组合式白腐真菌反应器处理实际染料废水过程中生态结构和功能解析
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摘要
白腐真菌因其极强的降解能力和特殊的代谢类型,其在难降解有机污染物的降解上发挥着十分重要的作用。但白腐真菌工艺由于各种因素的影响,其对实际染料废水的综合处理能力难以进一步提升,并影响到其实际应用价值和效益。
本论文主要依托于国家科技部863计划项目《污水的高效微生物处理技术》(2006AA06Z331),利用白腐真菌对难降解有机废水——实际染料废水进行处理技术研究,同时后期在上海染化八厂开展现场中试实验研究,论文的重点研究内容有以下几个方面：①四种白腐真菌菌株产酶(MnP和LiP)优化筛选研究；②P.C. (MnP和LiP)的固定化载体研究；③组合式白腐真菌反应器的构建及运行参数的确定；④组合式白腐真菌反应器用于实际染料废水的小试及中试研究。本论文的目标是通过白腐真菌菌株优化、固定化载体筛选、组合式白腐真菌反应器构建及小试和中试研究,为组合式白腐真菌反应器处理实际染料废水的特性、效能及存在问题提供一定的科学依据。主要得出如下结论：
(1)四种白腐真菌菌株产MnP和LiP优化筛选研究
四种白腐真菌紫芝G. sinense、云芝C. versicolor、韩芝G. lucidum HG及黄孢原毛平革菌P.C.产MnP及LiP酶活差异明显,其中以P.C.的两种酶活为最高,其MnP和LiP酶活分别于第5天和第6天达到最大值225.67 U/L和110.35 U/L而P.C.在氮限制培养时要明显高于碳限制培养条件下,通过统计分析软件spss18.0分析得知,P.C.在氮、碳限制培养条件下MnP和LiP的酶活均呈显著性差异,其p值均低于0.05。
对应P.C.的起始pH介于4.0～4.5之间时,P.C.产MnP及LiP的酶活相对较高,而当起始pH为4.5时两种酶活为最高；当装液量在500 mL/L时对应的MnP(高于240 U/L)和LiP(高于110 U/L)酶活均达到最高值；而孢子的接种量则以1.3×106个/mL时获得的MnP及LiP均分别达到180 U/L及130 U/L的最高水平；摇床转速介于120 r/min和140 r/min之间,其所获得的MnP及LiP酶活均较高。因此,对于四种白腐真菌而言,以P.C.所产MnP及LiP酶活为最高。
(2)P.C.经六种不同的固定化载体固定化后的产酶情况
P.C.经植物废料甘蔗渣和玉米芯固定化后其MnP和LiP酶活较其它四种载体固定化所获得的酶活高,通过spss18.0进行分析得知,甘蔗渣和玉米芯所产MnP与LiP酶活与其他四种载体具有极其显著的差异,其对应的p=0.000<0.01。虽经甘蔗渣固定后的MnP酶活要略低于玉米芯组,但甘蔗渣的LiP酶活均值要高于玉米芯组,综合考虑后采用甘蔗渣做为后续小试及中试的固定化载体。
固定化载体甘蔗渣的投加量在6g/L时其所获得的MnP为最高,达157.1 U/L,同时其对应的对100 mg/L活性艳红X3-B脱色效果也最佳,至实验结束时可达99.6%,利用spss分析得知,不同的甘蔗渣的投加量对脱色率具有极其显著的相关性,其相关系数r=0.373,p值为0.00<0.01。而且该投加量下甘蔗渣能有效抑制其他杂菌的生长,同时利用SEM观察得知甘蔗渣能较好的作为P.C.生长、附着的载体。
(3)利用正交优化实验确定了组合式白腐真菌反应器的多项重要参数,并成功运行了该反应器
利用正交优化实验的直观分析、极差分析及方差分析对前两级白腐真菌反应器的三项重要参数T、DO及pH进行确定,从方差分析得知T、DO和pH三因素的FA=190.37,FB=41.82,Fc=0.8530,也就是对实际染料废水脱色效果的影响依次为T>DO>pH,试验表明T为30℃、DO为5mg/L和pH为5时所获得的脱色率为最高。构建的组合式白腐真菌反应器的主体反应器P.C.—组合填料反应器在处理实际染料废水时,其色度和CODCr在0～24 h内降低较为迅速,二者的去除率分别可达53.41%和29.50%,据此确定组合式系统的HRT为24h。组合式白腐真菌反应器的染料废水与培养基的配比为4∶1时,系统对CODCr及BOD5的去除效果为最佳,其对NH3-N的去除平均可达到国家一级排放标准即<15mg/L,对实际染料废水的色度也达到了国家排放标准的50以下,经检测,出水的生物毒性仅相当于平均浓度为0.058mg/L HgCl2的毒性,为低毒水平。对应的真菌微生物群落的多样性指数基本稳定在5.60左右,反应器内P.C.始终为优势菌种,其中白腐真菌—组合填料反应器在运行期间其微生物群落结构基本稳定。对染料废水稀释并不能有效提高系统的CODCr、BOD5去除率以及对实际染料废水生物毒性的削减。对系统内微生物群落结构进行解析发现当该系统采用稀释染料废水时,反应柱内微生物群落多样性指数波动不大,基本稳定在5.48左右。但低温下系统对CODCr、BOD5、生物毒性及脱色率的去除效果较常温下要低,反应器内微生物群落多样性指数也较常温下低,其平均值为5.20,而随着系统的运行,在低温工况后期其多样性指数呈上升趋势,说明系统运行后期有其他菌的侵染,它们并不是优势种,同DGGE图谱分析结果相一致。DGGE测序结果显示,三种工况下Neosartorya fischeri、Sordaria macrospora、Penicillium chrysogenum及P.C.四种菌均存在,而其他几种菌均较少,不属于优势种。
(4)组合式白腐真菌反应器用于实际染料废水的中试研究
现场中试研究得知,表明该组合系统对NH3-N、CODCr及BOD5均有良好的去除效果,该组合系统对可生化性差的实际染料废水仍然具有一定的生化降解能力。同时该组合系统对实际染料废水的生物毒性物质也有良好的去除效果,出水毒性检测表明其对应的HgCl2平均浓度已降至0.08 mg/L,属于低毒水平。
本论文通过对白腐真菌菌株的优化、黄孢原毛平革菌产酶固定化载体的筛选、组合式白腐真菌反应器工艺技术参数选择及应用该反应器进行的小试及中试等方面的研究结果,为该组合式白腐真菌反应器处理染料废水并保障其稳定、高效运行提供了可靠的依据,具有一定的理论意义和应用价值。
White rot fungi is capable of oxidizing persistent organic pollutants (POPs) including dyes because of their strong biodegradability and unique metabolic type. However, It's hard to promote the comprehensive ability of treating actual dye wastewater, which will affect its applications and effectiveness. In addition, there are many factors influencing the growth and ligninases production of white rot fungi, as well as the enzymatic reaction, which raise new challenge to the technical process of white rot fungi applied to treating actual dye wastewater.
This thesis is based on the project of effective microorganism techniques in wastewater treatment supported by Chinese National Programs for High Technology Research and Development (863Program). In the current study, the ligninases production of four white rot fungi was studied. The six carriers for immobilization of P. C. were also researched. Then the small-scale of integrated white rot fungi reactor was constructed and the performance of this reactor was researched with different conditions. And finally, the pilot-scale field tests were carried by Shanghai No.8 Dyestuff Chemistry Plant. The purpose of this thesis is to provide certain scientific references for the construction of white rot fungi integrated reactor and its stable and effective running.
Four white rot fungi, G. Sinense, C. versicolor, G. lucidum HG and P.C., were significant different in enzyme activity of MnP and LiP. Both MnP and LiP of P.C. had the highest enzyme activity, for the peak activities of MnP and Lip were 225.67 U/L (detected at 5th day) and 110.35 U/L (detected at 6th day) respectively. The enzyme activities of P. C. under N-limited culture condition were much higer than under C-limited culture condition. Statistical analysis using spss18.0 indicated that the MnP and LiP enzyme activities of P. C. under Nitrogen and Carbon limited culture conditions exhibited significant differences, which p value less than 0.05.
The enzyme activities of MnP and LiP of P.C. were comparatively high under pH value 4.0 to 4.5, and the two enzyme activities reached peak at pH value 4.5. MnP (more than 240 U/L) and LiP (more than 110 U/L) were at the peak activites when medium volume was 250mL. MnP and LiP reached the peak of 180 U/L and 130 U/L respectively when inoculation amount of spore suspension was 1.3×106 cell/mL. Speed between 120r/min and 140r/min was favorable for higher activities of MnP and LiP. Consequently, with respect to the four white rot fungi, MnP and LiP produced by P.C. had the highest enzyme activities.
P.C. immobilized on bagasse and corn cob which was two agricultral wastes exhibited higher activities of MnP and LiP than on other four immobilized carriers. Through analysis by spss18.0, the activities of MnP and LiP produced on bagasse and corn cob were dramatically different from the situations of others, with corresponding p=0.000<0.01. The activites of LiP on bagasse were all higher than those on corn cob, despite the activities of MnP on bagasse were slightly less than those on corn cob. Taken these factors into consideration, bagasse was employed as immobilized carrier for the following lab-scale and pilot-scale experiments. When the immobilized carrier bagasse was 6 g/L, the maximum MnP could be 157.1U/L. Meanwhile, its decoloration was optimal, reaching 99.6%at the end of the experiment. Accroding to spss analysis, the dosage of bagasse and decoloration rate were extremely significant correlated, with correlation coefficient r is 0.373, p value less than 0.01. In addition, other bacterials were inhibited effectively under the above bagasse dose. SEM observation indicated that bagasse could be used well as carriers for P. C. adherence and growth.
Using the orthogonal optimization test to determine the three important parameters (T, DO and pH) of intergrated white rot fungi reactor. The variance analysis showed that T has the grwatest impact on reactor performance, followed by DO, pH. When T, DO and pH was respectively 30℃,5 mg/L and 5, the system obtained the highest decolorization efficiency. However, when integrated white rot fungi treating the dyestuff wastewater, the colority and CODCrwas reduced rapidly within 24 h. According to the analysis, the HRT was determined 24 h.
The removal efficiency of CODCr and BOD5 was the best when the ratio of dyestuff wastewater and the N-limited liquid medium was 4:1. The average removal of NH3-N can reach the national top standard, lower than 15 mg/L. The colority of the actual dyestuff wastewater was also reach the national standard lower than 50. Meanwhile, the average concentration of HgCl. was 0.058 mg/L, belonging to low toxicity level. The corresponding fungal microbial community diversity index stabilized around 5.60 and the P.C. is the dominant species all the time. Whether diluting or not, the reactor can't effectively improve the removal efficiency of CODCr, BOD5 and reduce the biotoxicity of the actual dyestuff wastewater. Microbial community diversity index of the reactor was stable at around 5.48. Under the condition of low temperature, the removal efficiency of CODcr, BOD5, biotoxicity and decolorization rate was lower than normal. Microbial community diversity index of the reactor was lower than normal temperature about 5.20. Meanwhile, with the system running, microbial community diversity index was increasing in the latter of the condition of low temperature. In accordance with DGGE profiles, it indicated that other bacteria intruded the system in the latter run, but not the dominant species.
The pilot study indicated that the intergrated system have good removal efficiency of NH3-N, CODCr and BOD5. Meanwhile, the intergrated system also had good removal efficiency on the actual toxicity of wastewater. The average concentration of HgCl2 in the effluent had been reduced to 0.08 mg/L, of low toxicity level.
本论文主要依托于国家科技部863计划项目《污水的高效微生物处理技术》(2006AA06Z331),利用白腐真菌对难降解有机废水——实际染料废水进行处理技术研究,同时后期在上海染化八厂开展现场中试实验研究,论文的重点研究内容有以下几个方面：①四种白腐真菌菌株产酶(MnP和LiP)优化筛选研究；②P.C. (MnP和LiP)的固定化载体研究；③组合式白腐真菌反应器的构建及运行参数的确定；④组合式白腐真菌反应器用于实际染料废水的小试及中试研究。本论文的目标是通过白腐真菌菌株优化、固定化载体筛选、组合式白腐真菌反应器构建及小试和中试研究,为组合式白腐真菌反应器处理实际染料废水的特性、效能及存在问题提供一定的科学依据。主要得出如下结论：
(1)四种白腐真菌菌株产MnP和LiP优化筛选研究
四种白腐真菌紫芝G. sinense、云芝C. versicolor、韩芝G. lucidum HG及黄孢原毛平革菌P.C.产MnP及LiP酶活差异明显,其中以P.C.的两种酶活为最高,其MnP和LiP酶活分别于第5天和第6天达到最大值225.67 U/L和110.35 U/L而P.C.在氮限制培养时要明显高于碳限制培养条件下,通过统计分析软件spss18.0分析得知,P.C.在氮、碳限制培养条件下MnP和LiP的酶活均呈显著性差异,其p值均低于0.05。
对应P.C.的起始pH介于4.0～4.5之间时,P.C.产MnP及LiP的酶活相对较高,而当起始pH为4.5时两种酶活为最高；当装液量在500 mL/L时对应的MnP(高于240 U/L)和LiP(高于110 U/L)酶活均达到最高值；而孢子的接种量则以1.3×106个/mL时获得的MnP及LiP均分别达到180 U/L及130 U/L的最高水平；摇床转速介于120 r/min和140 r/min之间,其所获得的MnP及LiP酶活均较高。因此,对于四种白腐真菌而言,以P.C.所产MnP及LiP酶活为最高。
(2)P.C.经六种不同的固定化载体固定化后的产酶情况
P.C.经植物废料甘蔗渣和玉米芯固定化后其MnP和LiP酶活较其它四种载体固定化所获得的酶活高,通过spss18.0进行分析得知,甘蔗渣和玉米芯所产MnP与LiP酶活与其他四种载体具有极其显著的差异,其对应的p=0.000<0.01。虽经甘蔗渣固定后的MnP酶活要略低于玉米芯组,但甘蔗渣的LiP酶活均值要高于玉米芯组,综合考虑后采用甘蔗渣做为后续小试及中试的固定化载体。
固定化载体甘蔗渣的投加量在6g/L时其所获得的MnP为最高,达157.1 U/L,同时其对应的对100 mg/L活性艳红X3-B脱色效果也最佳,至实验结束时可达99.6%,利用spss分析得知,不同的甘蔗渣的投加量对脱色率具有极其显著的相关性,其相关系数r=0.373,p值为0.00<0.01。而且该投加量下甘蔗渣能有效抑制其他杂菌的生长,同时利用SEM观察得知甘蔗渣能较好的作为P.C.生长、附着的载体。
(3)利用正交优化实验确定了组合式白腐真菌反应器的多项重要参数,并成功运行了该反应器
利用正交优化实验的直观分析、极差分析及方差分析对前两级白腐真菌反应器的三项重要参数T、DO及pH进行确定,从方差分析得知T、DO和pH三因素的FA=190.37,FB=41.82,Fc=0.8530,也就是对实际染料废水脱色效果的影响依次为T>DO>pH,试验表明T为30℃、DO为5mg/L和pH为5时所获得的脱色率为最高。构建的组合式白腐真菌反应器的主体反应器P.C.—组合填料反应器在处理实际染料废水时,其色度和CODCr在0～24 h内降低较为迅速,二者的去除率分别可达53.41%和29.50%,据此确定组合式系统的HRT为24h。组合式白腐真菌反应器的染料废水与培养基的配比为4∶1时,系统对CODCr及BOD5的去除效果为最佳,其对NH3-N的去除平均可达到国家一级排放标准即<15mg/L,对实际染料废水的色度也达到了国家排放标准的50以下,经检测,出水的生物毒性仅相当于平均浓度为0.058mg/L HgCl2的毒性,为低毒水平。对应的真菌微生物群落的多样性指数基本稳定在5.60左右,反应器内P.C.始终为优势菌种,其中白腐真菌—组合填料反应器在运行期间其微生物群落结构基本稳定。对染料废水稀释并不能有效提高系统的CODCr、BOD5去除率以及对实际染料废水生物毒性的削减。对系统内微生物群落结构进行解析发现当该系统采用稀释染料废水时,反应柱内微生物群落多样性指数波动不大,基本稳定在5.48左右。但低温下系统对CODCr、BOD5、生物毒性及脱色率的去除效果较常温下要低,反应器内微生物群落多样性指数也较常温下低,其平均值为5.20,而随着系统的运行,在低温工况后期其多样性指数呈上升趋势,说明系统运行后期有其他菌的侵染,它们并不是优势种,同DGGE图谱分析结果相一致。DGGE测序结果显示,三种工况下Neosartorya fischeri、Sordaria macrospora、Penicillium chrysogenum及P.C.四种菌均存在,而其他几种菌均较少,不属于优势种。
(4)组合式白腐真菌反应器用于实际染料废水的中试研究
现场中试研究得知,表明该组合系统对NH3-N、CODCr及BOD5均有良好的去除效果,该组合系统对可生化性差的实际染料废水仍然具有一定的生化降解能力。同时该组合系统对实际染料废水的生物毒性物质也有良好的去除效果,出水毒性检测表明其对应的HgCl2平均浓度已降至0.08 mg/L,属于低毒水平。
本论文通过对白腐真菌菌株的优化、黄孢原毛平革菌产酶固定化载体的筛选、组合式白腐真菌反应器工艺技术参数选择及应用该反应器进行的小试及中试等方面的研究结果,为该组合式白腐真菌反应器处理染料废水并保障其稳定、高效运行提供了可靠的依据,具有一定的理论意义和应用价值。
White rot fungi is capable of oxidizing persistent organic pollutants (POPs) including dyes because of their strong biodegradability and unique metabolic type. However, It's hard to promote the comprehensive ability of treating actual dye wastewater, which will affect its applications and effectiveness. In addition, there are many factors influencing the growth and ligninases production of white rot fungi, as well as the enzymatic reaction, which raise new challenge to the technical process of white rot fungi applied to treating actual dye wastewater.
This thesis is based on the project of effective microorganism techniques in wastewater treatment supported by Chinese National Programs for High Technology Research and Development (863Program). In the current study, the ligninases production of four white rot fungi was studied. The six carriers for immobilization of P. C. were also researched. Then the small-scale of integrated white rot fungi reactor was constructed and the performance of this reactor was researched with different conditions. And finally, the pilot-scale field tests were carried by Shanghai No.8 Dyestuff Chemistry Plant. The purpose of this thesis is to provide certain scientific references for the construction of white rot fungi integrated reactor and its stable and effective running.
Four white rot fungi, G. Sinense, C. versicolor, G. lucidum HG and P.C., were significant different in enzyme activity of MnP and LiP. Both MnP and LiP of P.C. had the highest enzyme activity, for the peak activities of MnP and Lip were 225.67 U/L (detected at 5th day) and 110.35 U/L (detected at 6th day) respectively. The enzyme activities of P. C. under N-limited culture condition were much higer than under C-limited culture condition. Statistical analysis using spss18.0 indicated that the MnP and LiP enzyme activities of P. C. under Nitrogen and Carbon limited culture conditions exhibited significant differences, which p value less than 0.05.
The enzyme activities of MnP and LiP of P.C. were comparatively high under pH value 4.0 to 4.5, and the two enzyme activities reached peak at pH value 4.5. MnP (more than 240 U/L) and LiP (more than 110 U/L) were at the peak activites when medium volume was 250mL. MnP and LiP reached the peak of 180 U/L and 130 U/L respectively when inoculation amount of spore suspension was 1.3×106 cell/mL. Speed between 120r/min and 140r/min was favorable for higher activities of MnP and LiP. Consequently, with respect to the four white rot fungi, MnP and LiP produced by P.C. had the highest enzyme activities.
P.C. immobilized on bagasse and corn cob which was two agricultral wastes exhibited higher activities of MnP and LiP than on other four immobilized carriers. Through analysis by spss18.0, the activities of MnP and LiP produced on bagasse and corn cob were dramatically different from the situations of others, with corresponding p=0.000<0.01. The activites of LiP on bagasse were all higher than those on corn cob, despite the activities of MnP on bagasse were slightly less than those on corn cob. Taken these factors into consideration, bagasse was employed as immobilized carrier for the following lab-scale and pilot-scale experiments. When the immobilized carrier bagasse was 6 g/L, the maximum MnP could be 157.1U/L. Meanwhile, its decoloration was optimal, reaching 99.6%at the end of the experiment. Accroding to spss analysis, the dosage of bagasse and decoloration rate were extremely significant correlated, with correlation coefficient r is 0.373, p value less than 0.01. In addition, other bacterials were inhibited effectively under the above bagasse dose. SEM observation indicated that bagasse could be used well as carriers for P. C. adherence and growth.
Using the orthogonal optimization test to determine the three important parameters (T, DO and pH) of intergrated white rot fungi reactor. The variance analysis showed that T has the grwatest impact on reactor performance, followed by DO, pH. When T, DO and pH was respectively 30℃,5 mg/L and 5, the system obtained the highest decolorization efficiency. However, when integrated white rot fungi treating the dyestuff wastewater, the colority and CODCrwas reduced rapidly within 24 h. According to the analysis, the HRT was determined 24 h.
The removal efficiency of CODCr and BOD5 was the best when the ratio of dyestuff wastewater and the N-limited liquid medium was 4:1. The average removal of NH3-N can reach the national top standard, lower than 15 mg/L. The colority of the actual dyestuff wastewater was also reach the national standard lower than 50. Meanwhile, the average concentration of HgCl. was 0.058 mg/L, belonging to low toxicity level. The corresponding fungal microbial community diversity index stabilized around 5.60 and the P.C. is the dominant species all the time. Whether diluting or not, the reactor can't effectively improve the removal efficiency of CODCr, BOD5 and reduce the biotoxicity of the actual dyestuff wastewater. Microbial community diversity index of the reactor was stable at around 5.48. Under the condition of low temperature, the removal efficiency of CODcr, BOD5, biotoxicity and decolorization rate was lower than normal. Microbial community diversity index of the reactor was lower than normal temperature about 5.20. Meanwhile, with the system running, microbial community diversity index was increasing in the latter of the condition of low temperature. In accordance with DGGE profiles, it indicated that other bacteria intruded the system in the latter run, but not the dominant species.
The pilot study indicated that the intergrated system have good removal efficiency of NH3-N, CODCr and BOD5. Meanwhile, the intergrated system also had good removal efficiency on the actual toxicity of wastewater. The average concentration of HgCl2 in the effluent had been reduced to 0.08 mg/L, of low toxicity level.
引文
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