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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.2013.08.033

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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

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