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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 Xinshu Zhu Juan Du Jun Gao PII: DOI: Reference: To appear in: Received date: Revised date: Accepted date: S0003-2670(13)01126-4 http://dx.doi.org/doi:10.1016/j.aca.2013.08.033 ACA 232791 Analytica Chimica Acta 3-6-2013 8-8-2013 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 Chimica Acta (2013), http://dx.doi.org/10.1016/j.aca.2013.08.033 This is a PDF le of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its nal form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Submit to Analytica Chimica Acta ACA-13-1197Rev. Highlighted Simultaneous Determination of Amino Acids and Carbohydrates in Culture Media of Clostridium Thermocellum by Valve-switching Ion Chromatography
Yun Faa,*, Haiyan Yanga, Chengshuai Jib, He Cuic, Xinshu Zhud, Juan Due, Jun Gao a,*
Public Laboratory of Bioenergy and Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101, China b China University of Petroleum, No.66, West Changjiang Road, Qingdao Economic & Technological Development Zone 266580, China Technical Center of Shandong Entry-Exit Inspection and Quarantine Bureau, No. 70 Qutangxia Road, Qingdao 266002, China d Metabolomics group, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101, China
c a
* To
Fax: +86-532-80662695
Public Laboratory of Bioenergy and Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, 266101, China
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Tel.: +86-532-80662692
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College of Materials Science and Engineering, Qingdao University of Science & Technology, No. 53 Zhengzhou Road, Qingdao 266042, China
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Simultaneous Determination of Amino Acids and Carbohydrates in Culture Media of Clostridium Thermocellum by Valve-switching Ion Chromatography
Yun Faa,*, Haiyan Yanga, Chengshuai Jib, He Cuic, Xinshu Zhud, Juan Due, Jun Gaoa,*
Public Laboratory of Bioenergy and Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101, China
b
An improved method for the simultaneous determination of 20 amino acids and 7 carbohydrates using one-valve switching after injection, ion chromatography, and integrated pulsed amperometric detection is proposed. The resolution of the amino acids and carbohydrates in the cation trap column was investigated. In addition, parameters including flow liquid type, flow rate, concentration, and valve-switch timing were optimized. The method is time-saving, effective, and accurate for the simultaneous separation of amino acids and carbohydrates, with a mean correlation coefficient of >0.99 and repeatability of 0.5% to 4.6% for eight replicates. The method was successfully applied in the analysis of amino acids and carbohydrates in aseptic media and in extracellular culture media of three phenotypes of Clostridium thermocellum.
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Abstract
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College of Materials Science and Engineering, Qingdao University of Science & Technology, No. 53 Zhengzhou Road, Qingdao 266042, China
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Technical Center of Shandong Entry-Exit Inspection and Quarantine Bureau, No. 70 Qutangxia Road, Qingdao 266002, China d Metabolomics group, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101, China
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China University of Petroleum, No.66, West Changjiang Road, Qingdao Economic & Technological Development Zone 266580, China
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Keywords: ion chromatography; valve switching; amino acids; carbohydrates; electrochemical detector
1. Introduction Lignocellulose is the most abundant, inexpensive, and renewable resource on earth.
biomass into regenerative fuels to address future energy needs [1-2]. Microorganisms such as Clostridium thermocellum that can directly convert cellulose into ethanol as
attention from many researchers [3-4]. To improve strains, analyze gene functions, and optimize cell systems, researchers aim to quantitatively understand the metabolism of bacteria and determine metabolic flux data. Amino acids and sugars are among the most important metabolites involved in
can reveal cellular metabolic differences under the interferences from cellular and environment. Furthermore, such analysis can explain the metabolic pathway [5]. Therefore, the accurate and efficient determination of amino acids and sugars is highly important. Cation-exchange chromatography with post-column derivatization and
spectrophotometric detection [6] as well as reversed-phase liquid chromatography with pre-column derivatization and spectrophotometric detection [7] are commonly
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fuel have an important value in the field of bio-energy, thereby attracting considerable
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52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73
used to determine amino acids. The methods for carbohydrate analysis include mainly liquid chromatography with refractive index detection [8]. The above methods cannot determine amino acids and carbohydrates of complex samples directly and simultaneously. Anion exchange chromatography and the integrated pulsed amperometric technique have been proven as selective and sensitive methods for determining amino acids and sugars directly without derivatization [9-11]. The bi-modal integrated amperometric detection can analyze mixtures of amino acids and carbohydrates [12]. However, these mixtures cannot be simultaneously determined in
complex sample, the presence of hydroxyl amino acids or other sugars need further identification. Valoran P. Hanko et al. and Yvonne Genzel et al. successfully determined amino acids in cell culture media [13-14]. However, the co-elution between amino acids and carbohydrates remains unsolved. Therefore, sugars need to
column with an optimized long gradient elution time [17]. They applied the condition to detect amino acids and sugars in green tea and rice wine [18-19]. Nevertheless, separating sugars from amino acids requires low [NaOH]. At this condition, the electrochemical response is lower, and the use of low [NaOH] probably causes a baseline drift [20]. Petr Jandik presented a two dimensional ion chromatography (IC) approach to separate amino acids and carbohydrates using three valves and a short time of 25 min to 40 min [21]. However, quantitative analysis is difficult to perform
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this manner because under the waveform of mode for carbohydrate detection in a
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74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95
because the extraction efficiency of the trap column decreases rapidly. At present, an in-depth quantitative analysis for real complex samples has not been reported. In this work, we achieved a precise quantitative and simultaneous analysis of amino acids and carbohydrates using valve switching with a mean correlation coefficient of >0.99 and repeatability of 0.5% to 4.6%. After injection, all additional procedures of
the system are carried out using a single 10-port valve. The resolution of the amino
acid from the carbohydrate on the trap column was investigated, and the optimum conditions for high trap efficiency were systematically studied online. The new
AS50 auto-sampler, and a 10-port valve. An HPLC system equipped with a high-pressure-double-piston pump and a refractive index detector (RID) (Yilite, Dalian, China) was used for pumping the trap solution and testing the resolution of glucose and aspartic acid on the trap column. Chromeleon software was used for system control. Carbohydrate Removal Cartridge (2 mm 15 mm, Dionex) was used as the cation-trap column. Chemical separation was performed using a CarboPac PA10 column (2 mm 250 mm, Dionex) with a guard column (2 mm 50 mm,
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method was successfully used to determine amino acids and carbohydrates in aseptic
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Dionex) and an AminoPac PA10 column (2 mm 250 mm, Dionex) with a guard column (2 mm 50 mm, Dionex). The gradient programs and the electrochemical waveforms used to separate the amino acids and the carbohydrates are listed in Tables 1 and 2. Gold electrodes were used in pH reference mode to detect the amino acids and in AgCl reference mode to
detect the carbohydrates. All amino acids and carbohydrates were separated at a flow rate of 0.25 mL min-1. The column temperature was 32.5 C.
Table 1 Gradient conditions Gradient conditions for the amino acids Time (min) 0 2 8 11 18 21 23 42 42.1 44.1 44.2 60 0 76 76 64 64 40 44 14 14 20 20 76 76 97 97 60 %H2O %NaOH (250 mM) 24 24 36 36 20 16 16 16 80 80 24 40 40 70 70 8 %NaAC (1 M) Curve
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24 3 3
20 30
40
40 45 50
60 10 10 20
40 70 70
50.1 52 52.1 60
80 80 3 3
20 97 97
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20 20
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8 5 8 8 5 8 8 5 8
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Wave form for amino acids Time (ms) 0 40 50 210 220 460 470 560 570 580 590 600 0 200 400 410 420 430 440 500 Potential (V) vs. pH 0.13 0.13 0.28 0.28 0.61 0.28 0.28 1.67 1.67 0.93 0.13 0.1 0.1 0.1 2 2 0.6 0.1 0.1 0.61
106 107 108 109 110 111 112 113 114 115
All the solutions were prepared in 18 M.cm water (Milli-Q) with a 0.22 m nylon membrane filter. Formic acid and acetic acid were purchased from Aladdin (Shanghai, China). Sodium hydroxide (50% w/w, certified grade) was obtained from Acros Organics (New Jersey, USA); sodium acetate (purity, >99%) was procured from Sigma-Aldrich (St. Louis, MO, USA); and the amino acid and carbohydrate standards were supplied by AccuStandard (St. New Haven, CT, USA). In this study, we selected L-norleucine and lactose as the internal standards. All the samples were obtained from cultures of C. thermocellum at 60 C.
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Approximately 1.0 mg mL-1 internal standards were added to the samples before pre-treatment. The cells and the extracellular matrix were delaminated by centrifugation at 12,000 rpm for 10 min at 4 C. The cells were treated for other analysis. The supernatants were diluted 10 times with water and filtered using 0.22m nylon membrane prior to IC analysis. 2.3. Valve-switching program
All the procedures of the system were carried out using a single 10-port valve and three pumps interconnected by a narrow poly (ether-ether-ketone) tubing system (Fig.
were 0.127 mm in inner diameter (I.D.). The other parts of the tubing system were 0.254 mm in I.D., and the volumes of loop1 and loop2 were 25 and 200 L, respectively.
The steps in the valve-switching program (Fig. 1) follow. Step 1 represents sample
phase, where amino acids and carbohydrates are eluted from the trap column and Loop 2, respectively; separated on anion-exchange columns; and detected by the EDs. Step 4 involves re-equilibration, where the trap solution is equilibrated in the trap column, and both anion-exchange columns are being re-equilibrated prior to their next analysis. The timing and the status of valves were listed in Table 3.
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trap column and carbohydrates are intercepted into Loop 2. Step 3 is the analysis
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loading. Step 2 is the sample injection stage, where amino acids are extracted on the
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1). T1 was 39.4 mm long, whereas T2 and T3 were both 5.5 mm long. T1, T2, and T3
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Fig. 1. Sketch map of the valve-switching program. Continuous black lines represent closed status of the path flow, and arrows indicate the flow direction. Table 3 The timing and the status of valves Procedure Step1 Step2 Step3 Step4 Time(min) -4.5 0 1.2 10.2-60.0 Status of injecting valve(6-port) load
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141 142 143 144 145 146 147 148 149 150
Status1: port1 connected to port2, port 3 connected to port4, port5 connected to port6, port7 connected to port8,
The method was validated for precision, sensitivity, linearity, and reproducibility. Repeatability (the relative standard deviation) was determined using eight replicates of 1.00 mg L-1 standard mixture. Reproducibility was determined using three replicates of three concentrations of 0.05, 0.20, and 1.00 mg L-1. Linear regression
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Status2: port1 connected to port10, port2 connected to port3, port4 connected to port5, port6 connected to port7,
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Status of switching valve(10-port) Status1a Status1 Status2b Status1
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151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172
calibration curves were calculated by plotting the peak area ratios of the external and internal standards versus the concentration of the external standards. Accuracy of the method was confirmed using an amino acid analyzer (Sykam 433D, Germany) with a LCA K07/Li column (4.6 mm 150 mm, Sykam) and a UV detector. 3. Results and discussion 3.1. The choice of trap solution
Water, buffer solution, and acids are used as eluent for cation exchange chromatography [22-24]. Using acid solution directly as the trap solution is beneficial
formic acid and acetic acid were tested online as trap solutions on a cation exchanger. Using acetic acid as trap solution, leucine and isoleucine co-eluted with a long tail on the methionine peak. Using formic acid as trap solution, a satisfactory chromatogram and >90% trap recovery of aspartic acid were obtained. Therefore, formic acid was
To obtain the highest trap efficiency of amino acids and complete separation of amino acids and carbohydrates, we studied their resolution on the cation exchange trap column.
Aspartic acid is acidic and easy to elute from the trap column because of its weak interaction with the cation-exchange resin. Therefore, the retention behaviors of aspartic acid and glucose were investigated as representative compounds. If baseline separation of aspartic acid and glucose is obtained, isolation on the trap column would
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the better choice as the trap solution given the analysis condition.
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for amino acid retention on the cation exchanger in hydrogen form [16]. In this work,
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be successful. We tested the resolution of 20 mg L-1 glucose and 10 mg L-1 aspartic acid on the trap column at different formic acid concentrations (0, 1.0, 3.0, 4.0, 5.0, 8.0, and 15.0 mM) and flow rates (0.02, 0.05, 0.10, 0.15, 0.20, 0.25, and 0.50 mL min-1) (Fig. 2). The 3.0 mM formic acid provided the best resolution. In addition, we checked the equilibration time of the trap column at 0.05, 0.10, and 0.15 mL min-1. The time values were 85, 45, and 25 min, respectively. Although the resolution
increased as the flow rate decreased, the most time-saving condition was at 0.10 mL min-1 with a resolution 2.0.
Fig. 2. Curve of resolution of aspartic acid and glucose with different flow rates and concentrations
3.3. Switching time of the 10-port valve We used 20 mg/L glucose as standard and a 200 L tube as Loop 2 to set the cut window (the switching time of the 10-port valve) on the optimum conditions in the section 3.2. We tested the peak area of glucose in different time (0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 2.2, 2.5min) on CarbonPac PA 10 column. The
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peak value of the area was at 1.20 min. In the experiment that followed, 1.20 min was used as the switching time. 3.4. Optimum conditions of the method As described above, the optimum analysis conditions include 3 mM formic acid solution as the liquid of the trap column at 0.10 mL min-1 flow rate, 1.20 min as the
switching time of 10-port valve. We tested the recoveries of 20 amino acids and 7 sugars (~1.0 mg mL-1 each amino acid and ~2.0 mg mL-1 each carbohydrate as standard mixture) under the optimum conditions. The results are satisfactory, with a
Fig. 3A1 shows severe co-elution in the direct injection of the same mixture onto a single column. Threonine and serine were found in the waveform for the carbohydrates as shown as Fig. 3B1. However, Fig. 3A2 and 3B2 displayed good
203 204
Fig. 3. Comparison of two methods for amino acid and carbohydrate standard mixture analysis. 12
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separation without interference between the amino acids and the carbohydrates.
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205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226
Chromatogram A1: direct injection of standard mixture on Amino Pac PA10 column. Chromatogram B1: direct injection of standard mixture on CarboPac PA10 column. Chromatogram A2 and B2: direct single injection of standard mixture by the new method on Amino Pac PA10 column and CarboPac PA10 column. Peak identities are given in Table 3.
The linearity of response was tested for the 25 L injections of 0.01, 0.05, 0.10, 0.25, 0.50, 1.00, 2.50, 5.00, and 10.00 mg L-1 standard mixtures. Moreover, the linear
Repeatability for the eight replicates of 1.00 mg L-1 standard mixture was 0.50% to 4.60%. Reproducibility for the three replicates of 0.05, 0.20, and 1.00 mg L-1 standard mixture was 0.67% to 5.45%.
The accuracy of this method was confirmed using post column derivatization and
data 1) with valve-switching IC and with post-column derivatization in an amino acid analyzer, respectively. A comparison among data 2, data 3, and data 1 in Table 3 shows almost similar results. The average relative errors were 4.34% and 2.84%, respectively. The relative error for the seven carbohydrates ranged from 0.50% to 5.00%.
Table 4 Repeatability, reproducibility, correlation coefficient, linear range and data comparison
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same sample (prepared using 19 amino acid standards which concentration listed in
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UV detection for 19 amino acids. Data 2 and data 3 were obtained by analyzing the
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reached 0.99. Repeatability and reproducibility were also investigated (Table 3).
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Serial number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 a b c d e f g
Correlation Analyte coefficient (n=6) Arginine Lysine Glutamine Asparagine Alanine Threonine Glycine Valine Serine Proline Isoleucine Leucine Methionine Histidine Phenylalanine Glutamic acid Aspartic acid Cytine Tyrosine Tryptophan Trehalose Arabinose Galactose Glucose Mannose Ribose 0.9844 0.9974 0.9996 0.9992 0.9994 0.9985 0.9991 0.9897 0.9984 0.9980 0.9985 0.9987 0.9979 0.9906 0.9986 0.9993 0.9981 0.9864 0.9985 0.9975 0.9995 0.9999 0.9999 0.9999 0.9999 Y=2.2647X+0.5309 Y=1.3588X-0.1135 Y=2.1601X-0.0425 Y=5.5032X+0.1239 Y=2.3712X-0.0567 Y=3.233X-0.1285 Y=2.4867X-0.1037 Y=1.8913X+0.0908 Y=2.1319X-0.0254 Y=3.293X-0.2051 Y=1.0801X0.0856 Y=0.8693X-0.0823 Y=2.7365X-0.187 Y=6.6014X+0.9758 Y=6.9946X+0.0203 Y=0.6773X+0.0186 Y=1.3429X-0.0511 Y=2.7317X-0.4972 Y=3.0703X-0.1335 Y=3.2886X-0.312 Regression equation
Linear range (mg L-1) 0.052.00 0.052.50 0.052.50 0.051.00 0.052.50 0.055.00 0.051.00 0.055.00 0.055.00 0.055.00 0.055.00 0.055.00 0.051.00 0.051.00 0.052.50 0.055.00 0.055.00 0.052.50 0.051.00
Repeatability (%,n=8) 2.26 2.05 2.44 2.42 2.53 2.91 2.30 4.69 2.66 2.65 3.35 2.88 2.43
Reproducibility (%) 4.42 2.60 1.59 1.61 1.32 2.12 1.82 3.61 1.89 2.77 3.13 3.77 2.77
Data1a (mg L-1) 2.00 2.00 2.00 2.00 2.00 2.00 2.00
Data2b (mg L-1) 1.93 1.98 2.09 1.94 1.89 1.90 1.90
Data3c (mg L-1) 1.93 1.99 2.05 2.08 2.07 2.02 1.99 1.95 1.99 1.96 1.90 2.12 2.01 1.97 1.96 2.05 2.02 -1.95 2.14 --------
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2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 -2.00 2.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00
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3.88 5.45 3.02 2.53 4.13 2.40 4.28 5.60 0.67 1.40 1.27 2.77 1.10 2.92
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1.98 2.59 2.47 2.61 2.42 2.41 3.40 3.87 0.42 0.50 0.51 1.05 0.48 3.44
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Y=3.5603X+0.0429 Y=1.4789X-0.0203
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0.9738
Y=1.4123X+0.4782 Y=1.7305X-0.0869
Cellobiose
0.9998
Data 1 is the actual concentration of the sample prepared using standards. Data 2 was obtained by the new method with valve switching for the sample mentioned above.
Data 3 was obtained by post-column derivatization using an amino acid analyzer for the same sample.
3.6. Sample analysis The proposed method was used to analyze amino acids and carbohydrates in aseptic
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1.86 1.90 1.89 1.87 1.88 1.88 2.01 1.90 1.91 2.03 -2.06 1.91 3.98 4.07 4.03 4.06 4.05 3.96 3.80
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234 235 236 237 238 239 240 241 242 243 244 245 246 247
medium and the corresponding medium of three phenotypes of C. thermocellum (CT). The three phenotypes included a wild-type strain (WT), an ethanol-tolerant strain with 0% ethanol addition (ET0), and an ethanol-tolerant strain with 3% ethanol addition (ET3), respectively. The chromatogram of the four samples obtained in the system (Fig. 4) demonstrates a complete separation of the carbohydrates from the amino
acids. Table 4 lists the average contents of amino acids and carbohydrates in four types of fermentation medium for three replicates. The data indicated that three phenotypes could consume glucose and release valine, etc. However, the consumption
ET0 and ET3 released arginine, which showed that most of the arginine is synthesized in ethanol-tolerant strains. The deduction is consistent with that reported in 2011 by Shao [25]. A similar case was observed in tyrosine, where ET3 released the most tyrosine. The concentration changes in glucose and cellobiose of the three phenotypes
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of arginine, tyrosine, and cellobiose was quite different. WT could use arginine, but
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Chromatogram A3 and B3: wild-type strain of Clostridium thermocellum by (WT-CT); Chromatogram A4 and B4: ethanol-tolerant strain with 0% ethanol addition (ET0); Chromatogram A5 and B5: ethanol-tolerant strain with 3% ethanol addition (ET3). i1 and i2 denote L-norleucine and lactose as the internal standards. Other peak identities and the analyte amounts are given in Table 4.
Table 5 Results of actual samples Serial number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 a Analyte Arginine Lysine Glutamine Asparagine Alanine Threonine Glycine Valine Serine Proline Isoleucine Leucine Methionine Histidine Phenylalanine Glutamic acid Aspartic acid Cytine Tyrosine Aseptic medium (mg L-1) 5.78 3.91 1.28 8.14 2.50 7.24 3.36 7.05 1.42 1.06 7.01 7.81 7.24 CT-WT (mg L-1) 4.93 9.40 2.73 9.94 24.89 6.24 3.20 CT-ET0 (mg L-1) 6.21
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6.05 2.14 9.44 21.04 7.07 3.49 16.46 19.14 15.50 10.13 12.41 11.53 ND 1.57 47.76 30.22 23.59
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1.01 ND 2.43 46.02 29.90 17.16
a
20.65
21.33
16.84
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10.71 17.40
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0.69
2.08
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20 b c d e f g
209.20 ND 98.75
192.80 ND 77.02
272.25
447.25
258 259
ND: Not detected 16
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CT-ET3 (mg L-1) 10.95 5.85 1.42 9.32 22.78 9.34 4.37 23.49 18.70 14.78 14.21 16.92 ND ND 1.36 46.01 26.85 12.94 538.87a 5.95 15.03 ND ND 248.92a ND 84.70 2156.80a
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260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278
CT-WT: wild-type strain of Clostridium thermocellum CT-ET0: ethanol-tolerant strain of Clostridium thermocellum 0% ethanol addition CT-ET3: ethanol-tolerant strain of Clostridium thermocellum 3% ethanol addition
4. Conclusions
for the simultaneous determination of amino acids and carbohydrates with no co-elution. The method has to do with a trapping column, trapping under the correct
analysis are satisfactory and highly valuable for metabolomics research. Acknowledgements
This project is supported by the National Special Fund for Major Research Instrumentation Development (No. 2012YQ090229), the Instrument Functional
2011SJGZ06, 2012SJGZ12, and 2012424012). The authors are grateful to Professor Shifen Mou, Engineer Renyong Li, and Engineer Xiangchun Li for the valuable discussions.
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yg2010072), and the Shandong Provincial Technology Development Plan Fund (No.
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conditions, valve timing, and two column separations. Results of actual sample
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278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299
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Graphical abstract Highlights A highly selective and sensitive IC method was developed and validated.
Only a single valve and cation-trapping column were used for condition optimization. 20 amino acids and 7 sugars were separated simultaneously without co-elution.
The method was applied to the medium of clostridium thermocellum successfully. The work built a new analysis platform for water-soluble metabolites.
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