COMPARATIVE STUDY OF GLUTAMIC ACID PRODUCTION BY WILD-TYPE AND MUTANT STRAINS OF CORYNEBACTERIUM GLUTAMICUM

ABSTRACT

Several different lignocellulosic biomass of agricultural origin hold remarkable potential for conversion into commodity products presenting dual advantage of sustainable resource supply and environmental quality. There is generally an increasing demand for amino acids especially L-glutamic acid as growth promoting factor, as well as flavour enhancer in foods. The present study was an investigation on comparative L-glutamic acid production by wild-type and a mutant strain of Corynebacterium glutamicum (CG^NTA) using rice husk pretreated with 1.0M H₂SO₄ and 1.0M KOH. The acid-treated and alkali-treated rice husk with high carbohydrate content of 64.25% and 76.37% respectively as determined, were used for the production of glutamic acid by submerged fermentation. The acid-treated and alkali-treated rice husk at concentration of 4% gave the highest glutamic acid yield of 27.84g/L and 15.72g/L respectively with the developed mutant strain (CG^NTA) under predetermined optimum fermentation conditions (30^oC, pH 7.0, 4% substrate concentration and 7% inoculum size). In contrast, lower yields of 10.40g/L and 9.08g/L respectively were obtained with the wild type strain under similar optimum culture conditions. Out of four parameters optimized, all were found to significantly (p˂0.05) influence glutamate production from both the acid and alkali-treated rice husk by the CG^NTA. Similarly, all parameters except variation in the concentrations of the acid and alkali-treated rice husk (p˂0.05) were found to be significant on the performance of the wild-type strain in glutamate production. Acid-treated rice husk hydrolysate was determined to be a better substrate for L-glutamate production by the CG^NTA mutant than the wild type strain of C. glutamicum. The mutant strain (CG^NTA) developed could, therefore, be useful in the industrial production of glutamic acid using rice husk as substrate pretreated with acid. This may perhaps form the basis of starting a microbial L-glutamate production industry from rice husk as substrate in this locality and Nigeria as a whole.

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3.3 Treatment of the Rice Husk (Substrate Pre-treatment) --------------------------------43

3.3.1 Alkaline Pre-treatment of Rice Husk--------------------------------------------------------43

3.3.2 Acidic Pre-treatment of Rice Husk----------------------------------------------------------43

3.4 Proximate Analyses of the Pre-Treated Rice Husk--------------------------------------44

3.4.1 Determination of Moisture Content---------------------------------------------------------44

3.4.2 Determination of Ash Content (AC) -------------------------------------------------------44

3.4.3 Determination of Crude Fibre Content (CFb) --------------------------------------------45

3.4.4 Determination of Crude Fat (CF) ----------------------------------------------------------45

3.4.5 Determination of Crude protein (CP) -----------------------------------------------------46-47

3.4.6 Determination of Carbohydrate (CHO) ---------------------------------------------------47

3.5 Isolation and Characterization of Corynebacterium glutamicum--------------------47

3.5.1 Media preparation for Isolation of Corynebacterium glutamicum --------------------47-48

3.5.2 Isolation of Corynebacterium glutamicum------------------------------------------------49

3.5.3 Identification of Corynebacteriumglutamicum-------------------------------------------49-54

3.6 Screening of the C. glutamicum Isolates for Glutamic Acid Production------------54

3.6.1 Screening Medium ----------------------------------------------------------------------------54

3.6.2 Preliminary Screening of the Isolates------------------------------------------------------54-56

3.6.3 Standard curve of L-glutamic acid------------------------------------------------------56

3.7 Production of Regulatory Mutant of the Selected C. glutamicum Isolate----------58

3.7.1 Mutation with Nitrous Acid-----------------------------------------------------------------58

3.7.2 Isolation of Regulatory Mutants Using a Toxic analogue ------------------------------58

3.8 Glutamic Acid Production from Rice Husk by Submerged Fermentation--------59

3.8.1 Inoculum preparation ------------------------------------------------------------------------59

3.8.2 Basal Medium---------------------------------------------------------------------------------59

3.8.3: Shake Flask Fermentation------------------------------------------------------------------59

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4.6.5 Effect of inoculum size on glutamic acid production by the mutant (CG^NTA) --------77

4.7. Comparative Production of Glutamic Acid by the Mutant and Wild-Type

Strains of C. glutamicum Under Optimum Parameters--------------------------------------83

4.8. Purity Determination of the Produced L-Glutamic Acid-------------------------------83

CHAPTER FIVE-------------------------------------------------------------------------------------87

5.0 DISCUSSION-------------------------------------------------------------------------------------87-94

CHAPTER SIX---------------------------------------------------------------------------------------95

6.0 CONCLUSION AND RECOMMENDATIONS------------------------------------------95

6.1 CONCLUSION----------------------------------------------------------------------------------95

6.2 RECOMMENDATIONS----------------------------------------------------------------------96

REFERENCES---------------------------------------------------------------------------------------97-103

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LIST OF TABLES

Table                                                                                  Title                                                                                  Page

4.1 Proximate Compositions of the Treated Rice Husk Substrates-------------------------------65

4.2 Cultural, Microscopic and Biochemical Characteristics of the Isolates---------------------66

4.3 Occurrence of Corynebacterium glutamicum in Various Soil Samples Collected

from Samaru Village, Zaria----------------------------------------------------------------------------67

4.4 Screening for L-Glutamic Acid Production by the C. glutamicum^aIsolates ----------------68

4.5 Comparative Glutamic Acid Production (g/L) by Mutant and Wild Strains of

C. glutamicum from Acid-treated Rice Husk Hydrolysates under Optimum

Fermentation Conditions-------------------------------------------------------------------------------84

4.6 Comparative Glutamic Acid Production (g/L) by Mutant and Wild Strains of

C. glutamicum from Alkali-treated Rice Husk Hydrolysates under Optimum

Fermentation Conditions-------------------------------------------------------------------------------85

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LIST OF FIGURES

Figure                                                                                Title                                                                                  Page

2.1 L-Glutamic Acid Chemical Structure------------------------------------------------------------20

2.2 Regulation of L-Glutamic acid biosynthesis in Corynebacterium glutamicum------------39

3.1 Standard curve of L-glutamic acid---------------------------------------------------------------57

4.1 Influence of different substrates concentrations (w/v %) on glutamic acid

production by wild-type C. glutamicum--------------------------------------------------------------72

4.2 Effect of incubation temperature on glutamic acid production by

wild-type C.glutamicum--------------------------------------------------------------------------------73

4.3 Effect of inoculum size on glutamic acid production by wild-type C. glutamicum-------74

4.4 Effect of initial pH on glutamic acid production by wild-type C. glutamicum-------------75

4.5 Trend of mutant (CG^NTA) growth observed at 600 nm after every 8hours------------------78

4.6 Effect of different concentrations of treated rice husk for the production of

Glutamic acid by CG^NTA at 30^oC and 96 hours of incubation time-------------------------------79

4.7 Effect of different temperature on the production of Glutamic acid with

4% substrate by CG^NTA and 96 hours of incubation time------------------------------------------80

4.8 Effect of pH for the production of Glutamic acid with 4% substrate by CG^NTA at

30^oC and 96 hours of incubation time----------------------------------------------------------------81

4.9 Influence of various sizes of inoculum for hyper production of Glutamic acid from

(CG^NTA) with 4% substrate for 96 hours of fermentation period at 30^oC and pH 7-----------82

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LIST OF PLATES

Plate                                                                                   Title                                                                                   Page

I Phenotypic Variations between the Wild-type and Regulatory mutant strains of

C. glutamicum-------------------------------------------------------------------------------------------71

II Spots of Glutamic acid on TLC plate for purity determination--------------------------------86

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Corynebacterium glutamicum is a rod-shaped Gram-positive aerobic bacterium, which can be found in soil, sewages, vegetables, and fruits (Eggeling and Bott, 2005). This bacterium is capable of utilizing various sugars as well as organic acids (Blombach and Seibold, 2010). Among others, C. glutamicum has the ability to metabolize glucose, fructose, and sucrose as well as lactate, pyruvate, and acetate (Blombach and Seibold, 2010); additionally, C. glutamicum has the ability to grow on mixtures of different carbon sources with a monoauxic growth (Wendisch et al., 2000) as opposed to diauxic growth observed for many other microorganisms such as Escherichia coli and Bacillus subtilis. Only a few exceptions have been reported as in the case of glucose-ethanol or acetate-ethanol mixtures, where preferential substrate utilization was observed (Zahoor et al., 2012). Since its discovery, C. glutamicum has become an indispensable microorganism for the biotechnological industry (Wendisch, 2014). With the development of amino acid market, a new era for the production of these amino acids by many companies and academic associations have enthusiastically arisen with the start of research and development in this field to increase the rate of amino acid production. This technological race has expedited the expansion of amino acid production by various methods. Thus, almost all the amino acids can be produced by any of the four methods which include; chemical synthesis, protein hydrolysis, enzymatic synthesis and fermentation. However, industrially, the most advantageous and economical method used for amino acids manufacture is microbial

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method, that is fermentation (Ikeda, 2003). For almost fifty years, amino acids have been produced through fermentation (Rastegari et al., 2013). Out of ten non-essential amino acids, glutamic acid is second to alanine in priority due to that it stands first in the list as it is commercially very important amino acid used as flavor enhancer in foods (Javaid et al., 2012).

Among all biochemical methods, fermentation is the most economical, practical and eco-friendly means of producing glutamic acid, with low temperature requirement and the possibility of using cheaper carbon sources such as agricultural residues (Ekwealor and Obeta, 2005).

Microorganisms have regulatory mechanisms to control the quantities and qualities of enzymes that are involved in the synthesis of amino acids. Therefore, it is necessary to use these regulatory mechanisms in order to get the mass production of the target amino acid. Moreover the titre of amino acid increases if the enzymes involved in the production of the required amino acid are found in large amounts under workable situations. For this purpose, strains of microorganisms are improved using several techniques to make this process possible (Kothari, 2009).

Microorganisms employed for amino acid production are categorized into four groups; including the wild-type, auxotrophic, regulatory and auxotrophic regulatory mutants. The species of Corynebacterium or Brevibacterium are widely used for glutamic acid production (Choi et al., 2004). Similarly, their mutant strains that are auxotrophic or resistant to certain chemicals result in enhanced production of glutamic acid (Anastassiadis, 2007).

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In most developing countries including Nigeria, hundred thousand tons of agricultural residues are produced annually (Khan et al., 2006). These residues can be utilized as substrates for generation of different value-added products such as amino acids. Rice husk is one of the highly utilized residues which have a perceptible amount of reducing and non-reducing carbohydrates.

Rice husk is the outer covering of rice that is separated from the starchy endosperm during the first stage of milling. It is rich in vitamin B, minerals, fiber, high level of carbohydrates and proteins (Sramkova et al., 2009). It is an agricultural waste that is produced as bulks in rice milling. It can also be used as cheapest source of energy for fermentation. Rice husk also contains about 10% of paddy and accessible in large amounts in major rice developing regions of the world (Ambreen et al., 2006).

Pre-treatment of rice husk, increases the availability of cellulose which can be hydrolyzed to glucose by microorganisms (Shafaghat et al., 2010).

L-Glutamate has a distinctive taste, known as “umami” that is not sweet, sour, salty, nor bitter (Nakamura et al., 2006) and is mainly used as a flavouring agent or enhancer. Globally, about 1.8 million tons of monosodium glutamate is produced annually by fermentation using coryneform bacteria (Nakamura et al., 2006). L-glutamate is a non-essential amino acid and is recently reported to act as neurotransmitter (Hawkins, 2009).

Corynebacterium glutamicum is a biotin auxotroph that secretes L-glutamic acid in response to biotin limitation; this process is employed in industrial L-glutamic acid production. Fatty acid ester surfactants such as Tween 40 and Tween 80 as well as

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penicillin also induce L-glutamic acid secretion, even in the presence of biotin (Nottebrock et al., 2003). However, the mechanism of glutamic acid secretion remains unclear.

1.2.                                                           Statement of Research Problem

There is generally an increasing demand for amino acids especially L-glutamic acid as growth promoting factor, as well as flavour enhancer in foods. This is of great importance worldwide (Wendisch, 2014)

One of the major problems affecting large scale synthesis and utilization of L-glutamic acid is the cost of raw materials or chemicals used (Nampoothri et al., 2002). The process is also tedious, and non-economical probably due to process inefficiency as a result of undesirable product formation due to side reactions (Mahmood, 1996). The use of chemicals used exerts serious health risks due to their mutagenicity and carcinogenicity as opposed to the use of microorganisms (Ahmed et al., 2013). Chemical synthesis of amino acid also produces a racemic (DL-glutamate) mixture, which requires additional optical resolution, since the amino acid in the L-isomeric form is the active form (Wendisch, 2014). Similarly, when chemicals are used for glutamic acid production, certain special vessels/ containers may be needed to avoid damage of the fermentor due to chemical corrosiveness usually accompanied by great economic loss (Hermann, 2003). Another disadvantage of using chemicals for amino acids production is the need for neutralizing agent where acidic or alkaline solutions are used in order to bring the pH to near neutrality. Hence, the need for microbial synthesis of amino acids including L-glutamic acid.

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Strikingly, utilization of expensive media for the microbial production of L- glutamic acid is often not cost effective. This in turn affects the market price of this particular amino acid worldwide (Mostafa and Ahmed, 2006).

The wild-type strains of C. glutamicum lack the ability to utilize the pentose fractions of lignocellulosic hydrolysates. Similarly, the intracellular accumulation of L-glutamic acid as opposed to the industrial need (secretion) is a common characteristic of the wild-type strains of C. glutamicum; which in turn affects the quantity of the L- glutamic acid produced by a wild strain of the Corynebacterium glutamicum (Nakamura et al., 2007)

Production of L-glutamic acid in Nigeria using the abundant agricultural residues as raw materials through fermentation processes will reduce the high importation cost and boost local industrial utilization which in turn has positive impact on the foreign exchange and economy of the country.

1.3                                                                              Justification of the Study

The search for new biological materials to be used as drugs and pharmaceuticals, flavours and food additives, resulted in a phenomenal growth of industrial Microbiology on one side, and fermentation engineering on the other hand. The increased industrial utilization of biological processes suggest that biotechnology will be the major growth industry in near future and this will affect the lives and welfare of people all over the world (Vijayalakshmi and Sarvamangala, 2011).

Apart from the role of microorganisms in the production of drugs and pharmaceuticals, it was observed that biotransformation could also be utilised for the production of food and feed materials including amino acids such as L-lysine and L-glutamic acid (Shagufta,

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2014). In the field of food Microbiology, the role of microorganisms in the preservation of raw and cooked food materials and improvement of flavours and colours has been fully established. However, with the establishment of the role of growth promoting substances such as vitamins, amino acids and gibberellins, much attention is now focussed on the utilization of microorganisms and their enzymes to produce these valuable substances. A large number of microbial strains were developed for the industrial production of these useful organics and the help of fermentation process came to the advantage of mankind (Shafaghat, 2010).

The demand of L-glutamate is still increasing in the field of foods, animal feeds, pharmaceuticals and chemicals. To meet these increasing and diversified demands, there is still room for strain improvement based on the knowledge of microbial physiology (Rastegari et al., 2013). Furthermore, studies on process optimization, especially for lowering the expense of carbon and energy sources are also desirable (Adnan et al., 2011).

L-glutamate may be produced either by isolation from natural materials (originally from the hydrolysis of animal or plant proteins) or by chemical, microbial or enzymatic synthesis. Although, the chemical synthesis of amino acid also produces a racemic (DL-glutamate) product, the amino acid in the L-isomeric form is required in all its applications. This technical problem is overcome by the microbial synthesis of L-glutamate, which however, gives rise to optically pure L-glutamate. This therefore makes the process advantageous over the synthetic one (Shafaghat, 2010).

It is also known that the wild-type of Corynebacterium glutamicum lacks ability to utilize the pentose fractions of lignocellulosic hydrolysates, but on the contrary, its certain mutants

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are able to grow with the pentoses such as xylose as well as with arabinose as sole sources of carbon and energy on media containing acidic rice straw (Damisa et al., 2008) or wheat husk hydrolysates for the production of L-glutamate. This may reveal that acid hydrolysates of agricultural wastes materials may provide an alternative feedstock for large scale amino acid production.

The success in industrial production of glutamic acid stimulated further interest in finding strains capable of over-producing glutamic acid and other amino acids as well. For the extracellular production of a desired amino acid, changes in cellular metabolism and/or regulatory controls are required (Pasha et al., 2011).

Most amino acids are produced nowadays by the use of mutants that contain a combination of auxotrophic and regulatory mutants (Wendisch, 2007). Even more prolific amino acid-producing strains have been obtained by eliminating the ability of the organism to degrade the product and by providing cell permeability in favour of excretion of the end product (Nakamura et al., 2007).

Interestingly, certain local materials such as sugarcane baggase, cassava, wheat bran, rice husk and maize cobs have shown promising ability to substitute the expensive media used in the L-glutamate production as substrates (Jyothi et al., 2005). Thus, this helps to significantly reduce the level of environmental land pollution that usually results from dumping of such wastes, and also save a lot of costs.

This research is therefore aimed to explore the local bacterial flora (Corynebacterium glutamicum) and to develop and isolate regulatory mutants resistant to feedback inhibition for an increased yield of L-glutamate. In addition, special emphasis will also be given on

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the exploration of the locally available raw material (rice husk) for the process design in the laboratory production of L-glutamate. Hence, this study was undertaken to utilize rice husk as carbon and energy sources, and 4-fluoroglutamate – a toxic analogue of L-glutamate to produce glutamic acid by liquid state fermentation.

1.4                                                                         Aim of the Study

The aim of this study was to isolate Corynebacterium glutamicum from soil and compare the level of L-glutamic acid produced by mutant and wild-type strains of the isolate using rice husk as substrate.

1.5                                                                  Objectives of the Study

The objectives of this study were to

1.      Determine the proximate composition of the pre-treated rice husk substrate.

2.      Isolate and Characterize Corynebacterium glutamicum from Different Soil Samples Using Conventional Cultural and Biochemical Methods.

3.      Screen the Corynebacterium glutamicum isolates for L-glutamic acid production.

4.      Produce Regulatory Mutant Strains of the C. glutamicum Isolate with the Best Potential for L-glutamic acid Production.

5.      Determine the Effects of Various Optimization Parameters on the Production of L-glutamic acid.

6.      Produce L-glutamic acid from the Pre-treated Rice Husk Using the Wild-type and the Regulatory Mutant of the C. glutamicum.

7.      Determine the Qualitative Characteristics of the glutamic acid Produced using Industrial glutamic acid as Control.