1、疏水性有機(jī)物污染的土壤的原位修復(fù)中，外源微生物在土壤介質(zhì)中的高效傳輸及在有機(jī)污染物上的富集可以提高修復(fù)效率在土壤修復(fù)過程中，生物表面活性劑增溶污染物的同時(shí)，有可能與微生物菌體發(fā)生作用，改變菌體的表面性質(zhì)，實(shí)現(xiàn)上述功能表面活性劑膠束溶解態(tài)污染物微生物細(xì)胞膠束溶解的污染物生物表面活性劑單分子土壤顆粒附著于土壤顆粒表面的污染物半膠團(tuán)本論文作為上述目的的應(yīng)用基礎(chǔ)研究，考察了生物表面活性劑鼠李糖脂在微生物菌體上的吸附規(guī)律以及由吸附所引起的菌體表面親水疏水性的變化規(guī)律方法產(chǎn)生：產(chǎn)生菌為Pseudomonas aeruginosa CCTCC AB93066，以20g/L的葡萄糖為碳源，在MSM溶液中，于3

2、0、200rpm條件下，發(fā)酵培養(yǎng)72h，獲得最大產(chǎn)劑量。分離和提純：首先通過離心將菌體從發(fā)酵液中分離，再采用酸沉降法將鼠李糖脂粗產(chǎn)品從發(fā)酵液中分離出來，將粗產(chǎn)品冷凍干燥并溶于氯仿后，過濾除去不溶性雜質(zhì)，濾液通過硅膠柱層析除去其它雜質(zhì)，并將單糖脂和二糖脂分離。二糖脂經(jīng)過兩次柱層析實(shí)現(xiàn)完全純化。表征：采用高效液相色譜-質(zhì)譜聯(lián)用對(duì)提純的鼠李糖脂單糖脂和二糖脂及其2-溴代苯乙酮的衍生物進(jìn)行化學(xué)成分表征。結(jié)果與討論鼠李糖脂單糖脂混合物外觀上呈棕色粘稠油狀；鼠李糖脂二糖脂混合物呈黃色膏狀或白色微黃的晶體狀。各部分鼠李糖脂的HPLC-MS圖譜如下。RT: 0.00 - 19.9902468101214161

3、8Time (min)05101520253035404550556065707580859095100Relative Abundance12.7515.2214.1910.0011.751.540.662.28 3.3518.786.114.3816.978.276.94NL:3.65E8TIC MS zhh03aR haC10C10R haC10C12-H2RhaC10C12zhh03 #778 RT: 12.76 AV: 1 NL: 1.56E8T: - c APCI corona Full ms 200.00-1200.00200300400500600700800900100011

4、001200m/z05101520253035404550556065707580859095100Relative Abundance1006.9503.01008.8538.9540.91009.81005.9332.9836.7606.71031.2651.11080.2920.11199.2bcdzhh04 #648 RT: 10.63 AV: 1 NL: 2.34E7T: - c APCI corona Full ms 200.00-1200.00200300400500600700800900100011001200m/z051015202530354045505560657075

5、80859095100Relative Abundance649.1684.7685.8711.8712.8479.1958.6515.3760.91128.1355.1290.3863.6646.5979.9zhh03 #867 RT: 14.19 AV: 1 NL: 1.14E7T: - c APCI corona Full ms 200.00-1200.00200300400500600700800900100011001200m/z05101520253035404550556065707580859095100Relative Abundance529.1564.91058.8566

6、.91060.8567.8886.8625.7333.11032.7499.1413.1694.6830.4201.51190.9914.91111.6zhh03 #930 RT: 15.22 AV: 1 NL: 1.57E7T: - c APCI corona Full ms 200.00-1200.00200300400500600700800900100011001200m/z05101520253035404550556065707580859095100Relative Abundance531.11062.9566.91063.9568.91064.9569.8616.8333.0

7、361.1944.7694.7499.1256.11074.0892.1755.21197.91044.8OHOOOOOCH3HOOHOHm/z鼠李糖脂單糖脂HPLC-MS圖譜。a) HPLC圖譜；b) 12.5-13 min m/z 1006.9 (RhaC10C10)2-H-；c) 14-14.5 min m/z 529.1 RhaC10C12-H2-H-；d) 15-15.5 min m/z 531.1 RhaC10C12-H-abcdm/z鼠李糖脂單糖脂的2-溴代苯乙酮衍生物的HPLC-MS圖譜。a) HPLC色譜圖；b) 14.5-15.0 min m/z 640.1 RhaC10C

8、10苯甲酰甲脂+NH4+；c) 15.5-16.0 min m/z 665.9 RhaC10C12-H2苯甲酰甲脂+NH4+；d) 16.5-17.0 min m/z 668.0 RhaC10C12苯甲酰甲脂+NH4+ bcdzhh08 #318 RT: 15.22 AV: 1NL: 1.48E7F: + c APCI corona Full ms 300.00-2000.00400600800100012001400160018002000m/z05101520253035404550556065707580859095100Relative Abundance814.2937.0816.2

9、938.0842.1505.21609.2993.9523.3317.0668.11613.51116.81789.8503.61474.71941.61363.31215.3aRT: 0.00 - 20.0002468101214161820Time (min)05101520253035404550556065707580859095100Relative Abundance14.8412.704.184.79 5.913.416.682.467.201.778.061.269.529.8611.3316.5615.7813.1213.3818.9718.54NL:7.18E7TIC F:

10、 + c APCI corona Full ms 100.00-1200.00 MS zhh12zhh12 #348 RT: 14.92 AV: 1 NL: 2.21E7F: + c APCI corona Full ms 100.00-1200.00100200300400500600700800900100011001200m/z05101520253035404550556065707580859095100Relative Abundance640.1641.2763.0654.1477.0668.1141.0494.21125.9765.0181.7307.0459.1720.034

11、0.8536.21004.8570.1949.9877.0zhh12 #370 RT: 15.86 AV: 1 NL: 2.22E6F: + c APCI corona Full ms 100.00-1200.00100200300400500600700800900100011001200m/z05101520253035404550556065707580859095100Relative Abundance665.9667.0141.0181.7680.0788.9502.9314.8705.9519.8640.0789.9182.7493.8707.9521.9332.8289.010

12、11.0942.01052.91178.8zhh12 #386 RT: 16.56 AV: 1 NL: 4.71E6F: + c APCI corona Full ms 100.00-1200.00100200300400500600700800900100011001200m/z05101520253035404550556065707580859095100Relative Abundance668.0669.0504.9682.1790.9181.5522.0683.1316.9709.0334.9523.0239.7487.0792.9640.11143.8846.6 938.1108

13、1.5acbdm/z一次柱層析得到的鼠李糖脂二糖脂的HPLC-MS圖譜。a) HPLC圖譜；b ) 1 0 . 5 - 11 m i n m / z 6 4 9 . 1 Rha2C10C10-H-；c) 12-12.5 min m/z 675.0 Rha2C10C12-H2-H-；d) 12.5-13 min m/z 503.0 RhaC10C10-H-；e) 13-13.5 min m/z 677.1 Rha2C10C12-H- aR ha2C10C10R ha2C10C1 2-H2Rha C10C10Rha2C1 0C12bcdezhh04 #648 RT: 10.63 AV: 1 NL:

14、 2.34E7T: - c APCI corona Full ms 200.00-1200.00200300400500600700800900100011001200m/z05101520253035404550556065707580859095100Relative Abundance649.1684.7685.8711.8712.8479.1958.6515.3760.91128.1355.1290.3863.6646.5979.9zhh04 #648 RT: 10.63 AV: 1 NL: 2.34E7T: - c APCI corona Full ms 200.00-1200.00

15、200300400500600700800900100011001200m/z05101520253035404550556065707580859095100Relative Abundance649.1684.7685.8711.8712.8479.1958.6515.3760.91128.1355.1290.3863.6646.5979.9zhh04 #747 RT: 12.25 AV: 1 NL: 1.34E6T: - c APCI corona Full ms 200.00-1200.00200300400500600700800900100011001200m/z051015202

16、53035404550556065707580859095100Relative Abundance675.0710.6733.7498.9412.8734.7206.7515.0252.0657.0828.2392.81001.0zhh04 #774 RT: 12.69 AV: 1 NL: 1.04E6T: - c APCI corona Full ms 200.00-1200.00200300400500600700800900100011001200m/z05101520253035404550556065707580859095100Relative Abundance503.0538

17、.8540.8412.7240.7736.6332.91006.7599.7498.8771.0691.0888.41166.4zhh04 #812 RT: 13.32 AV: 1 NL: 3.23E6T: - c APCI corona Full ms 200.00-1200.00200300400500600700800900100011001200m/z05101520253035404550556065707580859095100Relative Abundance677.1678.1712.7714.7739.8499.1514.9802.9250.2897.0309.8663.1

18、987.61156.6412.51085.5RT: 0.00 - 19.98024681012141618Time (min)05101520253035404550556065707580859095100Relative Abundance10.6013.3014.6812.2016.756.7516.936.560.793.522.0415.195.267.27 8.37 9.5819.36NL:5.47E7TIC MS zhh04OHOOOOOCH3HOOHOOHHOCH3OOHabcdem/z一次柱層析得到的鼠李糖脂二糖脂的2-溴代苯乙酮衍生物的HPLC-MS圖譜。 a) HPLC色

19、譜圖；b) 13-13.5 min m/z 786.1 Rha2C10C10苯甲酰甲脂+NH4+；c) 14-14.5 min m/z 812.2 Rha2C10C12-H2苯甲酰甲脂+NH4+；d) 14.5-15 min m/z 723.9 R h a C1 0C1 0苯 甲 酰 甲 脂+ ( C H3C H2)3N H+ 或 6 4 0 . 0 RhaC10C10苯甲酰甲脂+NH4+；e) 15-15.5 min m/z 814.2 Rha2C10C12苯甲酰甲脂+NH4+ bcdezhh08 #318 RT: 15.22 AV: 1NL: 1.48E7F: + c APCI coron

20、a Full ms 300.00-2000.00400600800100012001400160018002000m/z05101520253035404550556065707580859095100Relative Abundance814.2937.0816.2938.0842.1505.21609.2993.9523.3317.0668.11613.51116.81789.8503.61474.71941.61363.31215.3zhh08 #278 RT: 13.29 AV: 1 NL: 5.32E7F: + c APCI corona Full ms 300.00-2000.00

21、400600800100012001400160018002000m/z05101520253035404550556065707580859095100Relative Abundance786.11553.5909.0910.01555.5814.1477.11557.5 1676.5937.9306.81552.1495.11407.4640.41954.81063.7 1175.5zhh08 #300 RT: 14.35 AV: 1 NL: 9.71E6F: + c APCI corona Full ms 300.00-2000.0040060080010001200140016001

22、8002000m/z05101520253035404550556065707580859095100Relative Abundance812.2934.9814.1936.0840.11605.4503.1314.9961.9800.4666.31797.21941.81613.41093.7 1168.81604.41443.4zhh08 #310 RT: 14.83 AV: 1 NL: 2.41E6F: + c APCI corona Full ms 300.00-2000.00400600800100012001400160018002000m/z051015202530354045

23、50556065707580859095100Relative Abundance723.9828.0640.0414.9431.9738.1950.91429.41534.31976.6495.01199.21019.91345.21797.7358.71693.8zhh08 #318 RT: 15.22 AV: 1 NL: 1.48E7F: + c APCI corona Full ms 300.00-2000.00400600800100012001400160018002000m/z05101520253035404550556065707580859095100Relative Ab

24、undance814.2937.0816.2938.0842.1505.21609.2993.9523.3317.0668.11613.51116.81789.8503.61474.71941.61363.31215.3RT: 0.00 - 19.96024681012141618Time (min)05101520253035404550556065707580859095100Relative Abundance13.2915.2214.3518.8916.2811.4716.9610.8912.620.737.1510.130.923.224.376.487.634.95NL:1.58E

25、8TIC F: + c APCI corona Full ms 300.00-2000.00 MS zhh08aabdcem/z二次柱層析提純的鼠李糖脂二糖脂的HPLC-MS圖譜。a) HPLC圖譜；b ) 7 . 0 - 8 . 0 m i n m / z 6 2 1 . 1 Rha2C10C8-H-；c) 9.5-10.0 min m/z 647.1 RhaC10C10-H2-H-；d) 1 0 . 5 - 11 . 0 m i n m / z 6 4 9 . 0 Rha2C10C10-H-；e) 12.0-12.5 min m/z 675.0 Rha2C10C12-H2-H-；f) 1

26、3 . 0 - 1 3 . 5 m i n m / z 6 7 7 . 0 Rha2C10C12-H- aR ha2C10C10R ha2C10C1 2-H2Rha2C1 0C12bcdzhh04 #648 RT: 10.63 AV: 1 NL: 2.34E7T: - c APCI corona Full ms 200.00-1200.00200300400500600700800900100011001200m/z05101520253035404550556065707580859095100Relative Abundance649.1684.7685.8711.8712.8479.19

27、58.6515.3760.91128.1355.1290.3863.6646.5979.9RT: 0.00 - 19.94 SM: 3B024681012141618Time (min)05101520253035404550556065707580859095100Relative Abundance10.6912.2413.357.3614.641.099.734.548.945.253.573.1215.68 16.7117.15 18.62NL:4.28E7TIC F: - c ESI Full ms 100.00-1200.00 MS zhh14R ha2C10C8R ha2C10C

28、1 0-H2zhh14 #165 RT: 7.28 AV: 1 NL: 3.49E6F: - c ESI Full ms 100.00-1200.00100200300400500600700800900100011001200m/z05101520253035404550556065707580859095100Relative Abundance621.1703.0118.7706.0784.9479.1451.1844.0943.31181.2162.01071.6386.7981.5576.9282.3238.7zhh14 #221 RT: 9.73 AV: 1 NL: 8.39E5F

29、: - c ESI Full ms 100.00-1200.00100200300400500600700800900100011001200m/z05101520253035404550556065707580859095100Relative Abundance647.1648.1705.1729.0118.7730.1810.9788.8886.61050.1451.2905.61175.0178.6 212.6421.7548.3615.1280.7zhh14 #243 RT: 10.69 AV: 1 NL: 3.55E7F: - c ESI Full ms 100.00-1200.0

30、0100200300400500600700800900100011001200m/z05101520253035404550556065707580859095100Relative Abundance649.0650.0651.0479.0711.4480.0768.7zhh14 #279 RT: 12.24 AV: 1 NL: 1.30E7F: - c ESI Full ms 100.00-1200.00100200300400500600700800900100011001200m/z05101520253035404550556065707580859095100Relative A

31、bundance675.0479.0677.0479.9697.0 760.1275.1931.81179.3822.91138.5538.6zhh14 #305 RT: 13.35 AV: 1 NL: 9.97E6F: - c ESI Full ms 100.00-1200.00100200300400500600700800900100011001200m/z05101520253035404550556065707580859095100Relative Abundance677.0678.0479.0680.0 767.4480.0efabcdef小結(jié)采用銅綠假單胞菌CCTCC AB9

32、3066發(fā)酵培養(yǎng)產(chǎn)生了生物表面活性劑鼠李糖脂，并采用酸沉降和柱層析的方法分離和純化了得到的鼠李糖脂，獲得了較純的鼠李糖脂單糖脂和二糖脂。通過HPLC-MS對(duì)它們的成分進(jìn)行了鑒定，它們的主要成分均為Rha( 2 )C1 0C1 0、Rha(2)C10C12-H2和Rha(2)C10C12。本結(jié)果為后續(xù)研究的基礎(chǔ)。 背景abc生物表面活性劑具有表面活性劑的一般性質(zhì)，能夠在溶液中于不同條件下形成不同形態(tài)的聚集體，如圖中所示的膠束(a)、膠囊(b)和雙層膜結(jié)構(gòu)(c)。鼠李糖脂自身形成的聚集體可能影響到它在菌體上的吸附特征。另外鑒于本論文研究的應(yīng)用背景，有必要考察鼠李糖脂在近中性溶液中的聚集行為。Mon

33、orhamnolipidDirhamnolipidTotalRhaC10C10RhaC10C12-H2RhaC10C12TotalRha2C10C10Rha2C10C12-H2Rha2C10C12molecular weight510a504530532658a650676678Approximate mole fraction in mixture0.800.080.120.700.110.19MS major ions (M-H)1007b529531649675677molecular weight of derivative (M- phenacyl esterc)6226486507

34、68794796MS major ions of derivative (M- phenacyl ester+NH4+)640666668786812814a Average molecular weight.b Molecular weight of RhaC10C10 dimer-H.c Condensate of a rhamnolipid molecule and a Br-CH2-(C=O)-C6H5 by removal of HBr (molecular weight of phenacyl = 119).所用鼠李糖脂為完全提純的鼠李糖脂單糖脂和二糖脂，各包含三種主要成分材料和方

35、法鼠李糖脂CMC測(cè)定：通過溶液表面張力與鼠李糖脂濃度之間的關(guān)系曲線得到單糖脂和二糖脂在MSM中的CMC。聚集體的粒徑和表面zeta電位：鼠李糖脂單糖脂和二糖脂溶于MSM溶液中，在不同濃度和不同pH條件下，測(cè)定聚集物的粒徑分布和表面zeta電位，考察兩者隨濃度和溶液pH的變化規(guī)律。CMCRhamnolipid concentration, C (M).11101001000Surface tension, st (mN/m)20304050607080monorhanmolipiddirhamnolipidCMC鼠李糖脂單糖脂和二糖脂的臨界膠束濃度CMC分別為75M和106MRhamnolipi

36、d concentration, C (M)050100150200250300Aggregate diameter, d (nm)02004006004500500055006000a25 M50 M250 M100 MVolume contribution of aggregate (%)020406080100120bRhamnolipid concentration, C (M)050100150200250300Aggregate diameter, d (nm)02004006004500500055006000c25 M50 M250 M100 MVolume contribut

37、ion of aggregate (%)020406080100120dRhamnolipid concentration, C (M)050100150200250300Aggregate diameter, d (nm)0200400600450050005500e25 M50 M250 M100 MVolume contribution of aggregate (%)020406080100120fRhamnolipid concentration, C (M)050100150200250300Aggregate diameter, d (nm)0200400600450050005

38、500g25 M50 M250 M100 MVolume contribution of aggregate (%)020406080100120hRhamnolipid concentration, C (M)050100150200250300Aggregate diameter, d (nm)02004006004500500055006000i25 M50 M250 M100 MVolume contribution of aggregate (%)020406080100120j單糖脂在MSM中的聚集體共有三種形態(tài)，即使在鼠李糖脂濃度低于CMC（75M）時(shí)也檢測(cè)到了聚集體的存在。對(duì)于

39、同一pH下，隨著濃度的增加，一般地有大粒子c和中等粒子b的體積含量減小，而小粒子a的體積含量增加。一般地，隨pH的增加聚集體有也向小粒子轉(zhuǎn)化的規(guī)律。 Rhamnolipid concentration, C (M)0100200300400500600Aggregate diameter, d (nm)0100200300400500a10 M25 M50 M250 M500 M100 MVolume contribution of aggregate (%)020406080100120bRhamnolipid concentration, C (M)0100200300400500600A

40、ggregate diameter, d (nm)0100200300400500c10 M25 M50 M250 M500 M100 MVolume contribution of aggregate (%)020406080100120dRhamnolipid concentration, C (M)0100200300400500600Aggregate diameter, d (nm)0100200300400e10 M25 M50 M250 M500 M100 MVolume contribution of aggregate (%)020406080100120fRhamnolip

41、id concentration, C (M)0100200300400500600Aggregate diameter, d (nm)050100150200250300350g10 M25 M50 M250 M500 M100 MVolume contribution of aggregate (%)020406080100120hRhamnolipid concentration, C (M)0100200300400500600Aggregate diameter, d (nm)0100200300400500i10 M25 M50 M250 M500 M100 MVolume con

42、tribution of aggregate (%)020406080100120j鼠李糖脂二糖脂在MSM中也形成三種形態(tài)的聚集體，同樣即使在鼠李糖脂濃度低于CMC（75M）時(shí)也檢測(cè)到了聚集體的存在。在所有pH下，隨著鼠李糖脂濃度的增加，三種粒子的粒徑均有變小的一般規(guī)律。各濃度下，大粒子b的體積含量基本隨pH的增加而降低，小粒子a的體積含量在各pH下均為主要，且隨pH的增大而增加Rhamnolipid concentration (M)0100200300400500600zeta potential (mV)-30-25-20-15-10隨著液相中二糖脂濃度的升高，聚集體表面zeta電位先升

43、高后降低，最后趨于穩(wěn)定。MSM溶液的pH的升高也使聚集體表面zeta電位降低 （）pH 6.0；（ ）pH 6.5；（）pH 7.0；（ ）pH7.5；（）pH 8.0 小結(jié)單糖脂的CMC比二糖脂要小，認(rèn)為是單糖脂分子較大的疏水/親水比所致。在濃度低于和高于CMC時(shí)，單糖脂和二糖脂均能形成聚集體，但兩種聚集體結(jié)構(gòu)上可能存在差別。隨著濃度和pH的增加，聚集體的粒徑均有由大變小的一般規(guī)律，且小粒徑聚集體的含量增加，推測(cè)為聚集體結(jié)構(gòu)變化和表面分子電性變化共同作用的結(jié)果，而對(duì)于聚集體表面zeta電位的研究結(jié)果也有力支持該推測(cè)。聚集體有可能作為整體參與到鼠李糖脂與微生物體的相互作用過程中，因此在研究鼠李

44、糖脂在微生物體上的吸附時(shí)，它需要加以考慮。 背景表面活性劑在微生物菌體上的吸附過程是表面活性劑化學(xué)基團(tuán)和菌體表面化學(xué)基團(tuán)相互作用的過程，表面活性劑的化學(xué)結(jié)構(gòu)對(duì)吸附特征及菌體表面性質(zhì)的改變有影響材料和方法吸附質(zhì)為CTAB、Triton X-100、Tween 80、SDS以及一次純化后的鼠李糖脂二糖脂混合物。吸附劑為銅綠假單胞菌CCTCC AB93066菌體。菌體的表面親水疏水性通過菌體在親水性和疏水性玻璃珠表面的吸附率表征不同濃度表面活性劑溶液對(duì)數(shù)期菌體鼠李糖脂的吸附量、吸附前后菌體表面親水疏水性的變化at (min)0306090120150180210240qt, adsorption q

45、uantity (mol/1013Cap.)0306090120150bt (min)050100150200Log(qe-qt)-3-2-10123ct (min)0306090120150180210240t/qt05101520253035a) 吸附速率曲線；b) 一次速率方程回歸曲線；c) 二次速率方程回歸曲線。CTAB（）、Triton X-100（）、Tween 80（）、SDS（）和鼠李糖脂（）結(jié)果和討論一次速率方程：二次速率方程： 二次速率方程擬合得更好，根據(jù)吸附速率常數(shù)可以得到，表面活性劑的吸附速率從大到小依次為：CTAB、Triton X-100、Tween80、鼠李糖脂和

46、SDS tkqqqete303. 2log)log(1tqqkqteet1122aCe, equilibrium concentration (M)0100200300400500qe, adsorption quantity (mol/1013Cap.)030060090012001500blogCe0.0.51.01.52.02.53.0logqe-101234Freundlich等溫吸附方程：或： 這幾種表面活性劑在該銅綠菌體表面上的吸附符合Freundlich規(guī)律。CTAB和鼠李糖脂對(duì)應(yīng)的K值較大，說明細(xì)胞表面和這兩種表面活性劑之間的吸附驅(qū)動(dòng)力較大；細(xì)胞吸附Triton X-100的能

47、力最弱a) 等溫吸附曲線；b) Freundlich模型回歸曲線。 CTAB（）、Triton X-100（）、Tween 80（）、SDS（）和鼠李糖脂（）neeCKq/1eeCnKqlog1loglog表面活性劑吸附率 (%)親水性表面疏水性表面02.5mg/l25mg/l250mg/l02.5mg/l25mg/l250mg/lCTAB25.50.013.70.118.10.211.60.114.50.115.90.123.40.125.80.2Triton X-10015.60.514.80.318.70.516.50.216.50.027.30.3Tween 8014.10.111.7

48、0.116.20.021.80.020.90.124.20.0SDS10.20.15.40.019.20.016.80.110.20.119.20.0鼠李糖脂16.80.112.70.111.90.116.80.118.60.017.00.0用表面活性劑吸附處理過的銅綠假單胞菌在親水和疏水性玻璃珠上的吸附。CTAB、Triton X-100和Tween 80的吸附提高了細(xì)胞在疏水性，并減小了菌體在親水性。鼠李糖脂和SDS的吸附減弱了菌體表面的親水性。a) CTAB；b) Triton X-100；c) Tween 80；d) SDS；e) 鼠李糖脂。符號(hào)：30C，IS=0（）、30C，IS=2

49、0 mM（）、30C；IS=200 mM（）、40C，IS=0（）。隨著溫度的增加，Triton X-100、Tween 80和鼠李糖脂的吸附量都稍有減小，SDS的吸附量有了顯著的增加；隨離子強(qiáng)度的增大，Triton X-100，Tween 80和鼠李糖脂的吸附量增大，而SDS的吸附量有顯著增加，當(dāng)離子強(qiáng)度為200mM時(shí)，SDS的吸附率幾乎達(dá)到100%，和CTAB的吸附率相當(dāng)。 aCe (M)051015202530qe (mol/1013Cap.)0100200300400500600700bCe (M)04080120160200qe (mol/1013Cap.)0204060801001

50、20140cCe (M)01530456075qe (mol/1013Cap.)020406080100120140dCe (M)050100150200250qe (mol/1013Cap.)02004006008001000eCe (M)020406080100qe (mol/1013Cap.)050100150200250300小結(jié)五種表面活性劑在銅綠假單胞菌CCTCC AB93066菌體上的吸附動(dòng)力學(xué)規(guī)律與二級(jí)速率模型符合較好，而它們的等溫吸附規(guī)律則與Freundlich方程符合較好。由于這幾種表面活性劑的結(jié)構(gòu)和性質(zhì)上的差異，吸附驅(qū)動(dòng)力的類型和強(qiáng)弱有明顯差別。在靜電引力驅(qū)動(dòng)下，CTAB

51、吸附非?？欤椅铰式咏?00%。鼠李糖脂的吸附量大，可能是鼠李糖脂分子結(jié)構(gòu)與菌體表面化學(xué)結(jié)構(gòu)的相似性引起。SDS達(dá)到平衡所用的時(shí)間最長(zhǎng)，推測(cè)為靜電斥力導(dǎo)致。兩種非離子表面活性劑，達(dá)到吸附平衡較快，但吸附率較低，這與它們較大的極性基團(tuán)有關(guān)。五種表面活性劑的吸附對(duì)菌體表面的親水性均有減弱作用，同時(shí)CTAB、Triton X-100和Tween 80增強(qiáng)了菌體表面的疏水性。 背景有研究表明，生物表面活性劑對(duì)菌體表面性質(zhì)的改變與菌體性質(zhì)有密切關(guān)系。因此，為了更好的控制和利用生物表面活性劑調(diào)節(jié)菌體表面性質(zhì)這一功能，需要深入了解生物表面活性劑與不同菌體相互作用而改變其表面性質(zhì)的機(jī)制。本章以上一章節(jié)研究結(jié)

52、果為基礎(chǔ)，從微生物的一般性入手，繼續(xù)研究了鼠李糖脂二糖脂在四種微生物菌體表面的吸附行為以及吸附與菌體表面性質(zhì)的關(guān)聯(lián)。材料和方法吸附質(zhì)為一次純化后的鼠李糖脂二糖脂混合物。吸附劑菌種為一株銅綠假單胞菌，兩株枯草芽孢桿菌和一株解脂假絲酵母，它們分別作為革蘭氏陰性菌、革蘭氏陽性菌和酵母的代表菌。首先考察了它們菌體的表面性質(zhì)隨生長(zhǎng)時(shí)間的變化，接著考察了它們對(duì)數(shù)期菌體和靜止期菌體對(duì)鼠李糖脂的吸附特征以及菌體表面親水疏水性的變化。菌體的表面親水疏水性通過菌體在正十六烷相中的吸附率（BATH測(cè)試）表征結(jié)果與討論aIncubation time (h)01020304050Cell dwt (mg/ml).00

53、1.01.11Surface tension (mN/m)020406080100Adherence rate (%)05101520253035zeta potential (mV)-30-25-20-15-10-50lagexpstatbIncubation time (h)01020304050Cell dwt (mg/ml).001.01.11Surface tension (mN/m)020406080100Adherence rate (%)0102030zeta potential (mV)-30-25-20-15-10-50lagexpstatcIncubation time

54、(h)01020304050Cell dwt (mg/ml).001.01.11Surface tension (mN/m)020406080100Adherence rate (%)0102030zeta potential (mV)-30-25-20-15-10-50lagexpstatdIncubation time (h)01020304050Cell dwt (mg/ml).001.01.1110Surface tension (mN/m)020406080100Adherence rate (%)020406080100zeta potential (mV)-30-25-20-15

55、-10-50lagexplate expstat在整個(gè)生長(zhǎng)過程中，四種菌體均不產(chǎn)生生物表面活性劑。從對(duì)數(shù)期中后期到靜止期的過程中，三種細(xì)菌的表面變得更加親水，而解脂假絲酵母CCTCC AB92044的表面變得疏水，四種菌的表面zeta電位變化不大1101001000 bAdsorbed quantity, S (umol/gdwt)0.11101001000110100 c Equilibrium concentration, Ce (umol/L)0.111010010001101001000 a0.00.20.40.60.81.00510152025303540dadsorption ra

56、te (%)Initial concentration of RL, C0 (umol/L)0.00.20.40.60.81.0-30-25-20-15-10-50ezeta potential (mV)Initial concentration of RL, C0 (umol/L)銅綠假單胞菌 CCTCC AB93072a) 總鼠李糖脂在對(duì)數(shù)期細(xì)胞（）和靜止期細(xì)胞（）表面的平衡吸附曲線；b) 鼠李糖脂各成分在對(duì)數(shù)期菌體表面的平衡吸附曲線，RhaC10C10（），Rha2C10C10（），Rha2C10C12-H2（）和Rha2C10C12（）；c) 鼠李糖脂各成分在靜止期菌體表面的平衡吸附曲

57、線，符號(hào)含義同b)；d) 對(duì)數(shù)期菌體（ ）和靜止期菌體（ ）在不同濃度鼠李糖脂吸附處理后的BATH吸附率；e) 對(duì)數(shù)期菌體（）和靜止期菌體（）在不同濃度鼠李糖脂吸附處理后的表面zeta電勢(shì)。在以兩種菌體為吸附劑時(shí)，均發(fā)生了鼠李糖脂的兩步吸附現(xiàn)象，并且對(duì)數(shù)期菌體在這兩步吸附過程中均在較低的平衡濃度下達(dá)到吸附飽和（圖a）。對(duì)數(shù)期菌體有更強(qiáng)的吸附鼠李糖脂分子的能力。對(duì)于鼠李糖脂的各成分，它們的平衡吸附曲線的形狀和總表面活性劑平衡吸附曲線的形狀類似（圖b和圖c），然而對(duì)數(shù)期細(xì)胞對(duì)RhaC10C10的平衡吸附曲線中沒有第一個(gè)吸附平臺(tái)，表明該成分在該平衡濃度范圍內(nèi)的吸附受二糖脂成分的吸附影響較小。吸附使兩

58、種菌體表面親水疏水性向相反方向變化，且變化均在低鼠李糖脂濃度時(shí)最為顯著，表明菌體的表面親水疏水性對(duì)低濃度的鼠李糖脂的吸附更為敏感（圖d）。菌體表面電性對(duì)鼠李糖脂的吸附均不敏感（圖e） 1101001000 bAdsorbed quantity, S (umol/gdwt)0.11101001000110100 c Equilibrium concentration, Ce (umol/L)0.111010010001101001000 a0.00.20.40.60.81.00510152025303540dadsorption rate (%)Initial concentration of

59、RL, C0 (umol/L)0.00.20.40.60.81.0-30-25-20-15-10-50ezeta potential (mV)Initial concentration of RL, C0 (umol/L)枯草芽孢桿菌 CCTCC AB93108對(duì)數(shù)期菌體平衡吸附曲線包括在低濃度和高濃度時(shí)吸附量隨平衡濃度的增加部分和中間的吸附平臺(tái)。靜止期菌體的平衡吸附曲線包括低平衡濃度時(shí)的吸附平臺(tái)和高平衡濃度時(shí)吸附量隨平衡濃度增加而上升部分（圖a）。和銅綠假單胞菌CCTCC AB93072一樣，該菌對(duì)數(shù)期菌體的吸附能力高于靜止期菌體。鼠李糖脂各成分在對(duì)數(shù)期菌體上的平衡吸附曲線形狀與總表面活性劑

60、類似，不同的是RhaC10C10的吸附平臺(tái)不明顯，表明該單糖脂的吸附受二糖脂成分吸附的影響也較小（圖b）。但各成分在靜止期菌體上的吸附有很大差別。Rha2C10C10的吸附量是平衡濃度的凸函數(shù)，而具有更長(zhǎng)的脂肪烴鏈的Rha2C10C12和Rha2C10C12-H2兩種成分為凹函數(shù)（圖c），表明在吸附競(jìng)爭(zhēng)中，二糖脂三種成分中的強(qiáng)者由強(qiáng)疏水性的成分向相對(duì)弱疏水性成分的轉(zhuǎn)變。菌體對(duì)低濃度的鼠李糖脂的吸附敏感，對(duì)數(shù)期菌體BATH吸附率隨鼠李糖脂濃度的升高迅速降低，而靜止期菌體的表面親水疏水性在整個(gè)測(cè)試的鼠李糖脂濃度范圍內(nèi)經(jīng)歷的急劇的上升和下降（圖d）。表面zeta電勢(shì)對(duì)鼠李糖脂的吸附不敏感（圖e）枯草