Measuring methods for three kinds of polyhydroxyalkanoates from activated sludge by gas chromatograp

Journal of harbin Institute of Technology(New Series), Vol. 18, No 5, 2011Measuring methods for three kinds of polyhydroxyalkanoates fromactivated sludge by gas chromatographyCHEN Wei, CHEN Zhi-giang, LV Bing-nan, WEN Qin-xue, ZHANG Yun-hai陈玮,陈志强吕炳南,温沁雪张运海(I. State Key Lab of Urban Water Resource and Environment, School of Municipal and Environmental Engineering, Harbin Institute of TechnologyHarbin150090,China,ezq0521@tom.com;2.NationalEngineeringResearchCenterofUrbanWaterResources,Harbin150090,China)Abstract: Gas chromatography determination of polyhydroxyalkanoates has been common; however, the pre-reatment steps are often complex, and gas chromatography operation conditions are not given in detail. In thisstudy, gas chromatography is used for analyzing PHB, PHV and PH2MV, three majors of PHAs in activatedsludge, The sample was centrifuged at a speed of 4000 r/min for the separation of foe and supernatant, freezen,and dried for 12 h in vacuum freezing drier; and then transferred to the fridge for freezing to ice and dryingfor 12 h in vacuum freezing drier; then chloroform and a simple composition digestion solution including metha-nol, sulfuric acid and benzoic acid was added digested at 105 C for 6 h; cooled to room temperature, the lower solution of the result can be used for analyzing. Samples were analyzed by gas chromatography with Fid de-tector and auto sampler; the standard curve of standard material shows an excellent linear relationship withrelation coefficients larger than 0. 99; the relative standard deviation( RSD )of sludge samples is less than 1%The recovery rates of each sample are between 95%-105%. The GC analysis time of each PHA sample isshorter than 10 minutesKey words polyhydroxyalkanoates; activated sludge; gas chromatography measuring methCLC number. X703. 1Document code. AArticle ID:10059113(2011)05011306Polyhydroxyalkanoates ( PHAs), which have simi- sludge is a precondition for the research on PHAsnlar mechanical properties to polypropylene, are thermo- thesis by mixed bacteriaplastics synthesized by bacteria The basic units ofCurrent pha dePHA are long chain polymers 2). According to the dis- ning 8, ultraviolet spectrum, high performance liquidcrepancies of their branched chains, PHAs can be di- chromatography (HPLC), gas chromatography (GC)vided into polyhydroxybutyrate( PHB), polyhydroxy- infrared spectrum 9-12J, nuclear magnetic resonancevalerate(PHV ), polyhydroxyhexanoate( PHH ), poly NMR). Thereinto, the staining method can be on-(3-hydroxybutyrate-co-3-hydroxyvalerate( PHBV) and ly used in qualitative analysis; the ultraviolet spectrumso on. Actually, many kinds of bacteria are widely method is too complex in pretreatment and measurementdetected to synthesize PHA in cells(4, andd current in- for conventional analysis, and the infrared spectrumdustrialized productions of PHA are synthesized by a and NMR devices are too expensive to popularizespecial bacterium named Alcaligenes Eutrophus s, the Therefore, CC is an optimum choice for PHa measure-high-cost of the production process limits the populari- ment.zation of PHA in many fields 6) Fortunately, the polyThe principle of GC PHA measurement is thatmer can be synthesized by mixed bacteria such as acti- PHAs depolymerize in concentrated crotonic acid, andvated sludge, therefore, reduces the production ex- then react with methanol. The methyl esterificationpense of PHA. However, the activated sludge which is products can be separated and responded by GC withcharacterized as complex composition and various impu- FID detector 4. Previous researchers improved therities makes it difficult to extract and measure PHa method by freeze-drying and extraction, thus solventfrom the biomass. Moreover, the foreign substances peak and Pha peak are easier to be isolated[Is.Howmay interfere with the PHA measurement. Therefore, ever, the pretreatment steps for the actual GC detectionan effective and rapid extraction and measurement for methods are still tedious, including the separate disposroutine measurement of PHA from the cell in activated als such as sludge dissolved by solvent, methyl esterifim0amcm2m0 Stake Key lab of山中国煤化工m(cmCNMHG113·Journal of Harbin Institute of Technology( New Series), vol. 18, No 5, 2011cation, centrifugal separation and internal standard 's Firstly, 1.00 g benzoic acid was dissolved in 200 mLaddition[16-17,and too much steps lead to potential methanol, and then 30. 00mL 98 %(wt)H, SO4 werehuman errors. Moreover, the GC analysis condition added into the solution slowly, and transferred the so-was seldom presented in detail, and the precision, ac- lution into 1000 mL volumetric flask and added metha-curacy and recovery of standard addition were rarely nol to 1000 mL, then mixed homogeneously. The dimentioned. Based on the previous research, the pre- gesting solution could save one month under 5C.Thetreatment steps were simplified, and the standard benefit of mixing three materials in the solution was tocurves for PHB, PHV and 2-Hydroxycaproic acid reduce the error that may produce during the multi( 2HC)( instead of polyhydroxy-2-methylvalerate steps, ensuring the concentration of component con-PH2MV)) were given; GC with a flame ionizationstant in the experimentsdetector( FID) and auto sampler was used. The preci- 1.4 Sample Pretreatmentsion and accuracy of the measurement was discussedSample 4 mL activated sludge mixed lieuor anput it in, a 5 mL centrifuge tube to separate the sludge1 Materials and Methods1. 1 Gas Chromatographylower speed, separation may be incomplete so that theAnalysis of PHA was performed on an Agilent supernatant would be turbid; when the speed is higher6890n gas chromatography with a FID and an Agilent than 4000 r/min, the sludge might be adhered to the7683B series autosampler Agilent, USA). An Agi- tube wall and extended as a sheet; after freezing drylent 19091J-413 HP-5(5% Phenyl Methyl Siloxanethe sheet might break into piecescapillary column( Nominal length 30 m, Nominal diThe sludge sample after centrifuge was transferredameter 320. 00 um and Nominal film thickness 0. 25 to the fridge for freezing to ice and then transferred toum(Agilent, USA)was used. The column was elu- the vacuum freezing drier for drying for about 12 hted with a split mode( constant pressure 4.91 psi and The dry sludge sample was then weighed by analyticalthe split ratio 20: 1 )at a now rate of 44. 8 mL/min balance on smooth parchment paper to avoid adherwith hydrogen as carrier gas. The temperature of FIDencewas 300C, with a hydrogen flow rate of 40 mL minAccurate weighed sludge sample was finally transand an air flow rate of 450 mL/min. Nitrogen was used ferred into digesting tube; 2. 00 mL of chloroform andfor making up gas with a flow rate of 45 mL/min. The digesting solution were added. Shake the tube with osinjection volume was I uL. The GC was programmed collator to mix symmetrically, and then digested underfor 2 min isothermal time at 70 C, a ramp of 105 C for 6 h with tubes shake every 0.5-1h to25 C/min to 150 C. The injector temperature as 220 break up the sludge floc. After digestion, the sampleC and split time was 5. 2 min. After column separa- should be cooled down and 1.0 mL of distilled watertion the oven entered postrun step and hold on at 300 was added. Shake the digested sample acutely and sep-C for 2 min, under these conditions, the sample out- arate statically. Liquor from the bottom layer was theput rate was about six per hour, each sample running solution contained PHAmin1. 2 Other Equipments2 Results and discussionANKE TGL-16G centrifuge with an angle rotorwith the max speed 10000 r/min, equal to the max ac- 2.1 Standard Curve of PHB, PHv and 2HCceleration 8944g( Shanghai, China), BYK vacuumDraw the standard curve of Phb. phv and 2HCfreezing drier FD-1( Beijing, China )and EUROTECH with benzoic acid as internal standard substance, thenET3150B( HongKong, China ) digesting instrument measure the precision and accuracy of PHB, PHsed for2HC and retention times of activated sludge samples1. 3 Materialsand add standard material into sludge sample to meas-The standard PHA reagent including Poly (3- ure the reeovery ratehydroxybutyric acid-co-3-hydroxyvaleric acid), naturalStandard substances of PHB and PHv wereorigin, PHV content 5%(wt)( CAS No: 80181-31- ted and analyzed by the GC. Standard curves for PHB3), and 2-Hydroxycaproic acid 98%(wt)( CAS No: and PHV with benzoic acid as internal standard sub-636-36-2)instead of PH2MV were bought from SIG- stance were drawn. In case to avoid the effect of theMAL-ALDRICH. And there are some other materials, digesting solution and GC equipments, the standardsuch as Methanol, chromatographically pure, benzoic curves should be redrawn if the equipment status or diacid, chloroform and 98%H2sO4gesting solution waPrepare methanol, 3%(v/v)H,SO4 and 1000 GC chromatogram 4中国煤化工 typIcThe frontmg/L benzoic acid mixture as the digesting reagenttwo peaks showsYHCNMHGthe thirdJournal of Harbin institute of Technology( New Series), Vol 18, No. 5, 2011peak shows PHB and PHV, two types PHAs, the lasteak is internal standard benzoic acid3.676For the standard curve, the main error was weighting error, because only a small amount of the standardsubstances were used each time( from 1 to 10 mg),e1.64ven the humidity of air may lead to a significant error.To reduce the weighting error, parchment papers in2447stead of filter papers are recommended to be used. To2001Solution PHB PHv Benzoic acicproduce the standard curves, 0,1, 2, 3, 4 and 5 mgof the pure 95%(w/w)PHB-co-5%(w/w)PHV substance were taken respectively. The GC chromatogramresults for the standard substances are shown in TabStandard curves are shown in Fig. 2.Fig 1 Chromatographic elution peaks of PHB&PH\Tab 1 Analysis results of PHB, PHV and benzoate in GC chromatogramArea of phbArea ofWeight ofeight ofArea of phvbenzoic acidArea benoic scidArea bennie acidHB/mgPHⅤ/r318.2116726.289211512.649050.2103671500.017379583,1.0450.055578.5965650.932740.3900354920.0343340730.10573.520631536.828610.5377000240.0478391862.8500.15011371580.628300.7195918420.0640750903.8000.2001409.75452133.339811571.782710.8969143830.0848334884.6550.245PHB in the mixed avtive sludge liquor; for PHV withy=5.255lx-0.0218the range of quantification of 0-0. 4 mg, equals to0--125 mg/L PHV in the mixed avtive sludge liquor.The range of quantification for PHA can satisfy generalPHa analysis in the activated sludg2HC, the isomeric compound of PH2MV2as the standard substance for the standard curve ofPH2MV due to the lack of 3 H2MV( PH2MV's mono-mer)standard reagent. Under the GC analysis condi-tion the retention time of 2HC and 3 H2MV varied a-040.6bout 0. 1-0. 2 min; in the actual sample analysis, it iseasy to distinguish PH2MV from impurity peaks according to the retention time and the regular variety of thepeak. The GC chromatogram results for 2HC are listedy=2942x+0.004in Tab. 2, and the standard curve is shown in Fig. 3.R=09958Tab 2 Analysis results of 2HC and benzoate in GC chro-Area of 2HC Area of benzolc- Area 2Hc Weight of 2HCArea beavis acid-- PHV876.3032400.334161.10AHA544.67719889.598080.61227Fig 2 Standard curves for PHB and PHV868.064580.87121The linearty of the standard curves for PHB andl112.603276.254461.26973PHv was good withR>0. 99; for PHB with the range1349.60205中国煤化工5.70of quantification of 0--7 mg, equals to O-1750 mg/CNMHG115Journal of Harbin Institute of Technology( New Series), Vol. 18, No5,2011Compared with PHA producing bacteria in pure cul-ture, activated sludge has more impurity and complexy=3.8317x-0.0113cell content, which would give more peaks under theR2=09966GC condition. To distinguish the target peaks from im-purity peaks, both the height of the peaks and the re-tention time are important aspects in the analysisa sludge sample was taken at random from thesecond sedimentation tank of a brewery wastewater00.2040.60.81.01.2141.6treatment plant. The sludge was washed with clean dis-AaAmtilled water and pretreated followed the steps describedFig 3 Standard curve for 2HCin Section 1. 4, and then the sample was analyzed bythe GC. The analysis results are shown in Tab. 42.2 Analysis of Activated Sludge SampleRetention time of the object material is relative to Tab. 4 The data of the activated sludge sample from thethe accuracy of the equipment. By using the auto sam-GC chromatogrampler of the GC, the variety of the retention time of PHBPeakRetTime/ Type/ Width/Area/and PhV can be eliminated. Tab. 3 shows the retenmin (pA+s)tion time of phb and phv of the activated sludge sam0.02906.48E+04ples in twenty-five repeated experiments0.93230E+05Tab 3 Retention time of PhB and PhV of the sludge sa0.0234500.96994l.19PHBRepeat Times Retention Time/minPHV Retention Time0.01950.700061.396VB0.01760.67101.49021.4892.1870.023147.016511.6370.02420.5631.7000.04540.918052.1872.1880.02251.471272.1860.01831.837453.5090.0196813.428960.04495.501771.4882.1854.1900.03881.4882.185174.343PP0.0149l.4882.185punty peal.488are normally inconspicuous areas of peaks are less192.185than 10), and peaks of PHB, PHV and benzoic acidare easily to be distinguished according to their reten21tion time.the sludge sample taken fre2.186PHA producing systems would have more obvious peaks2.186in the chromatogram241.4882.18To investigate the accuracy and precision of theAverage2.186method, another experiment was progred. The same0.0007220.001021sludge sample was used to repeat the analysis for twen0.490.47%0ty times. The areas of PHB, PHV and 2HC wereRSD: relative standard deviationcorded, and the rsd of the results were calculatedlisted in Tab. 5中国煤化工As shown in the results the RSDs of the retentiontime of both Phb and Phv were less than 0. 5%CNMHGJournal of Harbin Institute of Technology( New Series), Vol 18, No 5, 2011Tab. 5 Precision of the method for PHB, PHV and 2HC analysiArea of phbArea of pHvArea of 2HCArea of benzoic Calculated weight Calculated weight Calculated weightTimesof PHB/ mgof PHV/mgof 2HC/ mg672.0013458.68740731.99023510.419567.9517069770.4480986585.4837224952679.0644559.16727511.420788.0188337390.450811213671.3571859.ll10735.9519510.2680179464999070.4513865985.51510345982.5946059.41585739.l8109512.561838.0423263540.451676l8l5.51451174960.1916747.75409518.058788.04266051756789691.5761160.60557749.66418519.629158.0373783840.4543878255.516658234689.1187760.67156754.17029522.723330.4522422145.51696731560.96957757.524l1524.90l188.031064780.4525699455.51851254961.43286764.31529.079358.043153480.4524l16345.524038500o12713.3189162.43722774.14581535.854618.0390319940.4539565475.524333071707.3551661.44757762.87219526.971928.1054906350.4542838635.535669885707.3181861.23681763.04626526.934758.1056370790.4527939275.537326949709.671861.79353765.87256528.772778.1043540465.538520366715.0031762.03551771.04779532.943608.1013668020.4535103645.532295639715.5910062.24396771.14172532.674568.1120104630.4552223005.53577123371L.3722561.9508767.38525530.467108.0978924630.4549704685.531720599721.7162562.77566778.13867537.820438.1032586610.4547300825.53254656218727.9426963.35267783.76605541.933658.1110522280.4554101655.53025729919729.1284263.1876842.827398.1108896300.4535221395.531722403681.6704159.44956738.68396512.568978.03l4409850.4519201715.510718490NAN/ANAN/A8.0549129270.4530929815.5225989150.0551981040.0018233300.01360910.40.25The RSDs of the three concerned substances are method. For one sample, PHB and PHV contents ofall lower than 1%, which means that the accuracy of the sample were measured directly for four times. Fcthe method can satisfy the general analysisthe other one; a quantitative amount of the PHB and2. 3 Recovery Rate AnalysisPHV standard substances were added in, and alsoRecovery rate is an important factor to analyze the measured for four times. The recovery rates were calaccuracy of methods. In this study, two parallel sludge culated, the results are listed in Tab. 6. The total ex-samples were used to analyze the recovery rate of the periment was separately carried out for two timesTab.6 Recovery rate analysis of PHB and PHVSeriesAddition ofAddition ofTotalPHy instandardPHV/· rate of plvSample Repeat timesPHB/mgrate of PHB Sludge/mgPHV/analysis/%5.0263.998.8860.2670.210.481101.82623.993.8.8396.0170.27399.7245.03095.4710.2750.484Average100.7523.23100.6120.170.43424.6983.237.931l00.0780.2580.4344.7197.94799.9370.254.708100.1700.2600.436100.20104.820d in the study, and the mixed solt3 Conclusionstions simplify the operation procedure, and the benefitof mixing three materials in the solution was to reduceA mixed digesting solution containeanol the error that may中国煤化工sepsmethyl esterification solution ) H2 SO4 maintain a-cidic condition)and benzoic acid( internal standards culture, activatedYHCNMHGandJournal of Harbin Institute of Technology( New Series),Vol. 18,No5,2011plex cell content. Therefore, it can be accepted whencryobiology,I982(44):238-241.some experimental substance such as ultra pure water [9]Sun SQ, Tian W D. A rapid method for detecting bacterialwas replaced by distilled water in sludge sample analy-polyhydroxyalkanoates in intact cells by Fourier transformsis. And by using an auto sampler, the variety of theinfrared spectroscopy. Applied Microbiology and Biotechretention time of target substances can be eliminated.logy,l999(51):523-526The calculated recovery rates varied between 95%[10 ]Sun SQ, Zhou Q, Hou W J, et al. Study on mechanism ofdifferent PHAs during heating by FrlR. Spectroscopy andand 105%, and the Pha analysis method described inSpectral Analysis, 2000, 20(5 ): 677-678this study was proved to be effective and reliable for [ 11]Tian G, Wu Q, Sun S Q, et al. Study of thermal meltingroutine detectionbehavior of microbial polyhydroxyalkanoates using two-dimensioual fourier transform infrared FT-IR correlationReferencesspectroscopy. 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