Accepted Manuscript

Title: Production and purification of glutamic acid: A critical

review towards process intensification

Author: Ramesh Kumar D. Vikramachakravarthi Parimal Pal

PII: S0255-2701(14)00088-9
DOI: http://dx.doi.org/doi:10.1016/j.cep.2014.04.012
Reference: CEP 6422

To appear in: Chemical Engineering and Processing

Received date: 5-2-2014

Accepted date: 25-4-2014

Please cite this article as: R. Kumar, D. Vikramachakravarthi, P. Pal, Production and
purification of glutamic acid: a critical review towards process intensification, Chemical
Engineering and Processing (2014), http://dx.doi.org/10.1016/j.cep.2014.04.012

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*Manuscript

Production and purification of glutamic acid: a critical review towards

process intensification
Ramesh Kumar, Vikramachakravarthi D, Parimal Pal*
Environment and Membrane Technology Laboratory, Chemical Engineering Department,
National Institute of Technology Durgapur, India- 713209

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ABSTRACT

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Amidst growing environmental awareness and stringent discharge regulations, chemical and

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allied process industries are now desperately seeking replacement of the conventional,

polluting processes by clean and green processes. In this context, production and purification

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of amino acids like L-glutamic acid assumes significance. Concerned conventional process

involves several steps like fermentation, centrifugation, carbon adsorption, evaporation,

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crystallization, ion-exchange and so on to get glutamic acid in desired concentration and

purification. Despite its tremendous potential for large scale use in a wide variety of
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applications, cost effective production of high purity glutamic acid has remained a challenge

for decades, mainly due to several downstream processing steps and the associated cost
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factors. With emergence of tailor-made membranes and modules, possibility of using

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membranes in downstream purification of glutamic acid appears imminent with expectation

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of a turnaround in amino acid manufacturing industry. The present manuscript through a brief
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yet comprehensive review of the very critical aspects of glutamic acid production and

purification, attempts to direct research efforts towards process intensification encompassing

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the concepts of green processing, compact and flexible design with promise of more

economically attractive production with better quality.

Keywords: L-glutamic acid, Membrane system, Green technology, process intensification

________________________________________________________________________
*Corresponding author
E-mail address: [email protected] (P. Pal); +91-9434788105/[email protected]

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1. Introduction

Konbu which is a kelp-like seaweed and traditionally used in Japan as flavour enhancer

was identified as L-glutamic acid (GA) [1]. This discovery led to industrial production of

monosodium GA by the Ajinomoto Company. Glutamic acid (GA) used to be produced in

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those days by acid hydrolysis of wheat gluten or soybean protein. But in the subsequent

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years, L-glutamic acid-producing micro-organisms were isolated [2] and research efforts

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culminated in development of fermentative process for production of GA. Introduction of

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fermentation technology provided a significant impetus to the development of microbial

production of primary metabolites. Subsequently, in a series of research initiatives, attempts

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were made to isolate wild strains or to derive genetic mutants for production of amino acids.

Thus amino acids are now commercially produced by fermentation.

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GA is one the most important amino acid products with a wide range of applications. Its

salt-derivatives can be used as nutrition elements and participate in body metabolism. Market
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for amino acids in general is observed to double every decade with scope for exploitation of
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new uses of amino acids and progress in production technology [3]. GA is used in seasoning i

throughout the world [4]. It is also used as a starting material for synthesis of various kinds of
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speciality chemicals [5]. At the same time, glutamic acid can improve the function of nervous
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centralize and cortical brain for neurasthenia patients [6]. Poly glutamic acid (PGA) is a

naturally occurring anionic polymer that is biodegradable, edible, and non-toxic towards
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human and environment [7-10]. It is a good candidate for various industrial applications

including thickener, bitterness reliving agent , Cryoprotectant [11], drug carrier [12], curable

biological adhesive [13], biodegradable fibers, highly water absorbable hydrogels,

biopolymer flocculants [14], and heavy metals absorbers [15].

Development of innovative products and processes is a big challenge to the chemical and

allied process industries all over the world for survival in an era of emaciated profit margins

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Page 2 of 55
amidst highly globalized trade competition and fast growing environmental constraints [16,

17]. Thus process intensification through revolutionary development of new products and

processes that ensure reduced material and energy consumption and reduced environmental

impacts while offering greater flexibility in scale of operation are the need of the hour.

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Production of monomer grade glutamic acid which has traditionally been used in food,

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cosmetics, medicine and agriculture has over the last few decades, attracted attention of the

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world researchers. To separate amino acids from fermentation broths, properties of the

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product, such as solubility, molecular size, and affinity to adsorbent and charge

characteristics may be utilized. Membrane separation is expected to be one of the most

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promising candidates in successfully separating target amino acids from large amounts of

other amino acids. Other methods like Electrodialysis that uses anion-exchange and cation-
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exchange membranes has generally not been successful due to significant pH changes in the

feed solutions, being undesirable for enzyme reactions when the electrodialysis separation
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process is incorporated into the reactors. In addition, an anion-exchange membrane was

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reported to be easily contaminated probably due to an oxidative reaction of a sulfhydryl

group present in the amino acids [18].

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In many chemical, pharmaceutical, food and biotechnological processes, the

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purification and recovery of amino acid plays pivotal role. Nanofiltration( NF) is a relatively

new class of the pressure-driven membrane processes and is considered a viable alternative to
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more traditional separation processes like extraction, ion-exchange, evaporation and

distillation [19,20]. The major technology barrier in cost-effective production of high purity

GA is its down-stream separation and purification. These are the fields where

membrane based processes are stepping in. Being modular in design, membrane-based

processes offer great flexibility in scale of production depending on market demand. By

virtue of high selectivity, membranes can ensure high levels of separation and purification.

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Page 3 of 55
As membranes of chosen selectivity and permeability can easily be integrated with

conventional fermentor, membrane-based processes permit simultaneous production and

purification in the same unit. This eliminates the need for separate purification units and

results in compact design with reduced capital investment. Membrane-based separation and

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purification (barring pervaporation) involves no phase change ensuring reduced energy

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consumption. Thus such processes can meet all the goals of process intensification. This

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paper first briefly discusses the traditional downstream processes to high-light the major

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problems associated with these processes and then critically takes up review of the

developments in membrane-based processes. The objective is to identify an environmentally

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benign, simple, economically viable and continuous manufacturing scheme capable of

producing monomer grade GA with high productivity.

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2. Upstream production of GA

2.1. Microbial strain

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Table 1 shows the number of wild strain that have been isolated as GA acid producing
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bacteria. Most of these GA producing bacteria are gram positive, nonspore-forming, non-

motile and require biotin for growth. Table 2 shows the different authors got the different
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yields of GA while using different processes and micro-organisms.

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2.2. Feed stock (carbon source)

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Carbon sources like glucose, fructose, sucrose, maltose, ribose or xylose can be utilized

by the GA producing bacteria as the substrate for cell growth and GA biosynthesis. A crude

feedstock has historically been avoided because high levels of extraneous materials can cause

troublesome separation problems in the recovery stage. In the late 1950, dextrose from corn

starch was the most commonly used feed stock [21]. Other feed stocks like concentrated

whey, hydrolysed potato, cellulosic material, sulphite liquor, and molasses were also used

[22-24]. Fermentation route from renewable resources like sugarcane juice with suitable

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Page 4 of 55
microorganisms has always been favoured to produce optically pure L-(+) glutamic acid as a

myriad of value-added products derived from biological origin are readily accepted by food

industries and consumers [25]. In addition to that sugarcane juice is easily available

throughout the year in some major sugar cane growing countries like India and Brazil [26].

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2.3. Others nutrients supplements

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Addition of nutrient supplementation like yeast extract, peptones and other micro-macro

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inorganic nutrients required for the growth of micro-organism in fermentation broth, leads to

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increased sugar utilization and reduced fermentation time but it also adds to residual

impurities in fermentation broth [27]. To produce pure and monomer grade GA efficient

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separation of those impurities is essential. Ammonium ion and urea are detrimental to both

cell growth and product formation and its concentration in the medium must be maintained at
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a low level. To neutralize the acid that is formed during fermentation, calcium carbonate,

calcium hydroxide and gaseous ammonia are typically used.

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2.4. Culture Conditions optimization

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Many techniques are available in the fermentation medium designer’s toolbox for media

optimization such as borrowing, component swapping, biological mimicry, one-at-a-time,

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statistical and mathematical techniques-experimental design and optimization, artificial

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neural networks, fuzzy logic, genetic algorithms, continuous fermentation, pulsed batch and

stoichiometric analysis [28]. Each technique has its advantages and disadvantages, and
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situations where they are best applied. While developing an industrial fermentation,

designing a fermentation medium is of critical importance because medium composition can

significantly affect product concentration, yield and volumetric productivity.

The design and optimization techniques are mostly in the tradition of Box and Wilson [29]

which includes steepest ascent and canonical analysis as components of response surface

methodology. During optimization process three phases of experimentation can be

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Page 5 of 55
distinguished: one for identifying important variables (screening); two for optimization and

three for central composite or Box-Behnken designs. In designing the perfect fermentation

medium, the first step is to integrate medium design and microbial screening to asses as wide

a range of microbial performance as possible as shown in Fig. 1.

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2.5. Microbial physiology and metabolic pathway of L-glutamic acid Fermentation

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The biosynthesis of GA is an aerobic process requiring oxygen throughout the

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fermentation (7 mg/L). Brevibacterium flavum, GA producing bacteria accumulate lactic acid

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and succinic acid when cultured under limited oxygen supply [30]. It was demonstrated that

the absence of ammonium ions, but with sufficient oxygen supply, resulted in the

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accumulation of α-ketoglutaric acid in place of GA. When the pH controlling agent was

switched from NH4OH to NaOH at the end of the growth phase, 18 g/L of α-ketoglutaric acid
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was accumulated at a yield of 0.20 g/g substrate in 72 h cultivation [31].

GA is converted into L-glutamine when the microbial culture is used in presence of

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excess ammonium chloride at a weakly acidic pH in the presence of zinc ions [32]. A key
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compound controlling GA fermentation is biotin. As shown in the Fig. 2, biotin is cofactor of

acetyl –CoA carboxylase, the first enzyme in the biosynthesis of oleic acid, and C16-C18
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saturated fatty acids inhibit the biosynthesis of oleic acid by repressing acetyl-CoA
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carboxylase [33,34].The accumulation of GA is at a maximum when the biotin concentration

is suboptimal for maximum growth. GA producing cells grown with limited biotin or grown
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with excess biotin and treated with either penicillin or Tween-60 excreted intracellular GA

when washed with phosphate buffer. Figure 3 shows the details about the regulatory pathway

for GA production. The properties of α-ketoglutarate dehydrogenase KD of GA producing

bacteria are favourable for the preferential synthesis of GA from α-ketoglutaric acid,

preventing the further oxidation of α-ketoglutaric acid to CO2 and H2O via succinyl-CoA.

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Page 6 of 55
3. Traditional production process

Manufacturing methods of amino acids are categorized as 1) extraction from hydrolysate

of animal or plant protein, 2) fermentation, and 3) enzymatic. Originally, GA used to be

manufactured by extraction from acid hydrolysate of plant protein. In the late 1950s,

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fermentation technology was established and was used for commercial production of GA.

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This was the beginning of modern amino acid production. Currently, most of the GA is

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produced by fermentation. In last decade, GA industry developed rapidly, and the quantum of

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GA production stood at 1.60 MT in China that is about 70% of the global production. More

than 200,000 t of GA is produced every year. Usually, separation of Glutamic Acid

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Hydrolysate (GAH) from its sodium salt is carried out by isoelectric crystallization method

with or without prior removal of biomass present in the fermentation broth. However, the
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presence of biomass reduces the crystallization process and favours the formation of p-form

of crystal [35]. The fermentation broth still contains 1–2% of GAH after separation in
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isoelectric supernatant [36]. The lower solubility of GAH in water compared to its salt is also
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a serious problem for achieving high separation and recovery. Large amount of mineral acids

and successive washing steps with water are required to remove the salts formed.
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All GA manufacturing industries have switched over to fermentation based technology as

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presented in Fig. 4. One of the methods for commercially producing glutamic acid relies on

the fermentation of relatively pure sugars with minimal amounts of nitrogenous nutrients.
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After the fermenter broth is filtered, activated vegetable carbon is used to bleach the calcium

glutamate for production of food grade acid. No activated carbon is used for the technical

grade. After that calcium glutamate is evaporated to a 37% concentration at temperature

around 70 oC and 0.57 atm. The next step is acidification with concentrated sulphuric acid

and the calcium sulphate precipitate is removed by continuous filter and sent back for reuse.

The filter acid is then treated with activated carbon for bleaching. Glutamic acid is

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Page 7 of 55
evaporated to concentrate it in 316 stainless steel evaporated. Heavy metals could be removed

by ion exchange which may also remove other amino acids. To get the higher grades of

product the liquor is cooled, crystallized and washed. Crystallization is presently performed

in a single unit and a stainless steel double effect evaporator is used. Liquid-liquid extraction

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is another method to get purer form GA by using immiscible solvent. The extraction solvent

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should have low water solubility, a high distribution coefficient for GA and a low distribution

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coefficient for impurities such as residual sugars.

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4. Immobilization of micro-organism for the production of GA

Immobilization has been considered as one useful technique in microbial production. The

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major advantages of this application are the long-term utilization of biocatalysts and

continuous operation of stabilized systems which lead to reduction of cost of bioprocessing.

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Disadvantages may also arise from the diffusional barrier created by the immobilization

matrix as well as the high cell density. Another disadvantage has been the failure of efforts to
d

create a versatile, general matrix capable of holding a variety of cells and functioning in
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differing bioprocesses. A number of amino acids have been produced using this

methodology. These include L-aspartic acid [37], L-isoleucine [38], L-serine [39], L-Lysine
p

[40] and L-glutamic acid [41]. Amin et al., [42] had studied the formation of by-products
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during glucose conversion to glutamic acid using Corynebacterium glutamicum immobilized

in polyurethane foam. Entrapment of protoplast of Brevibacterium flavum in matrices of

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agar-acetyl cellulose filtering is another attempt to produce GA [43]. Sunitha et al., 1998, co-

immobilized the whole cells of Micrococcus glutamicus and Pseudomonas reptilivora for the

higher yields 37.1 kg/m3 of GA. They found that the concentration of glucose, urea and biotin

in the production medium were proved to the most suitable medium constituents.

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Page 8 of 55
5. Limitations in conventional GA production

The traditional production process consists of a number of downstream treatment

schemes like precipitation, conventional filtration, acidification, carbon adsorption,

evaporation etc as shown in the Fig. 5. Thus the overall process plant scheme is quite

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complex involving energy-intensive and expensive steps releasing huge amount of

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wastewater and requiring relatively high manpower. The other major drawbacks of the

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existing technology is substantial demand for harsh chemicals like sulphuric acid, liquid

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ammonia and other chemical supplemental compounds that work to the overall economic

disadvantage in production of glutamic acid(GA). On the other hand, the GA extraction

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process also produces large amount of wastewater leading to environmental pollution [44,

45]. Ion exchange method is normally used for glutamic acid recovery from fermentation
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broth without precipitation. The ion exchange also involves use of large amount of acid and

base to regenerate ion-exchange resins. In addition to this, wastewater produced from the
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process in reactivating and washing the ion-exchange resins also causes a serious
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environmental pollution. Thus a new process is urgently required which will be eco-friendly

and economically more attractive. In last two decades, efforts have been made by GA
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enterprises and related scientific groups in solving the economic and environmental problems
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related to glutamic acid production [44,45]. Studies have revealed that the huge amount of

wastewater produced in GA extraction process is difficult to be handled with the traditional

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end-of-pipe treatment method. During the past half century, it has been recognized that end-

of-pipe treatment is hardly advisable in tackling environmental issues. Amidst tough

competition in the globalized market, cost of production needs to be brought down

significantly in the current regime of emaciated profit margin in chemical and allied

industries [46]. It has been observed that there is no substitute to clean production technology

in matters of controlling pollution and bringing down cost[47,48].

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Page 9 of 55
6. Membrane Processes

6.1. Concepts and principles

Membrane separation involves the use of a selective barrier (membrane) to regulate the

transport of substances, such as gases, vapours and liquids at different mass transfer rates

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[49]. The rates of mass transfer of different substances are controlled by the permeability of

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the barrier towards the feed components [49]. These membranes can play effective role in

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downstream purification of GA are microfiltration, ultrafiltration, nanofiltration, reverse

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osmosis and electrodialysis membranes. Nanofiltration (NF) membranes have been

developed recently and practiced for the separation of small neutral and charged solutes in

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aqueous solutions. NF membranes have two important features in their actual applications

[50,51].One is the intermediate molecular weight cut-off (MWCO) between reverse osmosis
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(RO) membranes and ultrafiltration (UF) membranes, which ranges from 200 to 1000; the

other is salt rejection caused by the charge effect due to their materials. They can be
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identified into the sieving (steric-hindrance) effect and the Donann (electrostatic) effect from
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viewpoint of membrane separation mechanism [52,53]. For continuous mode operation of a

fermentative process using renewal carbohydrate sources for GA production, the components
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that need to be continuously separated from fermentation broth are microbial cells, proteins,
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nutrients (yeast extract, salts of ammonium, potassium, phosphorus etc), unconverted carbon

sources, water and GA. Membranes suffer from fouling by microbial cells and proteins,
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though extent of fouling may be far less in nanofiltration/reverse osmosis membranes

compared to that in microfiltration membranes. However, there are some particular

membrane based modules which may be operated long without much fouling like flat sheet

cross-flow types (Fig. 6). To separate the microbial cells for their subsequent recycling to the

bioreactor to ensure high cell concentration and thus high productivity, microfiltration

membranes normally used with high pore size (0.1 0.45 µm) among the categories.

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Nanofiltration membranes being in between reverse osmosis and ultrafiltration membranes

with average pore size less than 1 nm are able to separate cells, proteins, nutrients, salts and

unconverted carbon sources from GA fermentation broth. Reverse osmosis normally known

as nonporous membrane where separation is based on solution diffusion mechanism can

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separate the same components from fermentation broth as nanofiltration membranes do but it

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required higher operating pressure than what is needed in nanofiltration [16]. Whereas

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ultrafiltration membranes with average pore size less than that of the microfiltration

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membranes can retain proteins along with the cells. Separation by microfiltration and

ultrafiltration membranes is based on size-exclusion and molecular weight cut off (MWCO)

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value should be ensured [54]. Solutes having larger molecular weight (MW) than the MWCO

of a membrane are rejected almost by the membrane and the ones having lower MW than the
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MWCO of a membrane will permeate easily through the membrane. That is so called the

sieving effect. Thus, solutes having different MWs can be separated basing on sieving effect.
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The Donnan effect of a membrane refers to the electrostatic interactions between ions and the
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membrane. The membrane is charged and mostly negatively charged since the thin films of

NF membranes are made of polyelectrolytes. Ions having the same sign of charge as the
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membrane charged are excluded, and ions having the opposite sign of charge can be attracted.
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Separation of electrolytes’ ions having different signs and valences can be manipulated

according to the rejection differences by the membrane [52,53].

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The modules of micro, ultra, nano or reverse osmosis membranes can be coupled with

fermenter permitting continuous removal of acid from the broth and separation and recycle of

cells, nutrients and unconverted carbon sources. The type of membrane used is the deciding

factor for the separation and recycle of the components. For example on use of microfiltration

membrane, microbial cells could be retained in the retentate side while permitting acids,

unconverted carbon sources, protein nutrients and water to pass to the permeate side. If an

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ultrafiltration membrane module is used in place of microfiltration module then proteins

along with cells get retained. This however, ensures continuous removal of acids from the

fermentor helping to arrest lowering of pH. In such continuous process, pH adjustment may

be redundant. If nanofiltration or reverse osmosis membrane is used in place of

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microfiltration then all the components barring acid solution are retained.

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6.2. Operating processes

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For operation of membrane, a membrane module, pump, pressure gauge, control valve,

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rotameter etc are required. The type of membrane used in the module is the deciding factor

for using the type of pump for feeding. For microfiltration membrane, low pressure (2–4

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kgf/cm2) pump may be used. For an ultrafiltration module, a pump of 4–7 kgf/cm2 is used.

Nanofiltration module demands high pressure pump (5–15 kgf/cm2) whereas reverse osmosis
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membrane system requires still higher pressure (>20 kgf/cm2) for filtration. The recycling

and permeation of the components depends on the types of membranes used in the module.
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The separation through electrodialysis membrane is based upon electromigration of ions

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through a stack of cation and an anion exchange membrane basically involves two steps-

conventional electrodialysis (CEP) and the bipolar electrodialysis (BED). The first step
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(CEP) separates and concentrates organic acid salt and the second step (BED) convert the salt
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form into acid form. The next section reviews the developments in the first stage of

separation and purification by microfiltration and ultrafiltration membranes and then moves
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over to nanofiltration, reverse osmosis and electrodialysis.

6.3. Microfiltration and ultrafiltration of fermentation broth

Microfiltration and ultrafiltration of fermentation broth are generally used for cell recycle

and sometime for the recovery of product. These may be applied to overcome the problem of

substrate-product inhibition during fermentation based batch production process [55,56]. In

addition to that the time loss for shut down and start up after every batch of production is

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major concerned. As the concentration of glutamic acid in the fermentation cell goes up,

microbial activities start getting reduced due to increase difficulty of survival of microbes in

low pH medium. Variation of pH-effects on bacterial growth results from the presence of

dissociated and undissociated forms GA in fermentation broth. The undissociated form is

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more inhibitory than dissociated form. At low pH undissociated form dominates and at high

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pH complete dissociated form of GA takes place. According to Milcent and Carrere [57], pH

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is a key parameter for studying the membrane based separation coupled to fermentation.

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Lowering of pH resulted in decrease of flux and vice-versa.

It was identified that critical fluxes were function of cross velocity. Due to irreversible

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fouling resulting from adsorption of molasses compounds used as a carbon source on the

membrane surface, cross flow velocity did not lead to an increase of flux. They did not reuse
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the separated cells resulting in low productivity. To ensure high productivity in a fermenter,

two important tasks are required at the outset, firstly, removal of acid (amino acid) from the
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fermentor medium to maintain optimum pH and secondly, recycle of the microbial cells at
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the late-logarithmic growth phase of the microbial cells. For that membrane a separation unit

should be coupled with a fermentor in an external unit permits continuous separation and
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removal of amino acids from the fermentation broth preventing lowering of medium pH to
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inhibition level and this simultaneously permits cell and recycle also. Like, Taniguchi et al.

[58] achieved 29-fold increase (compared to the system without filtration) by removing
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organic acid from the broth by a cross flow filtration and by recycling the cells retained on

the microfilter to the fermentor. This ensured high cell concentration (81.5 g dry cell/L) in the

fermentor. Cell harvesting by microfiltration or ultrafiltration for its subsequent recycling

leads to high cell concentration in the fermentor but often excessive build up increases

viscosity and causes lowering of flux. To overcome this problem Crespo et al. [59] suggested

cell bleeding.

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 Membrane fouling is a major barrier in the way of effective use of the MF/UF membrane.

During the micro or ultra-filtration of the fermentation broth, chances of membrane fouling

by microbial cells, proteins etc are obvious. If the membrane module operated is in dead-end

mode, concentration polarizations build up rapidly, resulting in decreased flux. However,

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build-up of concentration polarization is very much dependent on the mode of operation and

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type of membranes. Chances of concentration polarization are drastically reduced if the

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system is operated in a cross-flow module, due to sweeping of the fluid on the membrane

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surface. To overcome the problem of membrane fouling in microfiltration stage, Torang et al.

[60] suggested a shear-enhanced cross-flow ultrafiltration module for separation of cells and

an
proteins from fermentation broth. However, this fouling problem can be significantly reduced

with use of the cross-flow module, as the fluid travels parallel to the surface of the membrane
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imparting a sweeping action on the membrane surface and thus leaving very little scope for

the formation of the concentration polarization layer. Fouling problem which is the most
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serious obstacles in the application of membrane technology to the fermentation broth

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filtration is arises due to the formation of a deposited layer, which has a harmful effect both

on the permeation rate and the ease of membrane cleaning, on the membranes during
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ultrafiltration (UF) operation [61]. Although there are possibility to reduce fouling and delay
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its onset by adopting hydrodynamic and back-flushing technique [62,63], changing

membrane material and mode of operation [16], but it is impossible to eliminate the fouling.
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The membrane manufacturers have recommended majority of cleaning protocols which

include of a series of acid-alkali-cleaning cycles depending on the feed processed and

membrane material. The main way to restore and maintain the permeability and selectivity

performance of a membrane system is to periodical chemical cleaning. Another factor is

membrane materials which play vital role in fouling problem. Like use ceramic membranes

permitted easy disinfection, but ceramic membranes suffer from quick fouling and can retain

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Page 14 of 55
only cells where unconverted substrate gets lost. Polyamide membranes showed lower flux

reduction than Polysulfone types but direct ultrafiltration of broth without microfiltration

resulted in quick reversible fouling as the system was operated at cross flow velocity to

protect the microbes from shear.

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Hydrophobic (polyethersulfone) membrane (MWCO 25 kDa) retained 100% protein but

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due to the blocking of the pores by protein adsorbed on to the hydrophobic membrane surface

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the flux was higher for hydrophilic (regenerated cellulose acetate) membrane with MWCO of

20 kDa and flux of 12–85 L/m2 h. Hydrophilic membranes used had low protein binding

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tendency. The chemical stability and as well as protein separation was better for hydrophobic

an
membrane so microfiltration or centrifugation were suggested before the ultrafiltration to

make the ultrafiltration step more efficient by avoiding the fouling of the membrane by high
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molecular weight protein. The mechanism of flux decline by the broth of MF/UF membranes

are well understood and documented, there have been reported data on cleaning membranes
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fouled during broth filtration. The formulation of an optimal membrane-cleaning strategy

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through a systematic approach may lead to important process improvements including

optimized use of chemicals (hence minimized environmental impact), reduced loss of

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production time, improved permeate flux and quality control, and extended lifetime of the
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membranes which will add the economy to the overall processes. By controlling tangential

flow in the PVDF microfiltration membrane (Millipore) attached with a fermentor for
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downstream separation of cells, the problem of concentration polarization and fouling effect

was controlled [64].They have claimed that membrane was operated 155 h with on-line

sterilization and cleaning of the membrane using NaOCl and distilled water.

Moueddeb et al., [65] have designed a set-up in microfiltration membrane bioreactor that

consisted of two coaxial alumina tubes having alpha alumina membrane (pore size: 2.0 ×
7
10 m) on the inner wall of the inner tube and on the outer wall of the outer tube of the

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Page 15 of 55
tubular membrane, aimed at total substrate conversion in to product. The micro-organisms

were fixed in the macroporous support and confined in the annular space of two coaxial

porous tubes of a tubular membrane. The substrate solution was fed into the reactor inner

compartment whereas the liquid percolated in the radial direction across the two membranes.

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The organic acid was produced in the porous space between the two microfiltration layers.

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Though the new design focused on elimination of bacterial inhibition giving total substrate

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conversion, flux decreased rapidly due to membrane plugging by microbes which could,

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however, be reduced to some extent by sterilization of the ceramic membranes.

Feedback inhibition problem may be removed by direct removal of product from the

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fermentation broth. Giorno et al. [66] integrated one cross flow membrane module fitted with

microfiltration or ultrafiltration capillary membranes with a 2.5 L stirred cell. They have also
M
suggested that the ceramic membranes have the advantage of easy disinfection in comparison

to polymeric membranes. Ultrafiltration membranes can retain both cells and proteins. Xavier
d

et al. [67] coupled ceramic ultrafiltration membrane to a fermentor to separate both cells and
te

proteins for their recycling and simultaneous removal of acid from the medium.

6.4. Electrodialysis
p

Electrodialysis (ED) is an electro-membrane process in which ions are transported

ce

through ion permeable membranes from one solution to another under the influence of

potential difference across the electrodes. Therefore, ED can separate selectively ions on the
Ac

basis of their charge [68,69]. The main industrial application of this process is found in chlor-

alkali, water purification, and industrial effluent treatments [70,71]. The most recent

application of electrodialysis is in the separation and production of organic acids from

fermentation broths and enzymatically produced solutions [72-74]. Electrodialysis (ED) and

bipolar membranes electrodialysis (BMED) as well as combination of ED and BMED have

also been widely used for desalination, concentration, separation and purification in many

16
Page 16 of 55
fields. Like desalination of brackish water [75,76] and de-ashing of milk whey [77] are the

main application areas of ED. As fermentation based production requires constant

maintenance of near neutral pH for high productivity and this necessitates of alkali in most of

the cases where product is acidic in nature. Traditionally, GA is separated from fermentation

t
broth by isoelectric crystallization under low temperature with or without biomass. About 1-

ip
2% residual GA still remains in isoelectric supernatant, after separation through

cr
crystallization. Ion-exchange process was used for the removal of residual GA from

us
isoelectric supernatant [78]. Although large amounts of acid and base are consumed in order

to regenerate the ion-exchange resins, for the higher recovery for GA, which will increase the

an
operational cost. In addition, wastewater produced from the process to reactivate and wash

the ion-exchange resin will cause serious environmental pollution and increase the burden on
M
subsequent wastewater treatment. Zhang et al., [36] have used two electrodialysis processes:

two-compartment bipolar membranes electrodialysis and modified traditional electrodialysis,

d

to recover glutamic acid from isoelectric supernatant. Comparison of the two electrodialysis
te

processes indicated that the modified traditional electrodialysis was more efficient method

than two-compartment BMED. Higher recovery ratio of glutamic acid and lower energy
p

consumption were obtained by the modified traditional electrodialysis.

ce

Thus in the light of above facts, two routes of the membrane technologies can be used for

the recovery of GAH from GANa: (i) the BMED process, to convert GANa into GAH and
Ac

caustic soda and their recovery; (ii) acidification of GANa by sulphuric acid and subsequent

separation of GAH and sodium sulphate by ED using ion-exchange membranes. In recent

years, ED processes are also used in the production of organic acids, such as acetic acid,

propionic acid and lactic acid [79-82]. Due to its ions selective transport, organic acid can be

separated and concentrated. The problem of disposal of by-product gypsum associated with

conventional fermentation process can be largely overcome through electrodialysis. Datta et

17
Page 17 of 55
al. [83] studied the advantages associated with such electrodialysis process. For maintaining

pH, agents like NH4OH or NaOH used during fermentation can also be recycled by ED,

which will simultaneously reduce the environmental pollution [84-85].

Recently, bipolar membrane electrodialysis (BMED) has been developed for the

t
conversion of salts into corresponding acids and bases [86,87]. The bipolar membranes are

ip
able to split water at high applied potential, and generate the hydroxyl and hydrogen ions at

cr
the anode and cathode side. Several studies have shown that, the BMED process has

us
economic potential for recovery of inorganic, organic, and amino acids [88].

Energy consumption associated with ED processes is normally high and to make the

an
economically attractive, attempts have been made to increase energy efficiency. Shen et al.,

[89] were carried out experiments in the modified laboratorial electrodialyser consisting of
M
seven compartments with alternating cation- and anion-exchange membranes, which was a

self-made apparatus for the separation of glutamine from its fermentation broth. They
d

optimized energy consumption was 1.95 kW h kg 1sulphate under the operation of current
te

density of 204 A/m2 at a current efficiency of 81%. Kumar et al. [90] have developed an

electro-membrane reactor with four compartments (EMR-4) (anolyte, catholyte and comp. 1
p

and 2) based on in-house-prepared cation- and anion-exchange membrane (CEM and AEM,
ce

respectively) to achieve separation and recovery of glutamic acid (GAH) from its sodium salt

by in situ ion substitution and acidification.

Ac

Despite of several studies have been undertaken to establish the potential of bipolar

electrodialysis as an efficient and eco-friendly method of glutamic acid production,

concentration and purification, commercialization of bipolar membrane itself has been done

in very limited cases and electrodialysis fermentation (EDF) for glutamic acid has hardly

been commercialized. Due to poor conductivity of the organic phase power consumption in

electrodialysis process is always high.

18
Page 18 of 55
6.4 Nanofiltration and Reverse Osmosis

Purification and recovery of amino acids by NF is relatively new class of the pressure-

driven membrane processes and its application for such purposes is a viable alternative over

traditional separation processes like extraction, ion-exchange, evaporation and distillation.

t
NF membranes have been developed recently and adept for the separation of small neutral

ip
and charged solutes in aqueous solution. The performance of a nanofiltration system in terms

cr
of flux and retention of the target solutes will depend on solution pH, cross-flow rate,

us
membrane type, membrane module and presence of impurities. Nanofiltration (NF) is widely

used in water softening, dye recovery, bio-product separation, desalination and wastewater

an
treatment [91-95]. NF membranes can reject salts due to charge effects since the separation

layer is made of polyelectrolytes [96]. Nanofiltration applied to the separation of L-

M
Glutamine from fermentation broth in the place of traditional ion-exchange technology not

only can reduce acid and basic waste treatment but also can prevent the conversion of L-Gln
d

into L-Glu on the ion-exchange resin in the ion exchange separation process [97].
te

There are many studies have already been focused on the separation of amino acids by

NF membrane [98,99]. Tsuru et al., [98] firstly found that several commercial polymeric NF
p

membranes tested for single amino acids and peptides at their pI (net charge zero) showed a
ce

lower rejection than for amino acids and peptides with a net charge. They also showed that

the separation of amino acids and peptides could be manipulated by modifying the pH value
Ac

of the solution. The rejection difference between charged and non-charged amino acids was

mainly explained by occurrence of the Donnan effect [98]. During the NF of a mixture of

nine amino acids, acidic amino acids (anions) are separated at pH <3 and basic amino acids

(cations) are separated at pH >9 [99]. Whatever the solution studied, charge effects, repulsion

of co-ions and attraction of counter-ions, more than sieving effect, prevail in the behaviour of

the solute [100]. NF of amino acids was strongly influenced by the solute environment and by

19
Page 19 of 55
hydrodynamic parameters [101]. The selectivity of the separation of amino acids can be

manipulated by modifying the charged state of the amino acids by changing the pH value, or

by modifying the salt composition and concentration of the feed solution [102,103].

Nanofiltration of some amino acids like L-phenylalanine and L-aspartic acid aqueous

t
solution were carried by Wang et al., [104] by using two commercial NF membranes

ip
(ESNA2 and ES20). They found that the rejections to L-Phe and L-Asp by ESNA2

cr
membranes are about 0 and 90% respectively at the pF value ranging from 4 to 9, while ES20

us
membrane are almost 100% irrespective of pH value. They have concluded that these two NF

membranes are possible to concentrate and separate L-Phe and L-Asp effectively by choosing

an
proper condition such as the pH value of solution.

Separation of l-glutamine (L-Gln) from Gln fermentation broth by nanofiltration (NF)

M
was investigated with changing the experimental parameters such as transmembrane pressure,

pH and concentration of broth on the rejection of L-Gln and L-glutamate (l-Glu) showed that
d

NTR7450 was able to effectively separate l-Gln and l-Glu when the appropriate conditions
te

were chosen [105]. Kovacs and Samhaber, [106], have used the nanofiltration membrane for

the concentration of amino acid. To determine the permeate flux and amino acid rejection
p

from aqueous solutions diprotic amino acids (L-glutamic and glycine) as a function of
ce

increasing feed concentration and ionization state of the amino acids, Kovacs and Samhaber,

[106], have used numerous polymeric NF and tight UF membranes. The concentration of
Ac

amino acids in the whole range of their solubility was studied with a stepwise pH scan

ranging from 0 to 1 total net charge. Considerable higher rejection and flux drop over the

concentration was observed in higher pH range, where amino acids are present in dissociated

form. Membranes with different type of active layer material show similar concentration

dependent tendency in the permeation behaviour. This phenomenon can be explained by the

dissociation dependency of the osmotic pressure. In case of glutamic acid, at pH 8, where net

20
Page 20 of 55
charge is 1, a less pronounced rejection drop (95 75%) was measured over the

concentration than close to its isoelectric point (from 90 to 5%). McGregor [107] has used the

thin film composite reverse osmosis (RO) membrane to concentrate L-phenylalanine from

clarified bioreactor harvested media. He achieved the 100 g/L concentration at pH 10 and 50

t
0
C with flux from 17 to 119 L/(m2h). He suggested that applications of RO are likely to be

ip
case specific.

cr
A combined process of nanofiltration and reverse osmosis was developed by Li et al.

us
[108] for separation and concentration of lactic acid from cheese whey fermentation broth.

Five NF membrane (CK, DK, DL, HL, and GE) and two reverse osmosis membrane (DS 11

an
AG and ADF) were tested at different pressures. After nanofiltration reverse osmosis was

applied to concentrate lactic acid. 100% lactic acid retention was achieved at 5.5MPa
M
pressure for ADF membrane compared to DS11 AG membrane 96% at the same pressure.

Membranes suffered quick fouling in absence of prior filtration of microbial cells.

d

6.5. Chemistry of NF membrane for separation of solute

te

Estimation and use of effective membrane charge density for practical applications is
p

rather complex and very few quantitative description of the transport phenomena of amino
ce

acids has been reported so far. Teixeira et al., [109] have demonstrated the importance of

divalent hardness (CaCl2 and MgSO4) on the NF performance with pH in terms of both flux
Ac

and retentions of ions by using natural water. The steric hindrance and membrane solute

interactions are two major factors for separation of nanofiltration membrane [110,111]. For

the retention of uncharged molecules, steric hindrance and non-electrostatic membrane–

solute interactions (e.g. Van-der-Waals forces) are mostly responsible, and their transport

takes place by convection due to a pressure difference and by diffusion due to a concentration

gradient across the membrane [112,113]. Polarity decreases retention, which can be explained

21
Page 21 of 55
by electrostatic interaction directing the dipole towards the membrane [114]. Steric hindrance

and electrostatic interactions are responsible for separation for charged compounds [110]. In

addition to that another important parameter in the transport process through the membrane is

the membrane charge along the surface and through the pores [115]. When membrane surface

t
in contact with an aqueous solution acquire an electric charge by some mechanism like ionic

ip
surfactants and macromolecules, adsorption of polyelectrolytes, adsorptions of ions from the

cr
solutions and dissociation of surface functional groups [116].To maintain the

us
electroneutrality of the system, this charging mechanism takes place on the exterior

membrane surface and on the interior pore surface of the membrane, because of the

an
distribution of ions in solution [115].The ion separation resulting from the electrostatic

interactions between ions and membrane surface charge is based on the Donnan exclusion
M
mechanism [117].In this mechanism the co-ions (which have the same charge of the

membrane) are repulsed by the membrane surface and to satisfy the electroneutrality
d

condition, an equivalent number of counter-ions is retained which results in salt retention.

te

Retention of neutral solutes by size exclusion is dependent on two parameters of

membrane (effective pore radius, thickness-porosity ratio) and also on stoke radius of solute.
p

In addition to the solute transport, membrane properties like pore radius (rp), effective
ce

thickness- porosity ratio(∆x/Ak) are determined by comparison and convergence between

model and experiment rejection data of sucrose, undissociated glutamic acid as neutral
Ac

solutes. Ionic rejection is mainly based on Donnan effect and dependent on three parameters

(effective pore radius, thickness-porosity ratio and effective charge density of membrane).

6.6. Effect of pH of the solution on the NF membranes performance

The osmotic pressure of feed solution containing amino acid is greatly affected by the

pH. Amino acids are ionisable compounds. In neutral aqueous media, diprotic AA with non-

ionisable residual group is predominantly present in zwitter ionic form as a result of

22
Page 22 of 55
intermolecular proton transfer. If the pH of the solution is higher than their isoelectric point,

dissociation takes place, which results the anionic form. It has been reported in several

studies, that a considerably higher rejection can be observed for AA of anionic form than for

zwitterions [104]. As the amount of undissociated glutamic acid and glutamate salts present

t
in glutamic acid solution depends on the pH equilibrium, the Henderson-Hasselbalch

ip
equation may be incorporated in model development to correlate the pH effect on glutamic

cr
acid transport.

us
Apparent pore size of polyamide NF membranes can also vary with solution pH. The

protonation and deprotonation of the functional groups present in the membrane surface and

an
the molecules in the solution, are very much dependent on pH over its range. This will

change the membrane charge and the size of the membrane pores with consequences in the
M
NF and UF performance [118]. At the pore surface points of zero charge (isoelectric point),

the membrane functional group is minimal in charge and hence opens up, as the absence of
d

repulsion forces contribute to the widening of the membrane pores. At high or low pH value,
te

functional groups of membrane polymers can dissociate and take on positive or negative

charge functions. Interactions between these functions in the membrane polymer reduce or
p

close up membrane pores. At higher pH values, addition of sodium hydroxide leads to an

ce

increase in osmotic pressure and ionic strength, thus reducing the membrane permeability and

increasing rejection. Moreover, functional groups such as carboxyl and hydroxyl groups
Ac

present at the surface of the membrane become deprotonated at high pH. High pH results in

an increased thickness of the double layer of the charged functional groups over the surface

of the membrane thus reducing the apparent pore size and resulting in greater rejection of the

charged solutes [119]. In addition to that solution chemistry of natural waters like, pH,

alkalinity, salinity, TDS and hardness cations play a significant effect on the membrane

charge and on the characteristics of the molecules in the solution. The NF membranes interact

23
Page 23 of 55
with hardness cations during separation process, so they could have a marked effect on

fouling and NF performance [120]. Streaming potential measurements along surface and

through pores with several electrolyte solutions at different pH help to investigate the charge

of the membrane surface and pore.

t
7. New approach: Integrated Membrane System

ip
Membranes can be tailor-made in such a way that a high degree of selectivity can be

cr
ensured, which in turns means that very high degree of purity can be achieved during

us
downstream processing. Membrane fouling that is considered a major hindrance in membrane

separation can be overcome by using proper module (flat sheet cross-flow membrane

an
module). In the synthesis and down-stream processing of amino acids (AA), the purification

and recovery is a challenge, where NF is a promising separation tool. After fermentation,

M
glutamate is the most abundant free amino acid in bacterial cytoplasm and when micro-

organism overproduces glutamate in excess of their normal metabolic needs, it excretes into
d

culture broth. The major technology barrier in cost-effective production of high purity of
te

glutamic acid is its down-stream separation and purification from the fermentation broth. And

this is where; membrane-based processes are stepping in. Being modular in design,
p

membrane-based processes offer great flexibility in scale of production depending on market

ce

demand. By virtue of high selectivity, membranes can ensure high levels of separation and

purification. As membranes of chosen selectivity and permeability can easily be integrated

Ac

with conventional fermenters, membrane-based processes permit simultaneous production

and purification in the same unit. This eliminates the need for separate purification units and

results in compact design with reduced capital investment. Membrane-based separation and

purification (barring pervaporation) involves no phase change ensuring reduced energy

consumption. Thus such processes can meet all the goals of process intensification.

24
Page 24 of 55
 For downstream processing, conventional processes often require many chemicals that

may lead to environmental pollution. Many downstream units in conventional process are

also energy-intensive. Compared to such conventional processes, membrane based processes

involve very low energy consumption. With integration of membrane modules with

t
fermenter, continuous production can be ensured with recycle of cell and unconverted carbon.

ip
In such continuous mode of fermentation and purification, production can be ensured without

cr
any pH adjustment. Recycling of cells through the retentate of the microfiltration unit ensures

us
high sell concentration in the fermenter leading to high productivity. This may be a simple

production scheme involving a few steps only contrary to the use of a number of downstream

an
treatment steps of conventional production scheme. So many units in a conventional process

such as precipitation, filtration, acidification, extraction, neutralization, carbon adsorption,

M
crystallization and evaporation as shown in the Fig. 4 can be turned redundant. In

conventional production process, addition of lime for controlling pH leads to production of

d

calcium glutamate. Calcium glutamate is then separated from the microbial cells by filtration
te

and, further purified by activated carbon adsorption. In next phase, calcium glutamate is

evaporated and acidified by sulphuric acid to produce glutamic acid. Conventional batch
p

fermentation also suffers from low volumetric productivity due to both substrate and product
ce

inhibitions in addition to involvement of high labour cost following shutdown and start-up of

such batch processes [64]. Over the last two decades, several attempts have been made in this
Ac

direction of integration of traditional fermentor with membrane based separation and

purification. Literature shows that membrane integrated processes are rarely used for the

continuous production. In most of these studies, however, only a single stage of membrane

separation has been integrated with fermentor. In addition to that in these cases, studies have

been conducted with finished raw materials like glucose. Serious fouling is another problem

associated with these membrane modules used in these studies [16]. US Patent [121],claimed

25
Page 25 of 55
to have developed a membrane-integrated process with the combination of ceramic tubular

UF module in the first stage for cell separation and NF in the second stage for some organic

acid separation. In very few studies, two-stage membrane separation has been attempted.

Like Gonzalez et al. [122] recovered lactic acid from ultrafiltered whey by two types of

t
membranes – spiral wound DK 2540C and tubular AFC80 nanofiltration membranes. Some

ip
authors have produced organic acid by integrating membrane separation with conventional

cr
fermentation process and have shown the possibility of similar process intensification for

us
many other chemical process industries [123,124]. The situation improved when MF/UF of

the broth was done prior to nanofiltration or reverse osmosis. Two stage novel membrane-

an
integrated (micro and nano membrane) fermentor under non-neutralizing conditions. Dey and

Pal [125] have used novel two stage membrane integrated system for the simultaneously
M
production and purification of organic acid (lactic acid).

Membrane-integrated hybrid cell recycle bioreactors system with continuous

d

fermentation can mostly overcome these problems due to high cell density, much higher
te

productivity and higher acid concentration in a continuous process [16]. One of the most vital

criteria for continuous fermentation is steady state operation with prolonged exponential
p

growth phase and needs to be maintained with proper cell bleeding. The long term
ce

performance of such a membrane-integrated system ensures a largely fouling-free operation

to ensure a desired flux for commercial viability when operated by a properly selected
Ac

membrane module as demonstrated by Sikder et al. [126]. It can offer such fouling free long

hours of operation by virtue of sweeping flow of the fermentation broth over the membrane.

To improve glutamic acid concentration as well as productivity, studies on multistage

membrane cell recycle bioreactor may be exploited. The productivity and yield are based on

use of carbon source like glucose. Alternate renewal carbon source should be searched to

make the process more economic. To ensure long term operation, selection of membrane

26
Page 26 of 55
module is also very important, like involving expensive hollow fibre membrane modules that

accounted for 28% of the total capital cost. In this membrane module, additional purification

steps like precipitation through sulphuric acid, colour removal using adsorbent are necessary

to get the final product as pH adjustment is still done resulting in either sodium or ammonium

t
glutamate instead of glutamic acid directly. Multivalent ions and disaccharides are rejected

ip
by nanofiltration membranes [127]. Understanding these effects is essential to successful

cr
modeling and scaling up of the process. Thus for better understanding of the hydrodynamics

us
and transport phenomena in a more realistic setting where pure glutamic acid can be directly

obtained instead of glutamic acid salt like sodium glutamate which dominates most of the

an
reported studies in the available literature.

8. Discussion
M
The present review study aimed at investigating the feasibility of using nanofiltration for

separation of unconverted sugars from fermentation broth and purification of glutamic acid
d

solution under different operating conditions with provision of immediate separation of

te

glutamic acid from fermentation media eliminating the need of addition of caustic solution

for pH adjustment. Model solutions were also filtered along with actual fermentation broth to
p

find out the impact of very low pH on the transport phenomena as during actual fermentation
ce

without pH adjustment in a membrane-integrated system, low pH regimes are likely to

dominate.
Ac

9. Conclusion

It thus transpires that through the tireless efforts of early researchers on membrane-based

production schemes, world has definitely moved towards a better processes but serious

attention still needs to be paid to some areas to evolve a smaller, more compact, more

flexible, and less energy-intensive plant that could guarantee large scale production of a

highly demanding chemical product in an environmentally benign process. Such a plant, in

27
Page 27 of 55
other words, may be called to represent high degree of process intensification which modern

chemical process industries are desperately seeking for their survival in highly competitive

and environmentally conscious world market. Selection of appropriate membranes as well as

modules in cell separation and product purification along with provision of logical

t
sequencing of operations are essential in truly achieving such process intensification.

ip
Acknowledgment

cr
Authors are thankful to the Department of Science and Technology, Government of India

us
for financial support under Start-Up Research Grant for Young Scientist (SERB)

an
(SB/FTB/ETA-59/2013).
M
d
p te
ce
Ac

28
Page 28 of 55
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Tables and Figures

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Table 1 Different type of microbial strains producing L-glutamic acid

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________________________________________________________________________
Genus Species
________________________________________________________________________

cr
Corynebacterium C. glutamicum, C. lilium, C. callunae, C. herculis

Brevibacterium B. divaricatum, B. aminogenes, B. flavum, B. lactofermentum,

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B. saccharolyticum, B. roseum, B. immariophilum, B.

alanicum, B. aminoniagenes, B. thiogenitalis

Microbacterium

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M. salicinovolum, M. ammoniaphilum, M. flavum var.

glutamicum
M
Arthobacter A. globiformis, A. aminofaciens
___________________________________________________________________________
d
p te
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Ac

44
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 Table 2 Micro-organism used for the production of L-Glutamic acid
________________________________________________________________________________________________________________
Micro-organism Substrate (carbon source) Yield (g/L) References
________________________________________________________________________________________________________________

Micrococcus glutamicus Glucose 30 [128]

/Corynebacterium glutamicum
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Cephalosporium distiller’s soluble, corn steep liquor 1 to 31/2 - [129]
ce
Bacillus strain 14B22 glucose 12.5 mg/mL [130]

Arthrobacter globiformis
p glucose
te 0.45 M per moles of Glucose [131]

Brevibacterium divaricatum hydrolysed grain sugars 41.5 g/L [132]

Brevibacterium
d
Saccharide material 4 g/dl [133]

Micrococcus glutamicus Molasses

M 40 g/L [134]

Brevibacterium flavum Na/K acetate 15 g/L [135]

ATCC No. 13826
an
Brevibacterium roseum acetic acid 14.8 g/L [135]
ATCC No. 13825
us
Brevibacterium lactofermentus Na/K acetate 14.3 g/L [135]
ATCC No. 13869
cr
Corynebacterium acetoacidophilum acetic acid 7.3 g/L [135]
ATCC No. 13870
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 Corynebacterium hydrocarboclastus glucose 6.3 g/L [136]
(M-104)

Corynebacterium corn steep liquor 5 g/L [137]

Nocardia globerula Hydro-carbon 4 g/L [138]

ATTC15076
Ac
Bacillus thiogenitalis No. 653 Oleic acid 50 mg/mL [139]
ce
Corynebacterium alkanolyticum glycerol 40 mg/mL [140]
No 314
p
Brevibacterium flavum (AJ 3612) Polyoxy ethylene-sorbitan-mono-palmitate 52mg/mL [141]
te
Brevibacterium lactofermentum 2256 Beet molasses 2 g/dL [142]
d
Brevibacterium divaricatum NRRL 2311 Ethanol M 25 g/L [143]

Brevibacterium (mutant) glucose 52 g/L [144]

Arthrobacter globiformis glucose mineral salt 16.1 g/L [145]

an
Brevibacterium species glucose (2%) 6.68 mg/mL [146]

Brevibacterium species Sugar cane baggase (10% glucose) 80 mg/g [147]

us
dry baggase

Micrococcus glutamicus glucose 37.1 g/L [148]

cr
+ Pseudomonas reptilivora
ip
Brevibacterium Sp DSM. 20411 Cassava starch hydrolysate 21 g/L [149]
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 Corynebacterium glutamicum 2262, glucose (fed batch) 80 g/L [150]

Brevibacterium sp. Tc452 glucose 41.42 g/L [151]

Brevibacterium divaricatum Cassava starch (sub. culture) 3.86% [152]

Corynebacterium glutamicum lime citrus aurantifoliaswingle 13.7 g/L [153]

Ac
(ATCC 13032) + 2% glucose
ce
Brevibacterium roseum glucose medium 0.5 g/L [154]
p
Corynebacterium glutamicum (CECT 690) date waste juice 39.32 mg/mL [155]
_________________________________________________________________________________________________________
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Figures No: Captions

Figure 1 A co-ordination between medium design and microbes screening

process has promoted synergistic advantages as well as decreasing the

chance of missing a better performance system [28].

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Figure 2 Cell permeability of L-glutamic acid in relation to phospholipids

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contents in the membrane.

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Figure 3 Regulatory pathway for biosynthesis of glutamic acid

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Figure 4 Typical conventional fermentation-based glutamic acid production
scheme

Figure 5 Flow-sheet of conventional purification of glutamic acid

an
Figure 6 Schematic diagram of a cross-flow membrane separation process
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 t
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Figures 1

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Figure 2
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Figure 3

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Figure 4
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Figure 5
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Figure 6
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Research Highlights

PALRKVCCEP

Research Highlights

 Review of L-glutamic acid production processes

 Drawbacks of conventional processes reviewed critically
 Possibilities of new eco-friendly processes described
 Membrane-based processes examined for process intensification

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