Microbial Fermentation Produced Polymers Poly (γ-glutamic acid) with Corn in 300L Fermentor

Fengqing Wang1, Jinzhong Liang1, Yupin Song1

1. Key Laboratory of Food Science and Engineering,College of Heilongjiang Province. College of Food Engineering, Harbin University of Commerce, Harbin city, Heilongjiang

150076, PR China E-mail: ljz2050@126.com.

Abstract: Poly (γ-glutamic acid) (γ-PGA), which was first discovered as a capsule component of Bacillus anthracis, has extensive application because it is nontoxic towards human beings and the environment. In this paper, the suitable carbon sources and nitrogen sources were screened out for the high and cost-effective production of γ-PGA by Bacillus subtilis HCUL-B-115 which is a glutamic acid dependent bacterium. The effects of initial carbon sources, nitrogen sources and glutamic acid concentration on γ-PGA formation was researched in a 1L fermentor. The cultural condition were optimized and the synthesis rule of γ-PGA were investigated in a 300L fermentor. The results showed that the most suitable carbon source was 350mL/L corn saccharification liquid, the most suitable nitrogen source was 5g/L peptone and the optimum adding amount of monosodium glutamate was 70g/L. When the ventilation volume increased from 8m3/h to 16m3/h after 24h, the fermentation cycle shorten 36h than increasing ventilation volume after 72h, but the yield of γ-PGA was similar. Under the optimum cultural condition, the yield of γ-PGA was constant and could reach to 42g/L in a 300L fermentor. Keywords: Bacillus subtilis HCUL-B-115; Pliot Fermentation; Corn saccharification liquid; Poly (γ-glutamic acid). 1. Introduction

Poly (γ-glutamic acid) (γ-PGA) is a homo-polyamide of D- and L-glutamic acid units, could be synthesized by γ-PGA synthetase on membrane of microorganisms cell. It presents the linkage between α-amino and γ-carboxylic groups as opposed to peptide bond in proteins, so this polymer is resistant to proteases [1]. The molecular weight of γ-PGA varies from 100kDa to 5000kDa, and γ-PGA is water soluble, biodegradable, edible and nontoxic to human beings and the environment [2]. Therefore, potential applications of γ-PGA and its derivatives have attracted wide interest in a broad range of industrial fields such as food, cosmetics, medicine, agriculture, oil recovery, water treatment and so on [3-6]. But the cost of γ-PGA limited its application. In this paper, γ-PGA was produced by Bacillus subtilis HCUL-B-115 with cheap corn as material. The medium and cultural condition for γ-PGA formation were optimized in a 1L and 300L fermentor respectively.

2. Materials and methods 2.1 Microorganism

The strain was isolated, screened, and preserved in a fermentation engineering laboratory of Harbin University of Commerce. It was identified and then named as Bacillus subtilis HCUL-B-115, and collected by China General Microbiological Culture Collection Center (CGMCC2283). 2.2 Medium and culture condition Bacillus subtilis HCUL-B-115 was maintained by monthly subculture on 1.5-2%-agar slants containing (per litre): peptone 10g, beef extract 3g, NaCl 5g at pH 7.2-7.4. The medium for seed preparation containing (per litre): beef extraction 3.00g, glucose 20g, peptone 10g, NaCl 5g. The seed culture was incubated in 250 mL Erlenmeyer flasks containing 50 mL seed medium in a rotary shaker at 150 rpm and 37 ℃ for 24h. The medium for fermentor culture contained (per litre): corn saccharification liquid (sugar degree 200.00g/L) 250mL, CaCl2 0.42g, peptone 4.00g, monosodium glutamate 50g, NaCl 12g, MgSO47H2O 1.25g, KH2PO4 4g, MnSO4 0.08g. 2.3 Preparation of corn saccharification liquid The saccharification of corn starch was catalyzed by the double enzymes method. Corn powder, mixed with water in the tank by 1:3, were liquefied at 85 for ℃ 30min at pH 6.7-7.0 using the commercial thermo stable

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α-amylase with an enzyme activity of 20KU/g. The temperature was rapidly decreased to about 60℃, and then the pH of corn saccharification liquid was adjusted to 4.5 with hydrochloric acid. Monosaccharide was formed using the commercial glucoamylase with an enzyme activity of 50KU/g for 4h. The reducing sugars, cellulose, and protein (weight ratio about 70:11:4) are the main components in the corn hydrolysate. The glucose monomer fraction after pre-saccharification was close to 25%. The level of release of glucose and nutrients (including protein) depended on saccharification time. Completion of the starch hydrolysis required more than 7 h of pre-saccharification time. 2.4 Fermentation culture for γ-PGA production The γ-PGA fermentation culture was conducted in a 1L (Shanghai hundred Lun biological science and Technology Co Ltd) and 300L fermentor ( GB150-98 ), and it was made in the company of chemical printing and Dyeing Machinery in Yilida of Wuxi city) with two six-bladed disk turbines. The fermentor with 60%（v/v）fermentation medium was inoculated 2%（v/v）seed culture fluid. Dissolved oxygen in fermentation broth was adjusted by changing ventilation, tank pressure and stirring speed. The cultivation temperature was maintained at 36.5-37.5℃. 2.5 Analytical procedure The concentration of extracellular γ-PGA was determined by alcohol precipitation [7]. L-glutamic acid and glucose in the culture broth was measured with SBA-40D Bio-sensor (Shandong Science Academic Biological Institute, PR China). The value of pH was read from the monitor of fermentor. Dry cell weight was determined from 10 mL cell suspensions that were harvested by centrifugation, washed with distilled water, and dried at 80℃ for 24 h to a constant weight. The viscosity of the cell-free culture broth was measured by Digital Viscometer with a spindle SP-2 at 25℃(DNJ-5S, China).

3. Results and Discussion 3.1. Effects of different carbon sources and initial concentration on γ-PGA formation in a 1L fermentor Different carbon sources were added into the medium to investigate their ability in γ-PGA accumulation in a 1L fermentor. With corn saccharification liquid, glucose, citric acid, dextrin, sucrose, soluble starch, maltose and lactose as the carbon source, γ-PGA was detected in the culture broth. In addition to contain reducing sugar which is necessary for microbial growth, corn saccharification liquid also contain fat, various vitamins, linoleic acid, calcium, iron and other nutrients and so on. Thus, when corn saccharification liquid was used as the carbon source, higher γ-PGA concentration was achieved, as showed in Fig.1.LU Shu-Yun, YAO Jun [8] investigated γ-PGA production with a medium formulation containing glucose, monosodium glutamate and yeast extraction as main ingredients by Bacillus subtilis NX22, but they did not use corn saccharification liquid as energy and carbon sources during the process.

0

10

20

30

40

50

gluc

ose

citr

ic a

cid

dext

rin

sucr

ose

solu

ble

star

ch

mal

tose

lact

ose

Cor

nsa

ccha

rifi

catio

nliq

uid

γ-P

GA

(g/L

)

Fig.1 Effects of different carbon source on γ-PGA formation

In order to confirm the optimal added concentration, the effect of various concentration of corn

saccharification liquid on cell growth, cultrue broth viscosity and γ-PGA formation was assessed. As shown in

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the Table 1, the amount of γ-PGA increased gradually with increasing corn saccharification liquid concentration. But the cultrue broth viscosity began to reduce with the addition of 350mL/L of corn saccharification liquid to the medium. It means that the high concentration of carbon source was not conducive to harvest high molecular weight γ-PGA.

Table 1 Effect of corn saccharification liquid concentration on γ-PGA formation

corn

saccharification

liquid(mL/L)

CDW

（g/L）

γ-PGA

（g/L）

Residual glucose

（g/L）

Residual glutamate

（g/L）

Cultrue broth

viscosity

(mPa·s)

0 0.57 0 0 44.00 0

150 2.50 31.89 0 44.50 2350

200 2.56 33.26 0 41.50 2562

250 3.95 37.42 5.34 43.00 2950

300 4.55 43.02 17.80 41.50 3100

350 4.19 43.85 38.27 47.00 2870

400 3.55 43.18 50.73 46.00 2000

3.2 Effects of different nitrogen sources and initial concentration on γ-PGA formation in a 1L fermentor The effects of nitrogen sources on γ-PGA formation in B.subtilis HCUL-B-115 was carried out by using eight kinds of nitrogen sources, such as ammonium sulfate, ammonium chloride, yeast powder, beef powder, pancreas peptone, soybean peptone, casein peptone and ordinary peptone. The media with organic nitrogen sources were more suitable than inorganic nitrogen sources for cell growth and γ-PGA production as shown in Fig.2. The highest γ-PGA production were achieved by using peptone. The cost of peptone was seven dollars per kilogram, and it is cheaper than others. In addition, the color of the fermentation broth was lighter with peptone as the nitrogen sources, it was beneficial to the extraction and purification of γ-PGA. So 5g/L peptone was chosen to use industrial production of γ-PGA as economic type of nitrogen source according to the results in Table 2.

0

10

20

30

40

50

Ammoniu

m sulfa

te

Ammoniu

m chlo

ride

Yeast

powde

r

Beef p

owde

r

Pancre

as pe

pton

e

Soybe

an pe

pton

e

Casein

pepto

ne

Ordina

ry pe

ptone

γ-P

GA（

g/L）

Fig.2 Effects of different nitrogen source on γ-PGA formation

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Table 2 Effect of peptone concentration on γ-PGA formation

Peptone

(g/L)

CDW

（g/L）

γ-PGA

（g/L）

Residual glucose

（g/L）

Residual glutamate

（g/L）

0 2.48 5.60 2.61 46.02

1.0 3.01 37.94 0 43.13

2.0 3.4 39.16 0 42.78

3.0 3.5 39.78 0 43.15

4.0 3.78 41.62 0 44.00

5.0 4.11 44.06 0 41.98

6.0 4.08 43.97 1.10 44.01

7.0 3.7 42.63 2.00 40.86

8.0 3.2 43.25 2.23 43.59

3.3 Effects of sodium glutamate on γ-PGA formation in 1L fermentor The effect of glutamate on γ-PGA production, cell growth and cultrue broth viscosity were investigated with the concentration of sodium glutamate within the range 0~100g/L. As shown in the Table 3, Bacillus subtilis HCUL-B-115 depended on extracellular glutamate, and it would not produce γ-PGA without glutamate in medium. The amount of γ-PGA increased with increasing glutamic acid addition to the medium, while the cell growth and cultrue broth viscosity decreased. These results may be due to the substrate inhibition and the existence of γ-PGA depolymerase. Jun Yao [9] suggested that the γ-PGA depolymerase was present and active extracellularly during the batch production γ-PGA by Bacillus subtilis NX-2. In order to achieve high molecular weight γ-PGA, the concentration of sodium glutamate was chosen to add 70g/L in the production process.

Table 3 Effect of monosodium glutamate concentration on γ-PGA formation

monosodium

glutamate (g/L)

CDW

（g/L）

γ-PGA

（g/L）

Residual glucose

（g/L）

Residual glutamate

（g/L）

Cultrue broth

viscosity

(mPa·s)

0 1.99 0 48.10 0 0

10 3.12 12.33 0 3.10 450

20 3.26 16.78 0 11.07 700

30 3.18 20.75 0 20.95 1200

40 3.64 23.97 0 30.14 1700

50 3.90 32.09 1.02 37.23 2000

60 4.01 35.39 2.11 45.03 2870

70 4.17 45.80 6.30 54.86 2900

80 3.50 44.55 11.05 64.56 2200

90 3.22 43.02 15.13 71.29 1750

100 3.92 42.60 15.01 78.50 800

3.4 Effect of dissolved oxygen on the fermentation cycle and γ-PGA formation in 300L fermentor Dissolved oxygen is one of the essential condition for aerobic microbial growth, is one of the key environmental impact of material production and energy metabolism factors. And the difficulty in producing γ-PGA along the time course of cultivation is related to the increase in the medium viscosity that negatively affects oxygen transfer. Dissolved oxygen is often controlled by ventilation, stirring speed and tank pressure and fermented liquid properties of many factors in the fermentation process. Effect of dissolved oxygen on the

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fermentation cycle and γ-PGA formation in a 300L fermentor was investigated in this study. The ventilation was increased from 8m3/h to 16m3/h at different time in the process of fermentation. The content of γ-PGA and glucose in fermentation liquid were determined at certain time.

0

10

20

30

40

50

60

70

0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102 108 114

time(h)

gluc

ose(

g/L

)

72h48h24h

Fig.3 Effect of dissolved oxygen on the fermentation cycle in 300L fermentor

0

5

10

15

20

25

30

35

40

45

0 12 24 36 48 60 72 84 96 108 120

time(h)

γ-P

GA

(g/L

)

72h48h24h

Fig.4 Effect of dissolved oxygen on γ-PGA formation in 300L fermentor

Fig.3 and Fig.4 showed that the effect of dissolved oxygen on the fermentation cycle and γ-PGA formation

was conspicuous in a 300L fermentor. When the amount of dissolved oxygen in fermentation liquid in the process of fermentation after 24h increased, the fermentation cycle was shorten about 36h rather than 72h. But the yield of γ-PGA was consistent. It is significant to reduce the cost of production.

3.5 Fermentation rules of pH changing, substrate consumption and γ-PGA formation in 300L fermentor In order to evaluate the fermentation rules of pH changing, substrate consumption and γ-PGA formation in 300L stirred tank bioreactor, cultivation was performed. For the fermentor, the ventilation with 8m3/h and stirred agitation with 180rpm were used to improve aeration of the system and the oxygen depletion was avoided. The value of pH was monitored, as shown in Fig.6. Although pH played an important role in the γ-PGA production by Bacillus subtilis HCUL-B-115, the experiments in this study indicated that strict control of pH was not necessary. On the contrary, the rule of pH changing could be used to determine the fermentation was normal or not. In the process of fermentation, monosodium glutamate was consumed as the source of material for γ-PGA formation. Meanwhile, glucose in corn saccharification liquid was used as energy for microbial growth and material for product formation. The concentration of glucose rapidly decreased after 10h in bioreactor as shown in Fig.5. At this time, γ-PGA began to be excreted to the broth. That means γ-PGA produced was partially associated with cell growth, similar to what is reported [10, 11, 12].

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0

10

20

30

40

50

60

0 6 12 18 24 30 36 42 48 54 60 66 72 78 84

time(h)

γ-PG

A(g

/L),

gluc

ose(

g/L

),gl

utam

ic a

cid(

g/L

)

glutamic acid

glucose

γ-PGA

Fig.5 Fermentation rules of substrate consumption and γ-PGA formation in 300L fermentor

5.4

5.5

5.6

5.7

5.8

5.9

6

0 6 12 18 24 30 36 42 48 54 60 66 72 78 84time（h）

pH

Fig.6 Fermentation rules of pH changing in 300L fermentor

3.6 Stability test of 300L fermentor batch fermentation produced γ-PGA For the large-scale production of γ-PGA, yield stability is very important. In this study, three batches of fermentation were carried continuously out in 300L fermentor with 200L fermentation medium under following conditions: tank stirring speed 150r/min, ventilation 16m3/h, tank pressure 0.08MPa, culture temperatures between 36.5 ~37.5 .℃ ℃ Nutritional conditions of medium was the same with before. The yield of γ-PGA was stable in the pilot fermentation process, which could reach to 42g/L. 4.Conclusions Exploring biodiversity is a key step in the development of bioprocess for new or alternative sources of industrially important products. In this paper, corn saccharification liquid was used as the innovative source of energy and carbon to produce γ-PGA. With it, γ-PGA was produced in a 300L fermentor. Dissolved oxygen had great effect on the fermentation cycle. When increased ventilation volume from 8m3/h to 16m3/h after 24h, the fermentation cycle shorten 36h rather than increasing ventilation volume after 72h. But the yield of γ-PGA was similar. Initial fermentation at pH 6, the pH value decreased firstly and then increased, and finally tends to the initial value. The respective consumption of glutamate and glucose was about 10g/L and 60g/L during the whole fermentation process. These fermention rules could be used to determine the fermentation which is normal or not in the production process. Finally, it was proved that the yield of γ-PGA was constant by three batches of fermentation.

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Acknowledgment

This work was supported by the National Science and Technology Planning Program of China (no.2012BAD32B08). References [1] Shih IL, Van YT. The production of poly-(g-glutamic acid) from microorganisms and its various applications. Bioresour Technol 2001; 79:207–25. [2]Tian Guangming.Study of Glutamate Dehydrogenase and Poly-y-GIutamic Acid Hydrolase in Bacillus licheniformis.PhD DISSERTATION, 2014, 6. [3]Xing Nannan,Liang Jinzhong. Study on the Absorption of Mental Ions from Poly-γ-glutamic Acid.Food and Fermentation Industries, 2009, 2:61-65. [4]WANG Jing-xin;LI Zheng;ZHANG Qiu-ya.Decolorization of Methylene Blue withy γ-PGA hydrogel,Dyeing & Finishing, 2013,24:1-5. [5]LI Chao-ran;WU Kun;LIU Yan-qi.Effect of γ-PGA on the dough properties and noodle texture,Journal of Henan Agricultural University,2014,2:204-209. [6]Zhang Wen;Zhang Shuqing;Wang Xuejiang. The Microbial Synthesis of γ-polyglutamic Acid and Its Application in Agricultural Production. Chinese Agricultural Science Bulletin, 2014, 6 [7]LIANG Jin-zhong,WANG Feng-qing,ZHANG Lu-lu.Establishment of a kinetic model for γ-poly glutamic acid with batch fermentation. Science and Technology of Food Industry, 2013, 16: 174-177. [8]LU Shu-Yun ,YAO Jun ,XU Hong ,LI Sha.Main factors effecting the biosynthesis of poly ( glutamic acid).Chinese Journal of Bioprocess Engineering,2006,2:65-69. [9]Jun Yao, Jin Jing, Hong Xu, .Investigation on enzymatic degradation of γ-polyglutamic acid from Bacillus subtilis NX-2, Journal of Molecular Catalysis B: Enzymatic 56 (2009) 158-164. [10]Chen X,Chen S,Sun M,Yu Z,(2005)High yield of poly([gamma]-glutamic acid) from Bacillus subtilis by solid-state fermentation using swine manure as the basis of a solid substrate.bioresour technol 96:1872-1879. [11]Chen X,Chen S,Sun M,Yu Z,(2005)Medium optimization by response surface methodology for poly-γ-glutamic acid production using dairy manure as the basis of a solid substrate.Applmicrobiol Biotechnol 69:390-396. [12]Xu H,Jing M,Li H,Lu D,Ouyang P(2005)Efficient production of poly([gamma]-glutamic acid) by newly isolated Bacillus subtilis NX-2.Process Biochem 40:519-523.

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