2005 Du Effects of Glycerol on the Production of Poly(G-glutamic Acid)

7/28/2019 2005 Du Effects of Glycerol on the Production of Poly(G-glutamic Acid)

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Effects of glycerol on the production of poly(g-glutamic acid)by Bacillus licheniformis

Guocheng Dua, Ge Yanga, Yinbo Qub, Jian Chena,*, Shiyi Luna

aKey Lab of Industrial Biotechnology, Ministry of Education, Southern Yangtze University, School of Biotechnology,

Wuxi City, Jiangsu 214036, PR Chinab

State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, PR China

Received 11 February 2004; received in revised form 4 July 2004; accepted 16 August 2004

Abstract

The effect of glycerol on poly(g-glutamic acid) (g-PGA) fermentation was studied. The addition of glycerol as a co-substrate with L-

glutamic acid and citric acid enhanced the production ofg-PGA. The investigation on phospholipid composition of the cell membrane showed

that, among the five major phospholipids including phosphatidylethanolamine (PE), phosphatidyl serine (PS), phosphatidic acid (PA),

cardiolipin (CL) and phosphatidyl glycerol (PG), the syntheses of PG, PE, PS and CL were greatly influenced by the addition of glycerol. The

further investigation of phospholipids ester-linked fatty acids showed that the existence of glycerol in medium caused the obvious decrease in

C16:1 and C18:1 fatty acids and the increase in C10:0 and C12:0 fatty acids. It is concluded that the addition of glycerol led to an effective

excretion ofg-PGA resulting from the influences on the synthesis of cell membrane phospholipids and their ester-linked fatty acids.

# 2004 Elsevier Ltd. All rights reserved.

Keywords: Poly(g-glutamic acid); Bacillus licheniformis; Glycerol; Phospholipid; Fatty acids; Cell membrane

1. Introduction

poly(g-glutamic acid) (g-PGA) is an unusual anionic

naturally occurring homo-polyamide made up of D- and L-

glutamic acid units connected by amide linkages between a-

amino and g-carboxylic acid groups [1]. It is water soluble,

biodegradable, edible and non-toxic toward humans and the

environment. Therefore, potential applications of g-PGA

and its derivatives have been of interest in the past few years

in a broad range of industrial fields such as food, cosmetics,

medicine, plastics, oil recovery and water treatment [1,2].

g-PGA is an extracellular polymer produced by certainBacillus species [35]. Production of g-PGA was most

extensively studied and work has been carried out on the

nutritional requirements for cell growth, improving condi-

tions for g-PGA production [2,68] and variation in chain

[d]/[l]-repeat unit composition [9].

Glucose, glutamic acid, citric acid and/or glycerol were

usually used as carbon sources for the production ofg-PGA

[2,57,10]. Growth of the widely used strain Bacillus

licheniformis ATCC 9945A, usually employs glutamic acid,

citric acid and glycerol in media. Citrate and glutamate are

precursor substrates for polymer production [9,11] but it is

not clear as to the mechanism by which glycerol leads to

enhanced polymer formation, although observation has been

made that the production of g-PGA was stimulated in the

presence of glycerol [2,8]. In this paper, the effect of

glycerol on g-PGA fermentation was investigated. Phos-

pholipid composition of cell membrane and fatty acids wereanalyzed and compared with and without the addition of

glycerol in g-PGA production with B. licheniformis.

2. Materials and methods

2.1. Microorganism

B. licheniformis WBL-3, a mutant strain of B. licheni-

formis ATCC 9945A [12], was used in this study.

www.elsevier.com/locate/procbioProcess Biochemistry 40 (2005) 21432147

* Corresponding author. Tel.: +86 510 5888301; fax: +86 510 5888301.

E-mail address: [email protected] (J. Chen).

0032-9592/$ see front matter # 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.procbio.2004.08.005


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2.2. Media and culture conditions

B. licheniformis WBL-3 was maintained by monthly

subculture on 2.0%-agar slants containing (per litre): 10 g

citric acid, 10 g L-glutamic acid, 6 g NH4Cl, 1 g K2HPO4,

0.5 g MgSO47H2O, 0.02 g FeCl36H2O, 0.2 g CaCl2 and

0.05 g MnSO4H2O at pH 6.5. The same medium without

agar was also used for seed preparation. The seed culture

was incubated in 250 ml Erlenmeyer flasks containing 50 ml

seed medium in a rotary shaker at 200 rpm and 37 8C

for 24 h. The medium for flask culture contained (per litre):

6 g NH4Cl, 1 g K 2HPO4, 0.5 g MgSO47H2O, 0.02 g

FeCl36H2O, 0.2 g CaCl2 and 0.05 g MnSO4H2O at pH

6.5. The carbon source was added as indicated in the text.

The medium for fermenter culture contained (per litre): 10 g

citric acid, 20 g L-glutamic acid, 6 g NH4Cl, 1 g K2HPO4,

0.5 g MgSO47H2O, 0.02 g FeCl36H2O, 0.2 g CaCl2 and

0.05 g MnSO4H2O at pH 6.5. Glycerol was supplemented

as another carbon source if needed.

2.3. Shake flask and fermenter culture for g-PGA

production

10% (v/v) of seed culture was inoculated into a 500 ml

flask containing 50 ml fermentation medium. The culture

was incubated at 37 8C on a rotary shaker at 200 rpm for

96 h.

The g-PGA fermenter culture was conducted in a 5-l

fermenter (KF-5 l desk-top type fermenter) equipped with

two six-bladed disk turbines. The initial broth (3 l) was

inoculated with 10% (v/v) of seed culture. The aeration rate

was 1.5 l/min and agitation speed was 600800 rpm tomaintain the dissolved oxygen concentration at above 20%

of air saturation. The pH was automatically controlled at 6.5

by adding 3 mol/l NaOH or 3 mol/l H2SO4 solution. The

cultivation temperature was maintained at 37 8C.

2.4. Analytical procedures

Five millilitres of culture broth was centrifuged, washed

twice with distilled water and dried at 80 8C to a constant

weight for dry cell weight. Intracellular g-PGA concentra-

tion was determined according to the method by Cheng et al.

[13] after the cell pellets were resuspended in PBS buffer

solution and sonicated on ice bath for 5 min at the minimum

power setting with an ultrasonic cell disruptor (Branson

Model 250, Danbury, CT). The difference between dry cell

weight and intracellular g-PGA concentration was defined

as residual biomass. The concentration of extracellular g-

PGA was determined following the method by Cheng et al.

[13]. Glycerol and citric acid were measured according to

the method described by Yoon et al. [8]. L-glutamic acid in

the culture broth was measured with SBA-50B Bio-sensor

(Shandong Science Acamedic Biological Institute, PR

China). Residual sugar in the supernatant was measured

by a DNS method. Polysaccharides in the culture broth were

determined as follows. 5 ml culture broth was kept in

boiling-water bath for 1 h and centrifuged at 10,000 rpm to

discard the cell debris. 3 ml of 6 mol/l HCl solution was

added into 3 ml of the supernatant and kept in boiling-water

bath for 30 min. After cooling to room temperature, the

mixture was neutralized to pH 7.0 with 1 mol/l NaOH and

total residual sugar was measured with a DNS method andrecalculated according to the dilution. The difference

between total residual sugar and residual sugar was defined

as polysaccharide concentration. Phospholipids extraction

and analysis were done according to the methods by

Mikhaleva et al. [14]. Fatty acids in phospholipids were

prepared and analyzed according to Wollenweber and

Rietschel [15] and Sonesson et al. [16].

3. Results and discussion

3.1. Effects of different carbon sources on cell growth

and g-PGA formation in shake flask

Different carbon sources were added into the medium to

investigate their ability in g-PGA accumulation in shake

flasks. Table 1 shows the effects of different carbon sources

on cell growth and g-PGA production in the culture broth.

With acetic acid, L-malic acid, succinic acid and fumaric

acid as the sole carbon source, less residual biomass and g-

PGA were obtained, and polysaccharide was detected in the

culture broth. Although higher residual biomass and g-PGA

concentration could be obtained with soluble starch,

sucrose, maltose and glucose as the sole carbon source,

polysaccharide was also produced in each case. Ko andGross [2] investigated g-PGA production with a medium

formulation containing glucose as main carbon source and

trace amounts (0.5 g/l) of citric acid and glutamate by B.

licheniformis ATCC 9945A, but they did not mention if any

polysaccharide was produced during the process. When

citric acid, L-glutamic acid or glycerol was used as the sole

carbon source, however, higher g-PGA concentration was

achieved and no polysaccharide was detected in the culture

G. Du et al. / Process Biochemistry 40 (2005) 214321472144

Table 1

Effectof carbon sources on cell growthand g-PGA productionin the culture

broth

Carbon source g-PGA (g/l) Residualbiomass (g/l)

Polysaccharide(g/l)

Acetic acid 1.2 1.5 1.3

Succinic acid 2.7 1.8 1.1

L-Malic acid 3.0 1.8 1.3

Fumaric acid 2.4 1.2 1.6

Soluble starch 6.5 4.3 2.2

Sucrose 8.1 4.2 2.7

Maltose 8.2 4.6 2.9

Glucose 8.6 4.1 3.0

Citric acid 8.8 3.7 ND

L-Glutamic acid 8.7 3.8 ND

Glycerol 8.9 3.6 ND

The concentration of each carbon source is 25 g/l. ND: not detected.


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broth, which would be benefit for further purification of

g-PGA. Citric acid, L-glutamic acid or glycerol could be

used as the sole carbon source for the production ofg-PGA

with this mutant of B. licheniformis ATCC 9945A. In other

studies, however, glutamic acid and citric acid had to be

added into the media as precursor substrates for the

production of g-PGA with B. licheniformis ATCC 9945A[9,11]. Kunioka and Goto [7] reported that glutamate was a

good carbon source for g-PGA formation by Bacillus

subtilis IFO3335, but only a trace amount of g-PGA was

produced when citric acid was used as the sole carbon

source.

3.2. Effect of glycerol on cell growth andg-PGA

production

Glycerol was usually used in the medium to enhance the

formation ofg-PGA [2,68]. Table 2 shows the effects of

different glycerol concentrations (0110 g/l) on cell growth

and g-PGA production in the culture broth. It is interesting

to find that the increment of glycerol concentration from 0 to

70 g/l did not substantially increase cell growth, but the g-

PGA concentration in the culture broth was greatly

improved and a further increase in glycerol concentration

resulted in a decrease ofg-PGA production. The improve-

ment in g-PGA production via the addition of glycerol was

consistent with the investigation by Ko and Gross [2].

However, the mechanism by which glycerol functions on

enhancing g-PGA production remains unknown.

3.3. g-PGA fermentation with and without glycerol as

carbon source in a 5 l fermenter

Fig. 1 shows the time courses ofg-PGA production when

citric acid and L-glutamic acid were used as the substrates in

a 5 l fermenter. Residual biomass reached the highest value

of 3.4 g/l at 48 h and almost keep constant afterwards. No

polysaccharide was detected in the culture broth. The

concentration ofg-PGA in the culture broth (extracellular g-

PGA) and its concentration in the cells (intracellular g-PGA)

were measured at each sampling point during the cultivation

process. The concentration of extracellular g-PGA increased

and reached its highest value of 10.2 g/l at 96 h. While

intracellular g-PGAwas increased with time and reached the

highest value of 9.2 g/l at 84 h, and its concentration was

higher than that of extracellular g-PGA before 72 h. It is

assumed that the accumulation of intracellular g-PGA to

such a high concentration would inhibit its further synthesis

in the cells. The total concentration of g-PGA reached the

highest value of 19.3 g/l at 96 h.

To investigate the effect of glycerol on g-PGA

fermentation, 70 g/l glycerol was added into the medium

as a co-substrate. Results are shown in Fig. 2. Residual

biomass reached the highest value of 4.1 g/l at 56 h. The

concentration of extracellular g-PGA increased and reached

22.8 g/l at 88 h (Fig. 2A) that was much higher than the

value in Fig. 1 in which no glycerol was added. Interestingly,

the concentration of intracellular g-PGA reached its highest

value of 6.6 g/l at 80 h, which is much lower than that in Fig.

1 when only glutamic acid and citric acid were used as

carbon sources. It is suggested that more g-PGA could be

secreted into the culture broth with the addition of glycerol

and the existence of glycerol improved the permeability ofthe cell membrane for g-PGA. Total g-PGA was also

increased and reached the highest value of 29.4 g/l at 88 h,

indicating that the secretion ofg-PGA into the culture broth

improved the synthesis ofg-PGA in the cells.

Glycerol concentration decreased quickly and 67 g/l of

glycerol was consumed within 108 h of cultivation (Fig. 2B).

This observation was quite different from that obtained by

Yoon et al. [8], in which only 10 g/l of glycerol was

consumed during the culture of B. licheniformis ATCC

9945A for g-PGA production. It seems that the utilization of

glycerol was increased by the mutation of this strain.

G. Du et al. / Process Biochemistry 40 (2005) 21432147 2145

Table 2

Effect of different glycerol concentration on g-PGA production in the culture broth

Carbon sourcesa g-PGA (g/l) Residual biomass (g/l) Polysaccharide (g/l)

CA 10 g/l + GA 20 g/l + glycerol 0 g/l 9.7 4.3 ND








ND: not detected.a

CA: citric acid; GA: L-glutamic acid.

Fig. 1. Changes of extracellular, intracellular g-PGA concentration and

residual biomass with citric acid and L-glutamic acid as carbon sources.


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Glutamate and citrate were consumed completely at 96 h

(Fig. 2B).

3.4. Changes of phospholipid composition in cell

membrane of B. licheniformis with and without glycerol

as carbon source

Phospholipids are the most important components of cell

membranes and play an important role in substrate

absorption and metabolite secretion. Bacterial lipid meta-bolism has been intensively studied using the Gram-negative

model organism Escherichia coli and the Gram-positive

model organism Bacillus subtilis. Both organisms possess

phosphatidylethanolamine (PE), phosphatidylglycerol (PG),

and cardiolipin (CL) as their major membrane-forming

lipids [17]. However, the lipid composition of cell

membranes is not constant, and changes depending on the

carbon source and cultivation conditions [18].

To investigate the effects of glycerol on the phospholipid

composition of the cell membrane, the phospholipids were

analyzed during g-PGA fermentation with and without

glycerol as carbon source. Five phospholipids including

PE, PG, CL, phosphatidyl serine (PS), phosphatidic acid

(PA) were detected as the major phospholipids in the cell

membrane of B. licheniformis. The results are showed in

Figs. 3 and 4.

Without the addition of glycerol, PA decreased from 42.5

to 20.1% with time, while PE increased from 22.3 to 28.1%,

and PA and PE were the major phospholipids in the cell

membrane. CL increased and reached the highest value of

22.1% at 60 h then dropped to 18.0% at the end of

cultivation. PG increased from 10.2 to 19.3% and PS kept

almost constant in the process (Fig. 3). With the addition of

glycerol, however, PG increased rapidly from 10.7 to 49.2%,

which was much higher than that without glycerol addition.

PG became the major components of the cell membrane

after 40 h and it is assumed that PG played an important role

in the secretion of g-PGA. A key enzyme (SecA) in the

general secretory pathway in E. coli required PG for

membrane binding and ATPase activity in the secretion of

protein [19]. Even the process, in which M13 procoat protein

of E. coli was translocated across membranes in a signal

sequence-dependent manner, was independent of SecA, but

it still requires PG [20]. Therefore, it seems that PG plays a

dual role in the bacterial secretion pathway. PA decreased

from 42.5 to 19.0%, and almost the same tendency was

observed as that without the addition of glycerol. Interest-

ingly, PE decreased from 22.2 to 6.0%, while it increased in

the case without the addition of glycerol. PS decreased from

13.9 to 7.5% at the first 24 h and almost kept constant

afterwards. CL increased from 11.1 to 18.5% with time (Fig.

4). It is concluded that the synthesis of PG, PE, CL and PS in

the cell membrane was greatly influenced by the addition ofglycerol, resulting in the effective secretion of g-PGA.

These variations in phospholipids composition could be

either the result of adaptation of the cells to hyperosmotic

growth or a more general modification of the membrane in

response to different stresses.

3.5. Changes of fatty acids in cell membrane phospholipids

with and without glycerol as carbon source

To investigate the effects of glycerol addition on fatty

acids in phospholipids, phospholipid ester-linked fatty acids

G. Du et al. / Process Biochemistry 40 (2005) 214321472146

Fig. 3. Changes of phospholipids in the cell membrane ofB. licheniformis

without the addition of glycerol.

Fig. 4. Changes of phospholipids in the cell membrane ofB. licheniformis

with the addition of glycerol.

Fig. 2. g-PGAfermentation with L-glutamic acid, citric acid andglycerol as

carbon sources.


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were detected and the results were showed in Figs. 5

and 6. Without the addition of glycerol, C16:1 and C18:1

fatty acids slowly decreased with the similar tendency

from 28.7 and 25.5% to 18.7 and 17.5%, respectively.

While C16:0 and C18:0 fatty acids increased from 23.1 and

11.5% to 29.5 and 21.5%, respectively during g-PGA

fermentation. C10:0 fatty acid increased from 5.8% to its

highest value of 7.4% at 60 h and decreased afterwards.

C12:0 and C14:0 fatty acids almost kept constant in the

process (Fig. 5).

However, with the presence of glycerol in the medium,

C16:0 fatty acids increased quickly from 23.3 to 36.1% in

the first 60 h and kept constant later. While C18:0 fatty acid

increased from 11.3 to 18.9%, which was similar to the case

without the addition of glycerol. C16:1 and C18:1 fatty acids

decreased rapidly from 28.3 and 25.7% to 8.0 and 9.7%,

respectively and both were much lower than those in case

without the addition of glycerol. C10:0 and 12:0 fatty acids

increased from 6.0 and 3.7% to 12.8 and 11.6%,respectively. C14:0 fatty acid kept almost constant in the

process (Fig. 6). It is concluded that the addition of glycerol

resulted in the obvious decrease in C16:1 and C18:1 fatty

acids, and the increase in C10:0 and C12:0 fatty acids. With

oleic acid as the major component of C16:1 and C 18:1 fatty

acids, and their decreases in cell membrane may also

increase the permeability of cell membrane. Moreover, the

increase in C10:0 and C12:0 fatty acids may improve

the fluidity of cell membrane. Therefore, it is suggested that

the decrease in C16:1 and C18:1 fatty acids and the increase

in C10:0 and C12:0 fatty acids enhanced the secretion

of g-PGA, resulting in the higher extracellular g-PGA

concentration and lower intracellular g-PGA concentration

in g-PGA fermentation.

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Fig. 5. Changes of fatty acids in phospholipids without the addition of

glycerol.

Fig. 6. Changes of fatty acids in phospholipids with the addition of

glycerol.

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2005 Du Effects of Glycerol on the Production of Poly(G-glutamic Acid)