
10 5 acid has roused interest among researchers due to its potential application in seasoning
11 6 throughout the world.1 A wide variety of speciality chemicals are produced from GA as a
12
13 7 starting material.2 It has the ability to decrease or prevent nerve damage caused by anticancer
14
15 8 drug. GA can also improve the function of nervous centralize and cortical brain for
16 9 neurasthenia patients. Poly glutamic acid (PGA) is a naturally occurring polymer of GA that
17
18 10 is biodegradable, edible and non-toxic towards human and environment.3 GA has the greatest
19
20 11 demand of any single amino acid, exceeding 1.5 million tons per year on global basis.4
21 12 Manufacturing processes of amino acids are categorized as fermentative, enzymatic and
22
23 13 extractive (from acid hydrolysates of animal or plant protein). Currently, most of the GA is
24
25 14 produced by fermentation. Out of the two optically active forms of GA, only L (+) GA with
26 15 high optical purity can be used to produce monosodium glutamic acid and PGA.5 Some
27
28 16 researchers have tried to immobilize the whole microbial cells in calcium alginate or agar for
29
30 17 continuous production of GA6. But product concentration remains low due to inefficient
31 18 mass transfer, leakage of cells and lack of general matrix for immobilizing different cells. All
32
33 19 these make the process unattractive. The carbon source from renewable resources with
34
35 20 suitable micro-organisms has always been favoured to produce GA through fermentation
36 21 route as a myriad of value-added products derived from biological origin are readily accepted
37
38 22 by food industries and consumers.7 For fermentative production, the most widely used carbon
39
40 23 sources have been carbohydrates ranging from glucose to starch8. Materials like molasses,
41 24 corn starch and whey are considered as cheap carbon sources and have been frequently
42
43 25 examined in several studies. But these substrates demand additional treatments so to avoid
44
45 26 membrane fouling9. On the other hand; sugarcane juice has been tried very little though it
46 27 could be a very promising carbon source being clean, relatively cheap and renewable.
47
48 28 Throughout the year, sugarcane juice is easily available in some major sugarcane growing
49
50 29 countries like India and Brazil10. Addition of nutrient supplementation like yeast extracts
51 30 leads to increase sugar utilization and reduced fermentation time but it also adds to residual
52
53 31 impurities in fermentation broth11.To produce pure and monomer grade GA, efficient
54
55 32 separation of other impurities from the fermentation broth is essential. Conventional
56 33 purification scheme involves a number of downstream treatment steps like precipitation,
57
58 34 filtration, acidification, extraction, neutralization, carbon adsorption, crystallization and
59
60 2
1
2
3 1 evaporation.1 Traditionally, crystallization method has been used for GA separation from
4
5 2 fermentation broth at low temperature with or without biomass. But 1-2% GA still remains in
6 3 isoelectric supernatant. Ion-exchange process has been used for removal of residual GA from
7
8 4 isoelectric supernatant but substantial amounts of acid and base are required in regeneration
9
10 5 of ion-exchange resins7. In addition to that, conventional batch fermentation also suffers from
11 6 low volumetric productivity due to both substrate and product inhibitions. It also involves
12
13 7 high labour cost due to frequent shutdown and start-up of batch process. These problems may
14
15 8 be overcome by using membrane-integrated cell recycle bioreactors with continuous
16 9 fermentation which promise high cell density, much higher productivity and higher acid
17
18 10 concentration in the final product in a continuous process. To separate amino acids from
19
20 11 fermentation broths, product properties such as solubility, molecular size and affinity to
21 12 adsorbent and charge characteristics may be utilized12. The most vital criteria for continuous
22
23 13 fermentation are steady state operation with prolonged exponential growth phase which needs
24
25 14 to be maintained with proper cell bleeding13. Membrane separation is expected to be one of
26 15 the most promising candidates in successfully separating target amino acids from large
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28 16 amounts of other impurities such as fermentation medium14. Several attempts have been
29
30 17 made over the last two decades in this direction of integration of traditional fermentor with
31 18 membrane based separation and purification15-17. However, attempt has been made to
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33 19 separate product in a single stage resulting in serious membrane fouling problems after a
34
35 20 short period of operation.
36 21 It has been observed that mass transfer parameters of dissociated and un-dissociated
37
38 22 forms of any organic acid through NF membranes are better than those through reverse
39
40 23 osmosis membranes18. Ultrafiltration membrane modules have been used in some studies for
41 24 complete separation of proteins and cells, but not without encountering frequent problems of
42
43 25 membrane fouling19. Two-stage membrane separation has been attempted in very few cases.
44
45 26 Other methods like electrodialysis that use anion-exchange and cation-exchange membranes
46 27 have generally not been successful due to significant pH changes in the feed solutions, being
47
48 28 undesirable for enzyme reaction when the electrodialysis separation process is incorporated
49
50 29 into reactors20, 21. In addition, anion-exchange membranes have been reported to be easily
51 30 contaminated probably due to an oxidative reaction of a sulfhydryl group present in the
52
53 31 amino acids. Studies on multistage membrane cell recycle bioreactor also have been carried
54
55 32 out by a few researchers to improve concentration and productivity, but they have used pure
56 33 glucose only as carbon source22.
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58
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60 3
1
2
3 1 In many chemical, pharmaceutical, food and biotechnological processes, the purification
4
5 2 and recovery of amino acids plays pivotal role.23 A limited studies have been conducted on
6 3 the efficiency of NF membrane in the context of separation of amino acids. Even these
7
8 4 studies considered only model solutions of amino acids24. The major technological barrier in
9
10 5 cost-effective production of high purity GA is its downstream separation and purification.
11 6 And this is where membrane-based processes are stepping in in a big way. With integration
12
13 7 of downstream membrane-based purification in production of organic or amino acids,
14
15 8 possibility of development of better processes have certainly improved over the years but
16 9 serious attention still needs to be paid to some areas to evolve a smaller, more compact, more
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18 10 flexible and less energy-intensive plant that could guarantee large scale production of a
19
20 11 highly demanding chemical product in an environmentally benign process.25
21 12 Integration of appropriate membranes as well as modules in fermentor for cell separation
22
23 13 and product purification along with provision of logical sequencing of operations are
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25 14 essential in truly achieving such process intensification. This study develops a continuous,
26 15 GA production process under non-neutralizing conditions in a two-stage membrane
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28 16 integrated system. Judicious combination of microfiltration (MF) membranes in the first stage
29
30 17 followed by nanofiltration (NF) membranes in the second stage in flat sheet cross flow
31 18 membrane module with provision of instant separation of acid from the production media
32
33 19 eliminates the need for pH adjustment. To our knowledge, this kind of study related to
34
35 20 development of a green and continuous production process for GA in a fully membrane
36 21 integrated system has not been reported earlier.
37
38
39 22 2. MATERIALS AND METHODS
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41
23 2.1. Microorganism
42 24 Corynebacterium glutamicum (NCIM 2168) is a Gram positive, facultative anaerobic,
43
44 25 heterotrophic GA producing bacterium used throughout this work was brought from National
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46
26 Collection of Industrial Microorganism (NCIM), National Chemical Laboratory (Pune, India)
47 27 in lyophilized condition. The culture was maintained in nutrient agar slants at 4 oC and
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49 28 subcultured twice a month. Medium for preparing agar slant consisted of 2 g L-1 yeast extract,
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51
29 1 g L-1 beef extract, 5 g L-1 peptone, 5 g L-1 sodium chloride, 15 g L-1 agar at a pH of 7.0. The
52 30 flask was incubated overnight at 30 oC and used as primary inoculum. Seed culture medium
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54 31 was used with the composition (g L-1): urea, 5; yeast extract, 4; K2HPO4, 1; MgSO4·H2O, 0.5;
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56
32 FeSO4·7H2O, 0.02; MnSO4·H2O, 0.01; biotin (5µg L-1); thiamine HCl (80 µg L-1) and
57 33 aqueous medium was sugarcane juice containing 11.0% (w/v) fermentable sugar. Biotin,
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60 4
1
2
3 1 thiamine-HCl and urea were cold sterilized by membrane filter (0.2 µm, PALL Life
4
5 2 Sciences), whereas rest of the medium was sterilized separately by autoclaving at 15 psi (121
6 3 o
C) for 15 min. To reduce the lag phase of C. glutamicum (NCIM 2168) in fermentation, 2 L
7
8 4 inoculated sugarcane juice with this strain maintained at 30 oC overnight was used as
9
10 5 inoculum in membrane integrated reactor system.
11
12 6 2.2. Membranes
13
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15 7 Four different types of NF membranes namely, NF1, NF2, NF3 and NF20 were purchased
16 8 from Sepro Co. (USA) as flat sheets. These polyamide membranes are of thin-film composite
17
18 9 on polyester backing with a polysulfone substrate having different isoelectric points and
19
20 10 permeability characteristics. The detailed characteristics and properties of these membranes,
21 11 as provided by the supplier and collected from literature have been presented in Table 1.
22
23
24 12 2.3. Production medium preparation
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26 13 For the preparation of fermentation media, pure sugarcane juice was purchased from local
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28 14 farmers. Pure sugarcane juice contained 11.0% (w/v) fermentable sugar (97.7 g L-1, 7.2 g L-1,
29
30 15 and 5.35 g L-1 sucrose, glucose and fructose respectively) which was pre-filtered to remove
31 16 suspended particles like fibres and solids. The composition of production medium was same
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33 17 as the seed culture medium but the urea and biotin concentration were 8 g L-1 and 1 µg L-1
34
35 18 respectively and plus tween 80 (0.1 mL L-1). Other conditions like temperature, pH and
36 19 sterilization parameters were also same. Chemical reagents used were purchased from Sigma
37
38 20 Aldrich, Merck India Ltd. and Loba.
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40
21 2.4. Experimental equipment
41
42
43 22 Fermentation was carried out in a 30 L pilot plant fermentor which was fabricated with high
44
45
23 grade stainless steel (SS316) and was provided with water circulation system (Polyscience,
46 24 U.S.A.) and thermocouples for measuring and controlling temperature. There was also
47
48 25 provision for O2 gas purging system (Fig. 1). Agitation speed was set at 250 rpm whereas
49
50
26 temperature was maintained at 30 oC. Measurement and monitoring of pH and dissolved
51 27 oxygen (DO) concentration were done using pH-Ion meter (ORION, 4star, Thermo, U.S.A.)
52
53 28 and DO meter (TOSHBRO, India) attached to the fermentor. A flat-sheet MF membrane
54
55
29 fitted in a cross-flow module (having 0.01 m2 surface area) was attached to the fermentor. For
56 30 monitoring transmembrane pressure, two diaphragm pressure gauges were attached to the
57
58 31 inlet and outlet of each module. A peristaltic pump (Entertech, India) was used for circulating
59
60 5
1
2
3 1 the fermentation broth through the membrane module. The desired transmembrane pressure
4
5 2 was maintained during MF by controlling the attached diaphragm valve and pressure gauge.
6 3 For collecting the microfiltrate, a collecting vessel of 15 L was assembled with the system. A
7
8 4 series of flat sheet cross flow NF modules was arranged within the system in such a way that
9
10 5 the solution both from the fermentor and holding vessel could be directly filtered by the
11 6 respective module. A high pressure diaphragm pump (Hydra-cell pump, 2.2 kW,
12
13 7 Minneapolis, U.S.A.) was used between the holding vessel and NF module for circulation of
14
15 8 microfiltered fermentation broth under the desired transmembrane pressure. NF module was
16 9 fitted with three cross-flow membrane modules (each of 0.01 m2 surface area).
17
18 10 Microfiltration of the fermentation broth was carried out with a Nylon 0.45 µm membrane
19
20 11 (Membrane Solutions, U.S.A.). Composite polyamide NF1, NF2, NF3 and NF20 membranes
21 12 (Sepro Membrane, U.S.A.) were used in the NF unit. Before starting the experiment, the
22
23 13 fermentor was autoclaved at 121 oC and 15 psi pressure for 15 min and its tubing was
24
25 14 sterilized with ultrapure water at 60 oC till neutralization. Membrane cleaning was done after
26 15 each run with 0.1 N NaOH and 0.01 M HNO3 solution respectively. The membranes were
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28 16 also sterilized with 200 mg L-1 NaOCl solution followed by rinsing with Milli-Q water
29
30 17 (MILLIPORE Pvt. Ltd, India).
31
32 18 2.5. Fermentation
33
34
35 19 Fermentation was started with 10% (v/v) of inoculum in an 18 L total volume of prepared
36 20 medium in the membrane-integrated bioreactor of 30 L volume. Fermentation was carried out
37
38 21 at a temperature of 30 oC and pH of 6.5 under non-neutralizing conditions. In addition to
39
40 22 stirring at 250 min-1, the sterile air flow rate through the spurge was set at 0.1 vvm with the
41 23 help of a pump attached to 0.22 µm disc filter (Millipore). These conditions were optimum
42
43 24 for selective production of GA because very low oxygen transfer rate favours pyruvic acid
44
45 25 and lactic acid production whereas higher oxygen transfer rates favour production of α-
46 26 ketoglutaric acid and succinic acid 26
. After an initial start-up period of some 24 h, cell
47
48 27 recycling was initiated via microfiltration membrane module that separated cells and GA
49
50 28 from the fermentation broth. By this time period, culture grown inside the reactor was in the
51 29 last stage of exponential phase and that was the ideal time to make the system continuous
52
53 30 while maintaining the same active growth phase of the bacteria by adding new fermentation
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55 31 media. Through continuous removal of GA, product-inhibition could be minimised without
56 32 the need for adjusting pH with addition of caustic material. The working volume of the
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58 33 reactor was maintained at a constant level by supplying feeding medium in balance with
59
60 6
1
2
3 1 permeate flow from the membrane module. Effects of feed dilutions were investigated during
4
5 2 continuous fermentation. Dilution rate (h-1) is defined as the ratio of the feed stream inflow
6 3 rate to the working volume of the reactor. This is the rate at which the fresh media is added to
7
8 4 the fermentation broth of the reactor. Productivity (g L-1 h-1) is the final product concentration
9
10 5 of GA (g L-1) multiplied by the dilution rate (h-1) in the system. This indicates the plant
11 6 performance in terms of GA output per unit time. The cross-flow velocity (m s-1) through the
12
13 7 membrane may be computed by dividing volumetric flow rate of fluid through membrane
14
15 8 module (m3 s-1) by the cross sectional area of the inlet pipe of that module (m2). To minimize
16 9 membrane fouling problem, cross-flow flat sheet MF modules were operated for continuous
17
18 10 10 h in a constant permeate flux mode below the critical flux with partial cell bleeding. The
19
20 11 samples were collected and analysed at regular interval of time to check the physiological
21 12 properties of the broth and product. Retentate from NF was recycled back to holding vessel to
22
23 13 make solution properties inside the holding vessel constant. During continuous operation,
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25 14 permeate flow rate from MF unit was tuned in such a way that NF permeate flow rate
26 15 equalled the fresh feed flow rate to reactor.
27
28
29 16 2.6. Assay
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31 17 Samples were collected at regular intervals then ultra-centrifuged (Sigma Instruments) at
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33 18 12,000 rpm for 15 min and supernatants were collected for analysis of GA, sucrose, glucose
34
35 19 and fructose. Pellet was re-suspended with Milli-Q water with same volume and measured for
36 20 cell growth by taking optical density (OD) through UV spectrophotometer (Agilent, Cary 60,
37
38 21 U.S.A.) at 660 nm. Concentration of GA with all carbohydrates was measured using High
39
40 22 Performance Liquid Chromatography (Agilent, 1200 series). Analysis of GA was done by
41 23 MCI GEL CRS 10W column (Mitsubishi Chemical Corporation, Japan) with mobile phase
42
43 24 was 2 mM CuSO4 at flow rate 1 mL min-1 with Diode Array Detector (254 nm) at retention
44
45 25 time (RT) 16 min. The injected sample volume was 5 µL after filtration with 0.2 µm Nylon
46 26 syringe filter membrane (13 mm diameter). The measurement of all carbohydrates (sucrose,
47
48 27 glucose and fructose) was done by Refractive Index Detector with Agilent Zorbax
49
50 28 Carbohydrate Analysis Column at column temperature 30 oC with mobile phase acetonitrile:
51 29 water (Milli-Q) (75:25 ratio) at a flow rate 1.4 mL min-1 with sample injection volume 10 µL.
52
53 30 The RT of fructose, glucose and sucrose were 4.08 min, 5.32 min and 8.12 min respectively.
54
55 31 Minerals (Na+, K+ and Ca+) were quantified with Flame Photometer (Labard, India). Purity of
56 32 the nanofiltrated sample was determined by peak purity software tool of HPLC (Agilent,
57
58 33 series 1200).
59
60 7
1
2
3 1 3. RESULTS
4
5
6
2 3.1. Constant permeate flux filtration runs during fermentation
7
8 3 3.1.1. Effects of transmembrane pressure on flux during microfiltration
9
10
4 The cross-flow module integrated with flat-sheet MF membrane successfully separated cells
11
12 5 (as evident in cell analysis of the permeate) for recycling while generating clear permeate
13
14 6 from the fermentation broth without significant flux decline over 24 h. Filtration flux during
15
7 MF study varied with both transmembrane pressure as well as cross-flow velocities as shown
16
17 8 in the Fig. 2a. During MF, operating transmembrane pressure was varied from 0.5 bar to 3.5
18
19 9 bar and experiment was carried out at three cross-flow velocities of 0.55, 0.90 and 1.25 m s-1
20
10 with corresponding volumetric flow rates of 150, 250 and 300 L h-1 respectively. The cross-
21
22 11 flow velocities (m s-1) were calculated as volumetric flow rate (m3 s-1) of the retentate per
23
24 12 cross sectional area. Maximum fermentation broth flux during microfiltration as obtained at
25
13 2.5 bar in the case of 1.25 m s-1 cross-flow velocity was 135 L m-2 h-1. The permeate fluxes of
26
27 14 fermentation broth with increasing pressure up to 2.5 bar at 0.55 m s-1 cross-flow velocity
28
29 15 increased almost linearly but at higher cross-flow velocities like 0.9 and 1.25 m s-1 the
30
16 relationship curves turned quite sigmoid as shown in the Fig. 2a. The effect of time (up to 24
31
32 17 h) of operation had been investigated of fermentation broth flux at fixed transmembrane
33
34 18 pressure 2.5 bar at three cross-flow velocities of 0.55, 0.9 and 1.25 m s-1 as shown in the Fig.
35
19 2b. For over 24 h, the fermentation broth flux declined with the increase of cross-flow
36
37 20 velocities and it was minimum at 0.55 m s-1 lowest cross flow velocity. Moreover, the steady
38
39 21 state flux could be maintained for longer time at lower cross-flow rate than higher one at
40
22 fixed transmembrane pressure. Thus regulation was more prominent in case of higher cross-
41
42 23 flow rate. At higher operating cross-flow velocity, greater sweeping action resulted in higher
43
44 24 flux than that attained during the operation at lower cross-flow velocity under identical
45
25 transmembrane pressure. Higher cross-flow velocity reduces membrane fouling by higher
46
47 26 sweeping action up to certain level but beyond certain level, further increase in cross-flow
48
49 27 rate induces higher membrane fouling by virtue of higher hydrostatic pressure, adsorption on
50
28 membrane surface and pore blocking.
51
52 29 During MF run, it was essential to establish a continuous operational pattern by
53
54 30 maintaining the same reactor volume as well as same holding vessel volume under constant
55
31 permeates flux. To achieve a steady state condition of constant permeate flux below critical
56
57 32 flux was very convenient in clarification process involving two stage membrane filtration.27
58
59
60 8
1
2
3 1 The flux behaviour of the membrane modules at constant transmembrane pressure and at
4
5 2 different cross-flow velocities was investigated and it was observed that the steady state flux
6 3 could be attained after 3 h, 4 h and 6 h of operation for 0.55 m s-1 (dilution rate = 0.15 h -1),
7
8 4 0.9 m s-1 (0.17 h -1) and 1.25 m s-1 (0.18 h-1) cross-flow velocities respectively as shown in
9
10 5 Fig. 2b. The steady state fluxes were 76 L m-2 h-1, 84 L m-2 h-1 and 90 L m-2 h-1 for the above
11 6 mentioned three cross-flow velocities.
12
13 7 To operate the MF module at set flux rate, transmembrane pressure had to be adjusted at
14
15 8 regular interval of time for each cross flow velocities. In case of higher cross-flow velocity
16 9 like 1.25 m s-1 to maintain higher flux over a period of time, increase of transmembrane
17
18 10 pressure with time was quite sharper than the others as under higher operating pressure with
19
20 11 higher set flux, possibility of biomass adsorption on the membrane surface increases. Thus
21 12 high flux due to higher cross-flow velocity was translated to higher rate of membrane fouling
22
23 13 beyond a certain level. Cell viability and other physiological condition of C. glutamicum
24
25 14 (NCIM 2168) were not affected by variation of the operating conditions of three cross flow
26 15 velocities.
27
28
29 16 3.1.2. Effects of transmembrane pressure on flux during nanofiltration
30
17 Permeate collection and measurements were done after running the cross-flow NF membrane
31
32 18 module with four different types of NF membranes. The pure water flux and flux of micro-
33
34 19 filtered fermentation broth were measured in cross-flow membrane module over a
35
36
20 transmembrane pressure range of 2.5-15 bar at volumetric cross-flow velocity 4.44 m s-1. A
37 21 positive correlation between flux and transmembrane pressure has been observed in the Fig.
38
39 22 3. Transmembrane pressure (TMP) up to 15 bars, flux behaviour for fermentation broth was
40
41
23 in line with that of pure water flux. At lower levels of TMP, osmotic pressure difference
42 24 plays significant role in flux behaviour13. Pure water flux was found to vary linearly with
43
44 25 transmembrane pressure indicating absence of significant fouling. The NF2 exhibited highest
45
46
26 flux 350 L m-2 h-1 at 15 bar pressure whereas NF1 exhibited the lowest flux with the highest
47 27 attained value being 116 L m-2 h-1 at 15 bars transmembrane pressure indicating that NF2
48
49 28 membrane was the loosest membrane and NF1 was the tightest membrane (Table 1). Similar
50
51
29 trend was achieved during the NF of microfiltered fermentation broth with four different NF
52 30 membranes. At 15 bar transmembrane pressure, maximum and minimum fluxes obtained
53
54 31 were 220 and 70 L m-2 h-1 by NF2 and NF1membranes respectively. In this investigation,
55
56
32 operating pressure of 15 bars at cross-flow velocity of 4.44 m s-1 was optimum to maintain
57 33 system stability. Modules should be operated at their critical levels of cross-flow and this is
58
59
60 9
1
2
3 1 essential to minimizing membrane surface area as well as shear rate or cross-flow rate that
4
5 2 plays an important role in reducing fouling28.
6
7 3 1.3. Effect of transmembrane pressure on glutamic acid and sugar rejection
8
9 4 GA and sucrose rejection was calculated as R (%) = [1 – (Cp/Cf)] × 100, where R = rejection;
10
5 Cp = concentration of GA or sucrose in permeate; Cf = concentration of GA or sucrose in
11
12 6 feed. Fig. 4 (a) and (b) show GA and sucrose rejection as a function of transmembrane
13
14 7 pressure of microfiltered fermentation broth. It was observed that the rejection increased with
15
8 increasing transmembrane pressure for both components. GA rejection by NF membrane
16
17 9 increases linearly with increasing transmembrane pressure and the maximum rejection of
18
19 10 97% by NF1 is observed at 15 bar transmembrane pressure (Fig. 4a). During nanofiltration
20
11 with respect to sucrose, NF1 and NF20 membranes show maximum rejection whereas the
21
22 12 NF2 membrane retains the minimum (Fig. 4b). Significant differences are observed in
23
24 13 rejection behaviours of the used nanofiltration membranes for sucrose and glutamic acid
25
14 because of the neutral and ionic characteristics of the two major target solutes here as evident
26
27 15 in Fig.4a and 4b. NF20 membrane rejects very little GA (only 15%) allowing more than 85%
28
29 16 to the permeation side facilitating its recovery with the permeate stream. The same NF20
30
17 membrane at a transmembrane pressure of 15 bars, rejects sucrose by 95% for its continuous
31
32 18 recycle to the fermenter. As sugar is neutral solute, its retention is only due to steric effects.
33
34 19 Sugar retention depends on membrane pore radius and hydrodynamic radius or a combination
35
36
20 of the two. The variation of retention by the selected membranes was due to variation of the
37 21 mean pore sizes of the membranes. Sugar rejection was largely found to remain independent
38
39 22 of pH of the medium. However, rejection increased with the increase of transmembrane
40
41
23 pressure in all cases. This may be attributed to the solution diffusion mechanism that applies
42 24 to NF membrane. During the diffusion of solution through a NF membrane, solute flux and
43
44 25 solvent flux remain uncoupled and hence increased solvent flux at a higher transmembrane
45
46
26 pressure does not result in increase of solute flux. Rather, increased solvent flux results in
47 27 increased obstruction to the transport of solute through the membrane. Thus NF membrane
48
49 28 should be selected for downstream processing of fermentation broth in such a way that they
50
51
29 should offer low rejection of GA and a high rejection of sugars which makes NF20
52 30 membrane as the best choice here.
53
54 31 3.2. Continuous fermentation with cell recycles by microfiltration and nanofiltration
55
56
32 Fermentation was done at 30 oC in a 30 L pilot plant fermentor using 20 L of its working
57 33 volume (18 L fermentation media and 2 L inoculum, C. glutamicum NCIM 2168).
58
59
60 10
1
2
3 1 Microorganisms took some 24 h to reach an active stage of exponential growth phase. Lag
4
5 2 phase of microorganisms was not prominently observed during the considered period of
6 3 continuous fermentation (excluding the very initial 24 hours) because of direct use of pre-
7
8 4 inoculated (10% inoculums v/v) and nutrient-supplemented sugarcane juice as inoculums.
9
10 5 The changes in the bacterial growth (OD660), pH, GA concentration and total sugar
11 6 concentration with time have been presented in the Fig. 5. pH of the fermentation broth
12
13 7 dropped from 6.5 to 3.7 as the GA concentration reached 45 g L-1 during the initial stage.
14
15 8 Three continuous fermentation experiments were carried out at steady state fluxes of 76 L m-2
16 9 h-1, 84 L m-2 h-1 and 90 L m-2 h-1 respectively. To maintain the fermentor volume constant,
17
18 10 fresh feed was introduced at the same rates into the fermentor with dilution rate of 0.15 h-1,
19
20 11 0.17 h-1 and 0.18 h-1. After few hours of cell recycle with introduction of fresh medium with
21 12 different dilution rates to keep fermentor volume constant, the overall mass concentration
22
23 13 started to decrease, but this condition had again started to increase as microbes got adapted
24
25 14 themselves to the prevalent conditions as shown in the Fig. 5 a, b and c. The dry cell mass
26 15 measured during steady state at three different conditions were 2.95±0.5 g L-1, 3.55±0.6 g L-1
27
28 16 and 4.1±0.8 g L-1 at cross flow velocities of 0.55 m s-1, 0.9 m s-1 and 1.25 m s-1 respectively.
29
30 17 In attaining steady state conditions cell bleeding played significant role. Variation in pH was
31 18 insignificant during these three independent runs and only small variation 3.6 to 3.8 was
32
33 19 observed. With the onset of MF cell recycle at a relatively high dilution rate of D = 0.18 h-1,
34
35 20 total sugar concentration initially increased, but again decreased to finally settle at 50.4 g L-1
36 21 under steady state conditions. However, different sugar concentration profiles are observed in
37
38 22 Fig. 5 a and b for dilution rates of D = 0.15 h-1 and D = 0.16 h-1. Ultimately average total
39
40 23 sugar concentrations reached 52.3 g L-1 and 54.1 g L-1 respectively at steady state conditions.
41 24 Fig. 5 c shows a similar trend. Under steady state conditions, a higher GA concentration (55.6
42
43 25 g L-1) with maximum product yield of 95% was achieved at a cross-flow rate 0.55 m s-1. At
44
45 26 this cross-flow velocity, continuous operation could be easily sustained in the face of
46 27 changing operating conditions introduced with membrane cell recycle than higher cross-flow
47
48 28 velocity regime. In other words, to achieve steady state conditions quickly higher cross-flow
49
50 29 velocity should be avoided. Table 2 shows the product concentration, yield and productivity
51 30 achieved during three different individual runs. During continuous operation, the highest
52
53 31 productivity achieved was 8.39 g L-1 h-1 at u = 1.25 m s-1 and dilution rate D = 0.18 h-1. The
54
55 32 final concentration of GA in those three runs after nanofiltration were 55.6 g L-1, 50.4 g L-1
56 33 and 47.8 g L-1 at cross-flow velocities 0.55 m s-1, 0.9 m s-1 and 1.25 m s-1 respectively in
57
58 34 fully membrane integrated system. The overall productivities after nanofiltration were
59
60 11
1
2
3 1 computed as 8.3 g L-1 h-1, 8.1 g L-1 h-1and 8.6 g L-1 h-1 at the dilution rate 0.15 h-1, 0.16 h-1
4
5 2 and 0.18 h-1 respectively with >97% product purity. This is slightly less than the fermentative
6 3 productivity.
7
8
9 4 4. DISCUSSION
10
11 5 Glutamic acid with high purity could be produced continuously without any need for pH
12
13 6 adjustment in a fully membrane-integrated fermentation process using a cheap and renewable
14
15 7 carbon source. As membranes can be tailor-made, the new process will always have high
16 8 potential of ensuring high degree of purity of the acid product. Steady operation could be
17
18 9 attained for a constant fermentor capacity as well as constant transmembrane pressure. The
19
20 10 highest GA concentration achieved was 55.6 g L-1 with maximum product yield of 95% and
21 11 product purity 97% at a dilution rate of 0.15 h-1 although maximum productivity (8.6 g L-1 h-
22
1
23 12 ) was associated with a highest dilution rate of 0.18 h-1. Encouraging results were obtained
24
25 13 for definite improvements over the existing ones in terms of carbon source, product purity,
26 14 yield, concentration and process intensification. In the literature, yield of GA like 66.3 g g-1
27
28 15 (GA concentration 25 g L-1)20, and 48 g g-1 (GA concentration 41.4 g L-1)29 have been
29
30 16 reported in batch mode fermentation, which are comparatively low.
31 17 In place of a traditional multi-step process, this new eco-friendly scheme is quite simple as
32
33 18 well as flexible. Literature shows that membrane integrated processes are rarely used for the
34
35 19 continuous production of GA. Benefits of such a membrane-based process are many and
36 20 likely to percolate down to the cane-growing farmers also. Single stage membrane separation
37
38 21 integrated with fermentor has always suffered from severe membrane fouling. Thus two stage
39
40 22 membrane separation like MF and selective NF (NF20) with appropriate mode (flat-sheet
41 23 cross-flow) not only produces GA with reasonably high productivity, yield, concentration and
42
43 24 purity but also offers an industrially acceptable flux of 65-78 L m-2 h-1. For running the
44
45 25 system in continuous mode over a long period of time, set flux method adopted for both MF
46 26 and NF modules is quite effective.
47
48
49 27 5. CONCLUSIONS
50
51 28 The study culminates in the development of a novel, fully membrane-integrated hybrid
52
53 29 process for continuous and fast production of glutamic acid overcoming the difficulties of
54
55 30 substrate-product inhibitions from a renewable carbon source. Selection of appropriate
56 31 membranes as well as modules in cell separation and product purification along with
57
58 32 provision of logical sequencing of operations ensures fermentation under high cell density
59
60 12
1
2
3 1 and purification to a high degree by highly selective NF membrane. Such a plant represents
4
5 2 high degree of process intensification which modern chemical process industries are
6 3 desperately seeking for their survival in highly competitive and environmentally conscious
7
8 4 world market.
9
10
11
5 Acknowledgment
12
13 6 Authors are thankful to the Department of Science and Technology, Government of India for
14
15 7 financial support under Start-Up Research Grant for Young Scientist (Science and
16 8 Engineering Research Board) (SB/FTB/ETA-59/2013).
17
18
19 9 REFERENCES
20
21 10 1. Kumar, R.; Vikramachakravarthi, D.; Pal, P. Production and purification of glutamic
22
23 11 acid: A critical review towards process intensification. Chem. Eng. Process. Process
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25 12 Intensif. 2014, 81, 59-71.
26 13 2. Çalık, G.; Ünlütabak, F.; Özdamar, T. H. Product and by-product distributions in
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28 14 glutamic acid fermentation by Brevibacterium flavum : effects of the oxygen transfer.
29
30 15 Bioche. Eng. J. 2001, 9, 91–101.
31 16 3. Dutta, S.; Ray, S.; Nagarajan, K. Glutamic acid as anticancer agent: An overview.
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33 17 Saudi Pharm. J. 2013, 21, 337–343.
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35 18 4. Hermann, T. Industrial production of amino acids by coryneform bacteria. J.
36 19 Biotechnol. 2003, 104, 155-172.
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38 20 5. Shih, I.; Van, Y. The production of poly- (γ-glutamic acid ) from microorganisms and
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40 21 its various applications. Bioresour. Technolo. 2001, 79, 207-225.
41 22 6. Nampoothiri, K. M.; Pandey, A. Immobilization of Brevibacterium cells for the
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43 23 production of l-glutamic acid. Bioresour. Technol. 1998, 63, 101–106.
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45 24 7. Zhang, J.; Tang, L.; Zhang, H.; Yang, Y.; Mao, Z. A novel and cleaner technological
46 25 process of extracting L-glutamic acid from fermentation broth by two-stage
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48 26 crystallization. J. Clean. Prod. 2012, 20, 137–144.
49
50 27 8. Pal, P.; Sikder, J.; Roy, S.; Giorno, L. Process intensification in lactic acid production:
51 28 A review of membrane based processes. Chem. Eng. Process. Process Intensif. 2009,
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53 29 48, 1549–1559.
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55 30 9. Nayak, J.; Parimal Pal, P. Transforming Waste Cheese-Whey into Acetic Acid through
56 31 a Continuous Membrane-Integrated Hybrid Process. Ind. Eng. Chem. Res. 2013, 52,
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58 32 2977−2984.
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60 13
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3 1 10. Sikder, J.; Roy, M.; Dey, P.; Pal, P. Techno-economic analysis of a membrane-
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5 2 integrated bioreactor system for production of lactic acid from sugarcane juice.
6 3 Biochem. Eng. J. 2012, 63, 81–87.
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8 4 11. Dey, P.; Linnanen, L.; Pal, P. Separation of lactic acid from fermentation broth by
9
10 5 cross flow nanofiltration : Membrane characterization and transport modelling.
11 6 Desalination, 2012, 288, 47–57.
12
13 7 12. Timmer, J. M. K.; Speelmans, M. P. J.; van der Horst, H. C. Separation of amino acids
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15 8 by nanofiltration and ultrafiltration membranes. Sep. Purif. Technol. 1998, 14, 133–
16 9 144.
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18 10 13. Bouchoux, A.; Roux-de Balmann, H.; Lutin, F. Investigation of nanofiltration as a
19
20 11 purification step for lactic acid production processes based on conventional and bipolar
21 12 electrodialysis operations. Sep. Purif. Technol. 2006, 52, 266–273.
22
23 13 14. Li, S.; Li, C.; Liu, Y.; Wang, X.; Cao, Z. Separation of l-glutamine from fermentation
24
25 14 broth by nanofiltration. J. Membr. Sci. 2003, 222, 191–201.
26 15 15. Xu, G. et al. Development of a continuous cell-recycle fermentation system for
27
28 16 production of lactic acid by Lactobacillus paracasei. Process Biochem. 2006, 41,
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30 17 2458–2463.
31 18 16. Giorno, L.; Chojnacka, K.; Donato, L.; Drioli, E. Study of a Cell-Recycle Membrane
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33 19 Fermentor for the Production of Lactic Acid by Lactobacillus bulgaricus. Ind. Eng.
34
35 20 Chem. Res. 2002, 41, 433–440.
36 21 17. Torang, A.; Jonsson, A.; Zacchi, G. Separation of cells and proteins from fermentation
37
38 22 broth in a shear-enhanced cross-flow ultrafiltration module as first step in refinement of
39
40 23 lactic acid. Appl. Biochem. Biotechnol. 1999, 76, 144–157.
41 24 18. Wang, X.-L.; Shang, W.-J.; Wang, D.-X.; Wu, L.; Tu, C.-H. Characterization and
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43 25 applications of nanofiltration membranes: State of the art. Desalination 2009, 236,
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45 26 316–326.
46 27 19. Ward, R. A.; Klein, E.; Feldhoff, P. W.; Turnham, T. J. Ultrafiltration: separation
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48 28 enhancement by counter-current cascading. J. Membr. Sci. 1987, 33, 97–111.
49
50 29 20. Nampoothiri, K. M.; Ashok Pandey, A. Fermentation and recovery of L-glutamic acid
51 30 from cassava starch hydrolysate by ion-exchange resin column. Revista de
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53 31 Microbiologia 1999, 30, 258-264.
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55 32 21. Shen, J.; Duan, J.; Yu, L.; Xing, X.; Xu, P. Desalination of glutamine fermentation
56 33 broth by electrodialysis. Process Biochem. 2006, 41, 716–720.
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58
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60 14
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3 1 22. Russo, L. J.; Kim, H. Membrane-based process for the recovery of lactic acid by
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5 2 fermentation of carbohydrate substrates containing sugars, US Patent no. 5,503,750.
6 3 23. Leuchtenberger, W.; Huthmacher, K.; Drauz, K. Biotechnological production of amino
7
8 4 acids and derivatives : current status and prospects. Appl. Microbiol. Biotechnol. 2005,
9
10 5 69, 1-8.
11 6 24. Kov´acs, Z.; Samhaber, W. Contribution of pH dependent osmotic pressure to amino
12
13 7 acid transport through nanofiltration membranes. Sep. Purif. Technol. 2008, 61, 243–
14
15 8 248.
16 9 25. Wang, K.; Li, W.; Fan, Y.; Xing, W. Integrated Membrane Process for the Purifi
17
18 10 cation of Lactic Acid from a Fermentation Broth Neutralized with Sodium Hydroxide.
19
20 11 Ind. Eng. Chem. Res. 2013, 52, 2412-2417.
21 12 26. Çalık, G.; Ünlütabak, F.; Özdamar, T. H. (2001). Product and by product distributions
22
23 13 in glutamic acid fermentation by Brevibacterium flavum: effects of the oxygen transfer.
24
25 14 Biochem. Eng. J. 2001, 9, 91–101.
26 15 27. Carrère, H.; Blaszkow, F. Comparison of operating modes for clarifying lactic acid
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28 16 fermentation broths by batch cross-flow microfiltration. Process Biochem. 2001, 36,
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30 17 751–756.
31 18 28. Sikder, J. et al. Synthesis and characterization of cellulose acetate-polysulfone blend
32
33 19 microfiltration membrane for separation of microbial cells from lactic acid
34
35 20 fermentation broth. Desalination 2009, 249, 802–808.
36 21 29. Choi, S-U.; Nihira, T.; Yoshida, T. Enhanced Glutamic Acid Production of
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38 22 Brevibacterium sp. with Temperature Shift-Up Cultivation. J. Bioscie. Bioeng. 2004,
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40 23 98, 211-213.
41 24 30. Artug, G.; Hapke, J. Characterization of nanofiltration membranes by their
42
43 25 morphology, charge and filtration performance parameters. Desalination 2006, 200,
44
45 26 178-180.
46 27 31. Tanninen, J.; Manttari, M.; Nystrom, M. Effect of salt mixture concentration on
47
48 28 fractionation with NF membranes. J. Membr. Sci. 2006, 283, 57-64.
49
50 29 32. Sikder, J.; Chakraborty, S.; Pal, P.; Drioli, E.; Bhattacharjee, C. Purification of lactic
51 30 acid from microfiltrate fermentation broth by cross-flow nanofiltration. Biochem. Eng.
52
53 31 J. 2012, 69, 130–137.
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55 32
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60 15
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3
4
5 Table 1
6 Characteristics of membranes used in this work according to membrane manufacturer’s data, literature data and obtained in the present work.
7
____________________________________________________________________________________________________________________
8
9 Characteristics Membranes
10 _________________________________________________________________________
11 NF1 NF2 NF3 NF20
12 ____________________________________________________________________________________________________________________
13 Manufacturer Sepro Co. (USA) Sepro Co. (USA) Sepro Co. (USA) Sepro Co. (USA)
14 Geometry Flat-sheet Flat-sheet Flat-sheet Flat-sheet
15 Physical properties:
16 Membrane width (m) 1.02 1.02 1.02 1.02
17
Thickness including fabric backing (µm) 165 165 165 165
18
19 Roll length (m) 250 250 250 250
20 Material polyamide polyamide polyamide polyamide
21 Test pressure (bar) 10.3 10.3 10.3 10.3
22 Solute concentration (g L-1) 2 2 2 2
23 Solute rejection (%)
24 MgSO4 99 97 98 98
25 NaCl 90 50 60 35
26 Average molecular weight cut-off (Da) 150-300 150-300 150-300 150-300
27
pH range 3-10 3-10 3-10 3-10
28 o
29 Cleaning pH range (at 50 C) 2-11 2-11 2-11 2-11
30 Maximum operating temperature (oC) 50 50 50 50
31 Maximum operating pressure (bar) 83 83 83 83
32 Isoelectric point (pH) - <3a 2.7b 6.6c
-2 -1
33 Water permeability coefficient (L m h .bar) 7-9 23-25 10-12 8-10
34 d
Pore size (nm) 0.53 0.57 0. 55 0.54
35 Membrane surface area used (m2) 0.01 0.01 0.01 0.01
36 ____________________________________________________________________________________________________________________
37 a
See30; b See 31; c See 32; dSee 11
38
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42 16
43
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46 ACS Paragon Plus Environment
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48
1
2
3
4
5 Table 2.
6
7 Comparison of results obtained of three continuous fermentations associated with different operating conditions during microfiltration.
8
9
10 ___________________________________________________________________________________________________________________
11
12 Run Average glutamic acid Product yield (%) Productivity Product purity Operating conditions
13 -1 -1 -1
14 concentration (g L ) (product/substrate) × 100 (g L h ) (%)
15 ___________________________________________________________________________________________________________________
16
17 1. 55.6 95 8.34 96.8 u = 0.55 m s-1 & D = 0.15 h-1
18
19 2. 50.4 90 8.1 96.3 u = 0.9 m s-1 & D = 0.16 h-1
20 3. 46.8 84 8.6 95.5 u = 1.25 m s-1 & D = 0.18 h-1
21
22 ____________________________________________________________________________________________________________________
23
24 u = cross flow velocity; D = feed dilution rate
25
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46 ACS Paragon Plus Environment
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3 Figures No Captions
4
5
Figure 1 Schematic diagram of membrane-integrated hybrid reactor system for
6
7 continuous glutamic acid production
8
9
10 Figure 2 Effect of (a) increasing transmembrane pressure and (b) time on
11 microfiltration permeates fluxes of fermentation broth by Nylon 0.45
12
13 µm membrane with three different cross flow velocities.
14
15
Figure 3 Effect of increasing transmembrane pressure on fluxes of pure water
16
17 and microfiltered fermentation broth by four different types of NF
18
19 membranes.
20
21 Figure 4 Effect of increasing transmembrane pressure on (a) rejection of
22
23 glutamic acid and (b) rejection of sucrose by four different types of NF
24
25 membranes.
26
27 Figure 5 Effect of three cross flow velocities (u) and feed dilution rates (D) on
28
29 three different continuous fermentation process of glutamic acid
30 production.
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