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Title: Simultaneous Determination of Amino Acids and
Carbohydrates in Culture Media of Clostridium
Thermocellum by Valve-switching Ion Chromatography
Author: Yun Fa Haiyan Yang Chengshuai Ji He Cui XinshuZhu Juan Du Jun Gao
PII: S0003-2670(13)01126-4
DOI: http://dx.doi.org/doi:10.1016/j.aca.2013.08.033
Reference: ACA 232791
To appear in: Analytica Chimica Acta
Received date: 3-6-2013
Revised date: 8-8-2013
Accepted date: 20-8-2013
Please cite this article as: Y. Fa, H. Yang, C. Ji, H. Cui, X. Zhu, J. Du, J. Gao,
Simultaneous Determination of Amino Acids and Carbohydrates in Culture Media
of Clostridium Thermocellum by Valve-switching Ion Chromatography Analytica
http://dx.doi.org/doi:10.1016/j.aca.2013.08.033http://dx.doi.org/doi:10.1016/j.aca.2013.08.0337/30/2019 1-s2.0-S0003267013011264-main (1)
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Submit to Analytica Chimica Acta
ACA-13-1197Rev. Highlighted
Simultaneous Determination of Amino Acids and Carbohydrates
in Culture Media ofClostridium Thermocellum by
Valve-switching Ion Chromatography
Yun Faa,*, Haiyan Yang
a, Chengshuai Ji
b, He Cui
c, Xinshu Zhu
d, Juan Du
e, Jun Gao
a,*
a
Public Laboratory of Bioenergy and Biofuels, Qingdao Institute of Bioenergy and BioprocessTechnology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101, ChinabChina University of Petroleum, No.66, West Changjiang Road, Qingdao Economic &
Technological Development Zone 266580, ChinacTechnical Center of Shandong Entry-Exit Inspection and Quarantine Bureau, No. 70 Qutangxia
Road, Qingdao 266002, ChinadMetabolomics group, Qingdao Institute of Bioenergy and Bioprocess Technology,
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Simultaneous Determination of Amino Acids and Carbohydrates in1
Culture Media ofClostridium Thermocellum by Valve-switching Ion2
Chromatography3
4
Yun Faa,*, Haiyan Yanga, Chengshuai Jib, He Cuic, Xinshu Zhud, Juan Due, Jun Gaoa,*5
a
Public Laboratory of Bioenergy and Biofuels, Qingdao Institute of Bioenergy and Bioprocess6Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101, China7b
China University of Petroleum, No.66, West Changjiang Road, Qingdao Economic &8
Technological Development Zone 266580, China9cTechnical Center of Shandong Entry-Exit Inspection and Quarantine Bureau, No. 70 Qutangxia10
Road, Qingdao 266002, China11dMetabolomics group, Qingdao Institute of Bioenergy and Bioprocess Technology,12
Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101, China13eCollege of Materials Science and Engineering, Qingdao University of Science & Technology, No.14
53 Zhengzhou Road, Qingdao 266042, China15
16
Abstract17
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Keywords: ion chromatography; valve switching; amino acids; carbohydrates;30
electrochemical detector31
32
1. Introduction33
Lignocellulose is the most abundant, inexpensive, and renewable resource on earth.34
Great importance has been focused on the research on changing lignocellulose35
biomass into regenerative fuels to address future energy needs [1-2]. Microorganisms36
such as Clostridium thermocellum that can directly convert cellulose into ethanol as37
fuel have an important value in the field of bio-energy, thereby attracting considerable38
attention from many researchers [3-4]. To improve strains, analyze gene functions,39
and optimize cell systems, researchers aim to quantitatively understand the40
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used to determine amino acids. The methods for carbohydrate analysis include mainly52
liquid chromatography with refractive index detection [8]. The above methods cannot53
determine amino acids and carbohydrates of complex samples directly and54
simultaneously. Anion exchange chromatography and the integrated pulsed55
amperometric technique have been proven as selective and sensitive methods for56
determining amino acids and sugars directly without derivatization [9-11]. The57
bi-modal integrated amperometric detection can analyze mixtures of amino acids and58
carbohydrates [12]. However, these mixtures cannot be simultaneously determined in59
this manner because under the waveform of mode for carbohydrate detection in a60
complex sample, the presence of hydroxyl amino acids or other sugars need further61
identification. Valoran P. Hanko et al. and Yvonne Genzel et al. successfully62
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because the extraction efficiency of the trap column decreases rapidly. At present, an74
in-depth quantitative analysis for real complex samples has not been reported.75
In this work, we achieved a precise quantitative and simultaneous analysis of amino76
acids and carbohydrates using valve switching with a mean correlation coefficient of77
>0.99 and repeatability of 0.5% to 4.6%. After injection, all additional procedures of78
the system are carried out using a single 10-port valve. The resolution of the amino79
acid from the carbohydrate on the trap column was investigated, and the optimum80
conditions for high trap efficiency were systematically studied online. The new81
method was successfully used to determine amino acids and carbohydrates in aseptic82
media and in extracellular culture media of three phenotypes ofC. thermocellum.83
84
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Dionex) and an AminoPac PA10 column (2 mm 250 mm, Dionex) with a guard96
column (2 mm 50 mm, Dionex).97
The gradient programs and the electrochemical waveforms used to separate the98
amino acids and the carbohydrates are listed in Tables 1 and 2. Gold electrodes were99
used in pH reference mode to detect the amino acids and in AgCl reference mode to100
detect the carbohydrates. All amino acids and carbohydrates were separated at a flow101
rate of 0.25 mL min-1. The column temperature was 32.5 C.102
Table 1103
Gradient conditions104
Gradient conditions for the amino acids
Time %H2O %NaOH %NaAC Curve
(min) (250 mM) (1 M)
0 76 24
2 76 24
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Wave form for amino acids
Time Potential (V)
(ms) vs. pH
0 0.13
40 0.13
50 0.28
210 0.28
220 0.61
460 0.61
470 0.28
560 0.28
570 1.67
580 1.67
590 0.93
600 0.13
Wave form for carbohydrates
0 0.1
200 0.1
400 0.1
410 2
420 2
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Approximately 1.0 mg mL-1 internal standards were added to the samples before116
pre-treatment. The cells and the extracellular matrix were delaminated by117
centrifugation at 12,000 rpm for 10 min at 4 C. The cells were treated for other118
analysis. The supernatants were diluted 10 times with water and filtered using 0.22m119
nylon membrane prior to IC analysis.120
2.3. Valve-switching program121
All the procedures of the system were carried out using a single 10-port valve and122
three pumps interconnected by a narrow poly (ether-ether-ketone) tubing system (Fig.123
1). T1 was 39.4 mm long, whereas T2 and T3 were both 5.5 mm long. T1, T2, and T3124
were 0.127 mm in inner diameter (I.D.). The other parts of the tubing system were125
0.254 mm in I.D., and the volumes of loop1 and loop2 were 25 and 200 L,126
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cript136 Fig. 1. Sketch map of the valve-switching program. Continuous black lines represent closed status of the path flow,137and arrows indicate the flow direction.138
Table 3139
The timing and the status of valves140
Procedure Time(min)Status of injecting
valve(6-port)
Status of switching
valve(10-port)
Step1 -4.5 load Status1a
St 2 0 i j t St t 1
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calibration curves were calculated by plotting the peak area ratios of the external and151
internal standards versus the concentration of the external standards. Accuracy of the152
method was confirmed using an amino acid analyzer (Sykam 433D, Germany) with a153
LCA K07/Li column (4.6 mm 150 mm, Sykam) and a UV detector.154
3. Results and discussion155
3.1. The choice of trap solution156
Water, buffer solution, and acids are used as eluent for cation exchange157
chromatography [22-24].Using acid solution directly as the trap solution is beneficial158
for amino acid retention on the cation exchanger in hydrogen form [16]. In this work,159
formic acid and acetic acid were tested online as trap solutions on a cation exchanger.160
Using acetic acid as trap solution, leucine and isoleucine co-eluted with a long tail on161
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be successful. We tested the resolution of 20 mg L-1 glucose and 10 mg L-1 aspartic173
acid on the trap column at different formic acid concentrations (0, 1.0, 3.0, 4.0, 5.0,174
8.0, and 15.0 mM) and flow rates (0.02, 0.05, 0.10, 0.15, 0.20, 0.25, and 0.50 mL175
min-1) (Fig. 2). The 3.0 mM formic acid provided the best resolution. In addition, we176
checked the equilibration time of the trap column at 0.05, 0.10, and 0.15 mL min -1.177
The time values were 85, 45, and 25 min, respectively. Although the resolution178
increased as the flow rate decreased, the most time-saving condition was at 0.10 mL179
min-1 with a resolution 2.0.180
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peak value of the area was at 1.20 min. In the experiment that followed, 1.20 min was188
used as the switching time.189
3.4. Optimum conditions of the method190
As described above, the optimum analysis conditions include 3 mM formic acid191
solution as the liquid of the trap column at 0.10 mL min -1 flow rate, 1.20 min as the192
switching time of 10-port valve. We tested the recoveries of 20 amino acids and 7193
sugars (~1.0 mg mL-1 each amino acid and ~2.0 mg mL-1 each carbohydrate as194
standard mixture) under the optimum conditions. The results are satisfactory, with a195
mean value of 99.3% and scope of 91.3% to 109%.196
3.5. Evaluation of the new method197
Fig. 3A1 shows severe co-elution in the direct injection of the same mixture onto a198
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Chromatogram A1: direct injection of standard mixture on Amino Pac PA10 column.205
Chromatogram B1: direct injection of standard mixture on CarboPac PA10 column.206
Chromatogram A2 and B2: direct single injection of standard mixture by the new method on Amino Pac PA10207
column and CarboPac PA10 column.208
Peak identities are given in Table 3.209
The linearity of response was tested for the 25 L injections of 0.01, 0.05, 0.10,210
0.25, 0.50, 1.00, 2.50, 5.00, and 10.00 mg L -1 standard mixtures. Moreover, the linear211
range is described in Table 3. The mean correlation coefficient of the calibration212
reached 0.99. Repeatability and reproducibility were also investigated (Table 3).213
Repeatability for the eight replicates of 1.00 mg L-1 standard mixture was 0.50% to214
4.60%. Reproducibility for the three replicates of 0.05, 0.20, and 1.00 mg L-1 standard215
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Linear
rangeRepeatability Reproducibility Data1
a
Data2b
Data3c
Serial
numberAnalyte
Correlation
coefficient
(n=6)
Regression equation
(mg L-1) (%,n=8) (%) (mg L-1) (mg L-1) (mg L-1)
1 Arginine 0.9844 Y=2.2647X+0.5309 0.052.00 2.26 4.42 2.00 1.93 1.93
2 Lysine 0.9974 Y=1.3588X-0.1135 0.052.50 2.05 2.60 2.00 1.98 1.99
3 Glutamine 0.9996 Y=2.1601X-0.0425 0.052.50 2.44 1.59 2.00 2.09 2.05
4 Asparagine 0.9992 Y=5.5032X+0.1239 0.051.00 2.42 1.61 2.00 1.94 2.08
5 Alanine 0.9994 Y=2.3712X-0.0567 0.052.50 2.53 1.32 2.00 1.89 2.07
6 Threonine 0.9985 Y=3.233X-0.1285 0.055.00 2.91 2.12 2.00 1.90 2.02
7 Glycine 0.9991 Y=2.4867X-0.1037 0.051.00 2.30 1.82 2.00 1.90 1.99
8 Valine 0.9897 Y=1.8913X+0.0908 0.055.00 4.69 3.61 2.00 1.86 1.95
9 Serine 0.9984 Y=2.1319X-0.0254 0.055.00 2.66 1.89 2.00 1.90 1.99
10 Proline 0.9980 Y=3.293X-0.2051 0.055.00 2.65 2.77 2.00 1.89 1.96
11 Isoleucine 0.9985 Y=1.0801X0.0856 0.055.00 3.35 3.13 2.00 1.87 1.90
12 Leucine 0.9987 Y=0.8693X-0.0823 0.055.00 2.88 3.77 2.00 1.88 2.12
13 Methionine 0.9979 Y=2.7365X-0.187 0.051.00 2.43 2.77 2.00 1.88 2.01
14 Histidine 0.9906 Y=6.6014X+0.9758 0.051.00 1.98 3.88 2.00 2.01 1.97
15 Phenylalanine 0.9986 Y=6.9946X+0.0203 0.052.50 2.59 5.45 2.00 1.90 1.96
16 Glutamic acid 0.9993 Y=0.6773X+0.0186 0.055.00 2.47 3.02 2.00 1.91 2.05
17 Aspartic acid 0.9981 Y=1.3429X-0.0511 0.055.00 2.61 2.53 2.00 2.03 2.02
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medium and the corresponding medium of three phenotypes ofC. thermocellum (CT).234
The three phenotypes included a wild-type strain (WT), an ethanol-tolerant strain with235
0% ethanol addition (ET0), and an ethanol-tolerant strain with 3% ethanol addition236
(ET3), respectively. The chromatogram of the four samples obtained in the system237
(Fig. 4) demonstrates a complete separation of the carbohydrates from the amino238
acids. Table 4 lists the average contents of amino acids and carbohydrates in four239
types of fermentation medium for three replicates. The data indicated that three240
phenotypes could consume glucose and release valine, etc. However, the consumption241
of arginine, tyrosine, and cellobiose was quite different. WT could use arginine, but242
ET0 and ET3 released arginine, which showed that most of the arginine is synthesized243
in ethanol-tolerant strains. The deduction is consistent with that reported in 2011 by244
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Chromatogram A3 and B3: wild-type strain ofClostridium thermocellumby (WT-CT); Chromatogram A4 and B4:251
ethanol-tolerant strain with 0% ethanol addition (ET0); Chromatogram A5 and B5: ethanol-tolerant strain with 3%252
ethanol addition (ET3). i1 and i2 denote L-norleucine and lactose as the internal standards. Other peak identities and253
the analyte amounts are given in Table 4.254
255
Table 5256
Results of actual samples257
Aseptic medium CT-WT CT-ET0 CT-ET3Serial
numberAnalyte
(mg L-1) (mg L-1) (mg L-1) (mg L-1)
1 Arginine 5.78 4.93 6.21 10.95
2 Lysine 3.91 9.40 6.05 5.85
3 Glutamine 1.28 2.73 2.14 1.42
4 Asparagine 8.14 9.94 9.44 9.32
5 Alanine 2.50 24.89 21.04 22.78
6 Threonine 7.24 6.24 7.07 9.34
7 Glycine 3.36 3.20 3.49 4.37
8 Valine 7.05 20.65 16.46 23.49
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aData of samples that were diluted 250 times260
CT-WT: wild-type strain ofClostridium thermocellum261
CT-ET0: ethanol-tolerant strain ofClostridium thermocellum 0% ethanol addition262
CT-ET3: ethanol-tolerant strain ofClostridium thermocellum 3% ethanol addition263
4. Conclusions264
This study demonstrates an effective, accurate, and completely automated method265
for the simultaneous determination of amino acids and carbohydrates with no266
co-elution. The method has to do with a trapping column, trapping under the correct267
conditions, valve timing, and two column separations. Results of actual sample268
analysis are satisfactory and highly valuable for metabolomics research.269
Acknowledgements270
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References278
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[15] P. Jandik, J. Cheng, D. Jensen, S. Manz, N. Avdalovic, J. Chromatogr. B. 758 (2001) 196.300
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Graphical abstract314
Highlights315
A highly selective and sensitive IC method was developed and validated.316
Only a single valve and cation-trapping column were used for condition317
optimization.318
20 amino acids and 7 sugars were separated simultaneously without co-elution.319
The method was applied to the medium of clostridium thermocellum successfully.320
The work built a new analysis platform for water-soluble metabolites.321
322
323
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