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PHA测定

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2024年3月29日发(作者:廉德运)

PHA was analysed through the following procedure. Sludge samples from

lab-scale sequencing batch reactors (SBRs) were mixed with formaldehyde at a

ratio of approximately 1% formaldehyde per sample volume in order to inhibit

biomass activity in the sludge. The samples were centrifuged and the supernatant

was removed, then washed with a phosphate buffer solution, re-centrifuged, and

the supernatant decanted once more. All samples were then lyophilised through a

freeze drying unit (FTS, Queensland, Australia) operated at −54 ◦C and 0.1 mbar

for at least 20 h. Approximately 20 mg of lyophilised sludge was added to 2mL of

chloroform and 2mL of an acidified methanol solution (containing either 3%, 10%

or 20% sulfuric acid by volume, as well as approximately 100 mg/L of sodium

benzoate [17], used as an internal standard). Six standard solutions were

composed of 0–3 mgof a R-3-hydroxybutyric acid (3HB) and R-3-hydroxyvaleric

acid (3HV) copolymer (7:3) (Fluka, Melbourne, Victoria, Australia) and 0–3 mg of

2-hydroxycaproic acid (Sigma–Aldrich, Melbourne, Victoria, Australia). Due to the

unavailability of a direct standard for 3-hydroxy-2-methylvaleric acid (3H2MV),

itwas assumed that the relative response factor for 2-hydroxycaproic acidwould be

similar to that of 3H2MV for GC quantification purposes, since these two

molecules are isomers of each other. The samples and standards were then

digested in tightly sealed 10mL glass vials for either 2, 6 or 20 h at 100 ◦C, and

cooled to room temperature. Distilled water (1 mL) was then added and mixed

vigorously with each sample to remove particulate debris from the chloroform

phase and prevent degradation of the GC column [17]. After mixing, 1 h of settling

time was allowed to achieve phase separation. The chloroform (bottom) phase was

then transferred to another vial, dried with approximately 0.5–1 g of granular

sodium sulphate pellets, and separated from the solid phase. Three microlitres of

the chloroform phase was analysed with a Perkin-Elmer gas chromatograph. The

chromatograph was operated with a DB-5 column (30m length×0.25mm

I.D.×0.25_m film), a split injection ratio of 1:15 and helium as the carrier gas (1.5

mL/min). A flame ionisation detection (FID) unit was operated at 300 ◦C with an

injection port temperature of 250 ◦C. The oven temperature was set to 80 ◦C for 1

min, increased at 10 ◦C/min to 120 ◦C, and then to 270 ◦C at 45◦C/min and held for

3 min.

PHA:气相色谱法。取活性污泥混合液约50mL离心后用磷酸盐缓冲溶液清洗并再次

离心,样品在- 54℃、0.1mbar 条件下冷冻干燥20h ,称取约20mg冷冻泥加1,22二氯乙烷

2mL、含有浓盐酸的丙醇2mL (容积比为1∶4) ,在100℃下加热4 h ,加入2mL的去离子水,

剧烈振荡20~30 s ,使其分相,下层为含有PHA 的有机相,中间为活性污泥残渣,上层为水相;

取下层有机相用无水硫酸钠干燥。取1μL含有PHA的有机相用气相色谱仪测定,色谱仪型号

为安捷伦6890N色谱分析仪,色谱柱为DB25 (30 m Length ×012 mm I.D. ×0.25μm film),

气相色谱测定时氮气为载气,流速115mL/min ;分流比为1∶15 ;FID为探测器;柱子温度变化

程序为初始温度80℃,维持1 min ,后以45℃/min的速度增加到270℃,维持3 min ;检测器温

度为250℃,注射器温度为180℃。

PHA and glycogen were measured by triplicate. PHA was measured according

to Oehmen et al. (2005b) in a GC system operated with a Hewlett Packard 5890

column (30 m length _ 0.53 mm I.D. _ 1.00 mm film). 40 mg of lyophilized sludge

samples were digested and methylated with 4 ml of acidulated methanol (10%

H2SO4) and 4 ml of chloroform during 20 h at 100 _C. Benzoic acid was used as

internal standard. The calibration of the method was performed using a

3-hydroxybutyric acid and 3-hydroxyvaleric acid copolymer (7:3) as standards for

2024年3月29日发(作者:廉德运)

PHA was analysed through the following procedure. Sludge samples from

lab-scale sequencing batch reactors (SBRs) were mixed with formaldehyde at a

ratio of approximately 1% formaldehyde per sample volume in order to inhibit

biomass activity in the sludge. The samples were centrifuged and the supernatant

was removed, then washed with a phosphate buffer solution, re-centrifuged, and

the supernatant decanted once more. All samples were then lyophilised through a

freeze drying unit (FTS, Queensland, Australia) operated at −54 ◦C and 0.1 mbar

for at least 20 h. Approximately 20 mg of lyophilised sludge was added to 2mL of

chloroform and 2mL of an acidified methanol solution (containing either 3%, 10%

or 20% sulfuric acid by volume, as well as approximately 100 mg/L of sodium

benzoate [17], used as an internal standard). Six standard solutions were

composed of 0–3 mgof a R-3-hydroxybutyric acid (3HB) and R-3-hydroxyvaleric

acid (3HV) copolymer (7:3) (Fluka, Melbourne, Victoria, Australia) and 0–3 mg of

2-hydroxycaproic acid (Sigma–Aldrich, Melbourne, Victoria, Australia). Due to the

unavailability of a direct standard for 3-hydroxy-2-methylvaleric acid (3H2MV),

itwas assumed that the relative response factor for 2-hydroxycaproic acidwould be

similar to that of 3H2MV for GC quantification purposes, since these two

molecules are isomers of each other. The samples and standards were then

digested in tightly sealed 10mL glass vials for either 2, 6 or 20 h at 100 ◦C, and

cooled to room temperature. Distilled water (1 mL) was then added and mixed

vigorously with each sample to remove particulate debris from the chloroform

phase and prevent degradation of the GC column [17]. After mixing, 1 h of settling

time was allowed to achieve phase separation. The chloroform (bottom) phase was

then transferred to another vial, dried with approximately 0.5–1 g of granular

sodium sulphate pellets, and separated from the solid phase. Three microlitres of

the chloroform phase was analysed with a Perkin-Elmer gas chromatograph. The

chromatograph was operated with a DB-5 column (30m length×0.25mm

I.D.×0.25_m film), a split injection ratio of 1:15 and helium as the carrier gas (1.5

mL/min). A flame ionisation detection (FID) unit was operated at 300 ◦C with an

injection port temperature of 250 ◦C. The oven temperature was set to 80 ◦C for 1

min, increased at 10 ◦C/min to 120 ◦C, and then to 270 ◦C at 45◦C/min and held for

3 min.

PHA:气相色谱法。取活性污泥混合液约50mL离心后用磷酸盐缓冲溶液清洗并再次

离心,样品在- 54℃、0.1mbar 条件下冷冻干燥20h ,称取约20mg冷冻泥加1,22二氯乙烷

2mL、含有浓盐酸的丙醇2mL (容积比为1∶4) ,在100℃下加热4 h ,加入2mL的去离子水,

剧烈振荡20~30 s ,使其分相,下层为含有PHA 的有机相,中间为活性污泥残渣,上层为水相;

取下层有机相用无水硫酸钠干燥。取1μL含有PHA的有机相用气相色谱仪测定,色谱仪型号

为安捷伦6890N色谱分析仪,色谱柱为DB25 (30 m Length ×012 mm I.D. ×0.25μm film),

气相色谱测定时氮气为载气,流速115mL/min ;分流比为1∶15 ;FID为探测器;柱子温度变化

程序为初始温度80℃,维持1 min ,后以45℃/min的速度增加到270℃,维持3 min ;检测器温

度为250℃,注射器温度为180℃。

PHA and glycogen were measured by triplicate. PHA was measured according

to Oehmen et al. (2005b) in a GC system operated with a Hewlett Packard 5890

column (30 m length _ 0.53 mm I.D. _ 1.00 mm film). 40 mg of lyophilized sludge

samples were digested and methylated with 4 ml of acidulated methanol (10%

H2SO4) and 4 ml of chloroform during 20 h at 100 _C. Benzoic acid was used as

internal standard. The calibration of the method was performed using a

3-hydroxybutyric acid and 3-hydroxyvaleric acid copolymer (7:3) as standards for

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