Ecotoxicidade aguda, crônica e genotoxicidade de solo contaminado por óleo lubrificante usado e biorremediado para Eisenia andrei.

O objetivo do presente deste trabalho foi avaliar a toxicidade aguda, crônica e a genotoxicidade sobre E. andrei causadas por solo recém-contaminado com óleo lubrificante usado e após biorremediação por diferentes estratégias, após 22 meses, e paralelamente ao estudo de ecotoxicidade, foi conduzida uma investigação comparativa de três métodos de extração de HTP e HPA de solos para análise cromatográfica. A comparação das técnicas de extração evidenciou que para HTP, a técnica de extração acelerada por solvente-ASE foi a que melhor recuperou n-alcanos; já para as frações HRP e MCNR as técnicas soxhlet e micro-ondas-MARS não apresentaram diferenças significativas e foram melhores que ASE. Para HPA, a técnica de extração por soxhlet foi a que apresentou melhor recuperação em todos os solos. O teste de mortalidade apresentou, aos 14 dias, taxas crescentes de mortalidade de 10 6%, 20 0%, 73 25%, 93 12% e 100 0% para amostras de CONT (solo controle, sem contaminação artificial), BIOS (solo contaminado com 5% de OLU e biorremediado por bioestimulo), BIOA1 (solo contaminado com 5% de OLU e biorremediado por bioestimulo + bioaumento com adição de 10% de RSU maturado), e BIOA2 (solo contaminado com 5% de OLU e biorremediado por bioestimulo + bioaumento com adição de 10% de RSU semi-maturado) e OLU (solo contaminado com 5% de OLU), respectivamente. Aos 28 dias, entretanto, BIOS e OLU apresentaram taxas de mortalidade de 97 % 6 % e de 100 % 0 % respectivamente, valores estes significativamente superiores ao CONT. Foram observadas deformações anatômicas nos indivíduos mantidos em BIOS e OLU, assim como diminuição da biomassa em todas as amostras, evidenciando efeitos crônicos. O teste de reprodução, aos 28 dias, foram observadas grandes quantidades de indivíduos jovens nos solos biorremediados e recém-contaminado. No entanto, aos 56 dias houve uma diminuição dessas formas e o controle (CONT) exibiu uma quantidade maior de formas juvenis. O teste de densidade e viabilidade celular mostrou ser indicador sensível para toxicidade crônica apresentando queda nos solos BIOS e OLU em relação ao CONT com diferenças significativas (p <0.05). Não foram observados micronúcleos nos solos em estudo. Tal observação reforça a necessidade de testes de ecotoxicidade para avaliar a real eficácia de tecnologias de tratamento.

Ecotoxicidade aguda, crônica e genotoxicidade de solo contaminado por óleo lubrificante usado e biorremediado para Eisenia andrei.

O objetivo do presente deste trabalho foi avaliar a toxicidade aguda, crônica e a genotoxicidade sobre E. andrei causadas por solo recém-contaminado com óleo lubrificante usado e após biorremediação por diferentes estratégias, após 22 meses, e paralelamente ao estudo de ecotoxicidade, foi conduzida uma investigação comparativa de três métodos de extração de HTP e HPA de solos para análise cromatográfica. A comparação das técnicas de extração evidenciou que para HTP, a técnica de extração acelerada por solvente-ASE foi a que melhor recuperou n-alcanos; já para as frações HRP e MCNR as técnicas soxhlet e micro-ondas-MARS não apresentaram diferenças significativas e foram melhores que ASE. Para HPA, a técnica de extração por soxhlet foi a que apresentou melhor recuperação em todos os solos. O teste de mortalidade apresentou, aos 14 dias, taxas crescentes de mortalidade de 10 6%, 20 0%, 73 25%, 93 12% e 100 0% para amostras de CONT (solo controle, sem contaminação artificial), BIOS (solo contaminado com 5% de OLU e biorremediado por bioestimulo), BIOA1 (solo contaminado com 5% de OLU e biorremediado por bioestimulo + bioaumento com adição de 10% de RSU maturado), e BIOA2 (solo contaminado com 5% de OLU e biorremediado por bioestimulo + bioaumento com adição de 10% de RSU semi-maturado) e OLU (solo contaminado com 5% de OLU), respectivamente. Aos 28 dias, entretanto, BIOS e OLU apresentaram taxas de mortalidade de 97 % 6 % e de 100 % 0 % respectivamente, valores estes significativamente superiores ao CONT. Foram observadas deformações anatômicas nos indivíduos mantidos em BIOS e OLU, assim como diminuição da biomassa em todas as amostras, evidenciando efeitos crônicos. O teste de reprodução, aos 28 dias, foram observadas grandes quantidades de indivíduos jovens nos solos biorremediados e recém-contaminado. No entanto, aos 56 dias houve uma diminuição dessas formas e o controle (CONT) exibiu uma quantidade maior de formas juvenis. O teste de densidade e viabilidade celular mostrou ser indicador sensível para toxicidade crônica apresentando queda nos solos BIOS e OLU em relação ao CONT com diferenças significativas (p <0.05). Não foram observados micronúcleos nos solos em estudo. Tal observação reforça a necessidade de testes de ecotoxicidade para avaliar a real eficácia de tecnologias de tratamento.

Cadmium leachability and microbial population dynamics in a stabilized and bioaugmented model contaminated soil

This article presents a laboratory study on the consequences of the application of combined soil stabilization and bioaugmentation in the remediation of a model contaminated soil. Stabilization and bioaugmentation are two techniques commonly applied independently for the remediation of heavy metal and organic contamination respectively. However, for a cocktail of contaminants combined treatments are currently being considered. The model soil was contaminated with a cocktail of organics and heavy metals based on the soil and contaminant conditions in a real contaminated site. The soil stabilization treatment was applied using either zeolite or green waste compost as additives and a commercially available hydrocarbon degrading microbial consortium was used for the bioaugmentation treatment. The effects of stabilization with or without bioaugmentation on the leachability of cadmium and copper was observed using an EU batch leaching test procedure and a flow-through column leaching test, both using deionized water at a pH of 5.6. In addition, the population of hydrocarbon degrading microorganisms was monitored using a modified plate count procedure in cases where bioaugmentation was applied. It was found that while the stabilization treatment reduced the metal leachability by up to 60%, the bioaugmentation treatment increased it by up to 100% Microbial survival was also higher in the stabilized soil samples.

Effect of microbial activities on the mobility of copper in stabilised contaminated soil

Stabilisation, using a wide range of binders including wastes, is most effective for heavy metal soil contamination. Bioremediation techniques, including bioaugmentation to enhance soil microbial population, are most effective for organic contaminants in the soil. For mixed contaminant scenarios a combination of these two techniques is currently being investigated. An essential issue in this combined remediation system is the effect of microbial processes on the leachability of the heavy metals. This paper considers the use of zeolite and compost as binder additives combined with bioaugmentation treatments and their effect on copper leachability in a model contaminated soil. Different leaching test conditions are considered including both NRA and TCLP batch leaching tests as well as flow-through column tests. Two flow rates are applied in the flow-through tests and the two leaching tests are compared. Recommendations are given as to the effectiveness of this combined remediation technique in the immobilisation of copper. © 2005 Taylor & Francis Group.

Establishment and characterization of dual-species biofilms formed between a 3,5-dinitrobenzoic-degrading strain and bacteria with high biofilm-forming capabilities

In this study, the possibility of establishing a dual-species biofilm from a bacterium with a high biofilm-forming capability and a 3,5-dinitrobenzoic acid (3,5-DNBA)-degrading bacterium, Comamonas testosteroni A3, was investigated. Our results showed that the combinations of strain A3 with each of five strains with a high biofilm-forming capability (Pseudomonas sp. M8, Pseudomonas putida M9, Bacillus cereus M19, Pseudomonas plecoglossicida M21 and Aeromonas hydrophila M22) presented different levels of enhancement regarding biofilm-forming capability. Among these culture combinations, the 24-h dual-species biofilms established by C. testosteroni A3 with P. putida M9 and A. hydrophila M22 showed the strongest resistance to 3,5-DNBA shock loading, as demonstrated by six successive replacements with DMM2 synthetic wastewater. The degradation rates of 3,5-DNBA by these two culture combinations reached 63.3-91.6% and 70.7-89.4%, respectively, within 6 h of every replacement. Using the gfp-tagged strain M22 and confocal laser scanning microscopy, the immobilization of A3 cells in the dual-species biofilm was confirmed. We thus demonstrated that, during wastewater treatment processes, it is possible to immobilize degrader bacteria with bacteria with a high biofilm-forming capability and to enable them to develop into the mixed microbial flora. This may be a simple and economical method that represents a novel strategy for effective bioaugmentation.

厌氧产氢菌的分离筛选、诱变选育及其产氢特性的研究

本文从四川绵竹酒厂、成都市龙泉长安垃圾填埋场以及四川大学荷花池底的厌氧污泥中先后分离得到63株厌氧产氢菌，其中H-8、H-61、HC-10等16株产氢细菌产氢能力较高，HC-10的产氢能力最高，最大产氢量和最大产氢速率分别达到2840 ml H2/L培养基和25.39 mmol H2/g drycell·h，对HC-10进行生理生化鉴定和分子生物学鉴定，判定其为clostridium sp.，对HC-10的产氢条件进行了研究，结果表明，该菌的最适生长温度为35 ℃，最适生长初始pH为7，以葡萄糖为最佳碳源，以蛋白胨为最佳氮源，不利用无机氮源，其产氢发酵液相产物以乙醇和乙酸为主，其发酵类型属于乙醇型发酵。此外，以酒糟废液作为底物，进行了菌株HC-10的生物强化试验，研究表明，投加了HC-10的强化系统其产氢量比对照高出40.32%。 同时为了获得厌氧产氢菌的高效突变株，分别以产氢菌H-8和H-61为原始菌株进行微波诱变处理，对微波诱变参数进行了优化，考察了突变株的遗传稳定性、产氢特性及耐酸性。菌株H-8经过微波诱变得到5株高产氢突变株HW7、HW33、HW181、HW184、HW195，经多次传代表明HW195是稳定的高产突变株。突变株HW195具有较好的耐酸性，在pH值为2.8时仍能生长。通过间歇发酵实验，其最大产氢量和最大产氢速率分别达到2460 mL/L培养基和27.97 mmol H2/g drycell·h，比原始菌分别提高了50.75%和41.7%。菌株H-61经过微波诱变后选育得到的突变株HW-18，其最大产氢量和最大产氢速率分别达到2190 mL/L培养基和25.86 mmol H2/g drycell·h，比原始菌分别提高了23.03%和31.00%。 为了对比各种诱变方式对产氢菌产氢能力的影响，以厌氧产氢菌H-61为原始菌株，先后经亚硝基胍(NTG)、紫外(UV)诱变，选育得到1株高产突变株HCM-23。在葡萄糖浓度为10 g/L的条件下，其产氢量为3024 mL/L培养基，比原始菌株提高了69.89%；其最大产氢速率为33.19 mmol H2/g drycell·h，比原始菌株提高了68.14%。经过多次传代实验，稳定性良好。其发酵末端产物以乙醇和乙酸为主，属于典型乙醇型发酵。其最适产氢初始pH为6.5，最适生长温度为36 ℃，以蔗糖为最佳碳源。与原始菌株相比，突变株HCM-23的产氢特性发生了改变，如生长延滞期延长，可利用无机氮源等。 From anaerobic activated sludge, 16 strains of hydrogen producing bacteria were newly isolated. One of them named as HC-10 had the highest hydrogen producing capability, under the batch fermentative hydrogen production condition, the maximal hydrogen yield and hydrogen production rate was 2840 mL/L culture and 25.39 mmol H2/g drycell·h. It was identified as clostridium sp.HC-10 by 16S rDNA sequence analysis. Various parameters for hydrogen production, including substrates, initial pH and temperature, have been studied. The optimum condition for hydrogen producing of strain HC-10 were achieved as: initial pH 7.0, temperature 35 ℃, glucose as the favorite substrate, Moreover, using distiller's solubles wastewater as substrate, HC-10 strain was added in the biohydrogen producing system to research the bioaugmentation effection. The results showed that the hydrogen production of bioaugmentation system was 40.32% higher than the noaugmentation system. An anaerobic, hydrogen producing strain H-8 was irradiated by microwave to optimize the microwave mutagenesis condition, and to test the heredity, hydrogen-producing potential and aciduric of the mutants. An aciduric mutant named as HW195 with steady hydrogen-producing capability was obtained, which can grow at pH 2.8. Its capability of hydrogen production was tested in the batch culture experiments. The maximum hydrogen yield and hydrogen production rate was 2460 mL/L culture and 29.97 mmol H2/g drycell·h, which was 50.7% and 41.7% higher than those of the initial strain, respectively. When used the strain H-61 as original strain, a mutant named as HW18 was obtained. The maximum hydrogen yield and hydrogen production rate was 2190 mL/L culture and 25.86 mmol H2/g drycell·h, which was 23.03% and 31.00% higher than those of the initial strain, respectively. The results demonstrated that microwave mutagenesis could be used in the field of hydrogen producing microorganism. The hydrogen producing strain H-61 was used as an original strain which was induced by NTG and UV for increasing and the hydrogen production capability. One of the highest efficient H2-producing mutants was named as HCM-23 with its stable hydrogen production capability. which was tested in the batch culture experiments. With the condition of 10 g/L glucose, its cumulative hydrogen yield and hydrogen production rate was 3024 mL/L culture and 33.19 mmol H2/g drycell·h, 69.89％and 68.14％ higher than that of the original strain, respectively. The terminal liquid product compositions showed that the mutant HCM-23 fermentation was ethanol type, while the original strain H-61 fermentation was butyric acid type. Varieties of parameters of hydrogen production fermentation were studied, including time, carbon source, nitrogen source, glucose concentration, glucose utilization, initial pH and incubation temperature had been studied, indicated the optimum condition of hydrogen production for the mutant HCM-23 as initial pH6.5, temperature 36 ℃, and the favorite substrate was sucrose. The hydrogen production characters of the mutant and the original strain were different, such as, the growth lag phase and the utilization of inorganic nitrogen source, etc. This work shows a good application potential of NTG-UV combined mutation in the biohydrogen production. And the hydrogen production mechanism and metabolic pathway should be explored furthermore.

菌剂强化处理炼油废水及苯酚冲击下功能菌响应机制研究

近年来，我国炼油行业发展迅速，炼油能力全世界第二，炼油行业已成为污染大户。本研究针对炼油废水生物处理中存在的稳定达标难、抗冲击负荷能力差、建设投资与运行成本高等问题，就菌剂强化处理炼油废水中试与工程应用展开了研究，以期为菌剂的工程应用与推广提供理论参考与技术支持；并以炼油废水中的主要特征污染物苯酚为研究对象，考察了不同浓度苯酚冲击下功能菌的响应机制，并以此为指导研制功能菌激活促进剂，考察其对功能菌生物学指标的调控效果，以期为废水生物处理有毒污染物冲击调控提供理论依据与技术支持。 中试研究表明，菌剂强化处理炼油废水，出水COD、NH4+-N 平均值为86.7、7.6 mg/L，其平均去除率较常规生物处理系统分别提高了35.47%、59.28%，其耐受COD、NH4+-N 容积负荷分别高达2.42、0.139kg/(m3·d)，具有良好的耐冲击能力。工程应用研究表明，菌剂强化处理炼油废水，出水COD、NH4+-N 平均值分别为85.05、8.4mg/L，其去除率较常规生物处理系统提高了25.1%、28.7%，出水水质各项指标均达到了国家《污水综合排放标准GB 8978-1996》一级排放标准。技术经济分析表明，菌剂强化处理炼油废水在建设成本、运行成本上分别降低38%、49%，具有良好的技术经济优势。 苯酚冲击下功能菌响应机制研究表明：不同浓度苯酚冲击下，生物学指标生物量、脱氢酶酶活、1,2-双加氧酶酶活对冲击都有不同程度的响应，其响应敏感程度为脱氢酶酶活＞生物量＞1,2-双加氧酶酶活。1,2-双加氧酶酶活与COD 降解率相关性良好，可表征苯酚降解过程，确认为调控重点。以此为指导研制出苯酚降解功能菌抗冲击激活促进剂，可有效调控功能菌对有毒污染物苯酚的降解效果，1000mg/L 苯酚冲击下，经调控，其COD 去除率较对照提高20%，降解时间缩短16%以上。其对生物学指标的调控效果为1,2-双加氧酶酶活＞生物量＞脱氢酶酶活，验证了功能菌在苯酚冲击下的响应机制。研究表明菌剂强化处理炼油废水切实可行，具有良好的技术经济优势。有毒污染物冲击下废水生物处理系统响应机制研究为抗冲击调控提供了新的研究思路。 Currently, China’s oil refining industry is developing rapidly and has become the second largest all over the world. The oil refining industry is one of the major pollution industries in our country. The pilot scale study and engineering application research were conducted aiming at the problems in refining wastewater such as poor treatment stability and water quality, poor anti-shock capacity and expensive running cost, etc., so as to provide theoretical references and technological supports for the engineering application and popularization of microbial preparation in wastewater treatment. Also, the response mechanism of functional microbe under shock of different phenol concentrations, which is the main pollutants in refinery wastewater, was studied. Based on this result, functional microbe activation accelerator was developed, and the regulation effect of functional microbe biological index under phenol shocking were studied, in order to provide theoretical basis and technological support for regulation of toxic shocking of wastewater biological treatment. The result of pilot scale research indicated: for treatment of refinery wastewater in bioaugmention treatment system of microbial preparation, the COD and NH4+-N average value of effluent was 86.7 and 7.6 mg/L, Comparing with normal biological treatment system, the average removal rates of COD, NH4+-N increased 35.47%,59.28% separately by bioaugmention treatment system, which showed better anti-shocking capacity, the volumetric load r of COD and NH4+-N reached 2.42 kg/(m3·d) and 0.139 kg/(m3·d), respectively. The research on engineering application of refinery wastewater bioaugmentation treatment by microbial preparation indicated:the average concentrations of effluent COD and NH4+-N in the bioaugmentation treatment system were 85.05 and 8.4mg/L, which increased by 25.1% and 28.7% comparing with normal biological treatment system of refinery wastewater, And the effluent quality meets the first grade of discharging standard of National Integrated Wastewater Discharge Standard GB 8978-1996. The economic analysis of technology indicated: the demonstration project of bioaugmentation treatment of refinery wastewater by microbial preparation decreased by 38% in construction cost and 49% in running cost. This technology has economic benefits. The response mechanism of functional microbe under phenol shock indicated: biological index such as the biomass concentration, dehydrogenase and 1,2-dioxygenase had different responses under phenol shocking of different concentrations. The response sensitivity of different biological index under phenol shocking of different concentrations is: dehydogenase activity ＞ biomass ＞1,2-dioxygenase activity, and high correlation of 1,2-dioxygenase and COD degradation percentage is achieved, thus 1,2-dioxygenase could be used to reflect the degradation situation of pollutants. So, 1,2-dioxygenase is the keypoint of regulation. The anti-shock activation accelerator of phenol degradation functional microbe was primarily developed. The results indicated: the activation accelerator could regulate the degradation effect of toxic substance-phenol by functional microbe effectively. For the functional microbe treatment system under phenol shocking of 1000mg/L, the COD degradation rate increased by 20% and the degradation time reduced by more than 16% under regulation of activation accelerator. The regulation effects of biological index are: 1,2-dioxygenase ＞ biomass ＞ dehydrogenase. In this way, the response mechanism of functional microbe under toxic shocking is verified. The result indicated: the augmented microbial preparation treatment of refinery wastewater is applicable. It has many technical and economical advantages. The research results of responses mechanism of wastewater treatment system on toxic pollutants would offer a new idea for regulation of anti-shock.

功能菌剂的混合发酵研究

随着化工行业的发展，大量有毒有害难降解有机物随工业废水的排放进入环境，这些物质能够在环境中长期存在、积累和扩散，通过食物链对动植物的生存及人类的健康造成不良影响。本文以苯酚、对氯硝基苯、氯苯和十六烷为模拟污染物，以前期研制的功能菌剂为对象，经过紫外线线诱变筛选出优于出发菌株的功能菌，对诱变后功能菌的理化性能进行了研究，对菌种进行了鉴定，在此基础上，就其相互之间的微生态关系进行研究，为混合发酵提供理论基础，并就其最佳发酵条件及发酵参数进行了研究，最后对发酵产品的性能进行了检测。目前，国内外有关功能菌剂的研究还存在多方面的不足，主要包括：①由于多菌种混合发酵过程较为复杂，各菌之间存在复杂的相互作用，影响因素较多，关于菌种之间的相互关系研究得很少，环境功能菌剂的发酵方法大多采用单独发酵后混合的方式。单独发酵对原材料、设备和能源的利用率较低，对于多菌种制剂发酵，在设备、能源和原材料的方面造成的浪费更大，将会大幅增加菌剂的生产成本，影响多菌种功能菌剂的发展；②功能菌剂生产过程的质量控制方面研究得较少；③功能菌剂产品的稳定性、抗冲击性能研究得较少，对环境微生物制剂的研究主要集中在菌种选育和培养条件优化方面。 通过本论文研究，得到以下主要结论。 （1）在紫外线诱变处理中，用紫外线对发生一定程度退化的出发菌株进行诱变处理后，六株具有高效降解性能的菌株被筛选出来，诱变筛选出的菌株形态和ERIC-PCR指纹图谱与出发菌株相比发生了明显改变；而且诱变后的菌株对目标难降解底物的降解能力均得到改善，其中，FPN、FCB、F14、FEm对目标底物的降解率提高了20%以上；诱变后菌株经过7次连续传代接种后，对目标难降解底物的降解率无显著变化，具有一定的遗传稳定性。并对诱变后的功能菌进行了初步的鉴定，这6株菌都分别是芽孢杆菌。 （2）对诱变后的功能菌相互之间的微生态关系进行了研究，通过抑菌实验、生长量以及基质消耗量的比较，确定它们之间的生长关系是无害共栖关系，可以进行混合发酵。 （3）对该功能菌剂进行发酵培养条件研究，结果表明发酵培养基的最佳成分（g/L）：葡萄糖 31.0g/L、玉米粉10.0g/L、磷酸氢二钾1.0g/L、硫酸铵1.1g/L、硫酸镁0.55g/L。通过研究不同的培养条件对菌体生长和降解性能的影响，确定了最佳培养条件：培养基初始pH7.5；最适温度32℃；培养基装液量125mL（250 mL三角瓶），以及培养时间对降解性能的影响，培养20 h的产物对降解最为有利。通过研究添加不同目标污染物对菌体生长和降解性能的影响，确定了添加目标污染物的最佳量以及最佳时间：苯酚投加量：1.125 g/L，对氯硝基苯投加量：0.1 g/L；最佳投加时间为发酵培养开始后4 h。 （4）以摇瓶分批发酵最优条件为基础，对FPN、F10、FCB、FNa、F14 和 FEm进行了摇瓶分批发酵试验。以摇瓶分批发酵试验数据为依据，对功能菌剂分批发酵动力学进行了研究，建立了菌体生长和基质消耗的动力学模型，拟合模型能较好的反映功能菌剂分批发酵过程。 （5）功能菌剂和活性污泥协同作用，可以提高系统的生物降解能力，功能菌剂投加量为2%，新鲜活性污泥3500 mg/L，降解24 h条件下，功能菌剂和活性污泥的协同作用对COD的去除率和对照组相比，最多的提高了36.8%。功能菌剂和活性污泥协同作用以及活性污泥的单独作用，其生物降解过程均符合一级反应动力学过程，功能菌剂和活性污泥协同作用的生物降解动力学方程为：，相关系数97%。采用SBR运行方式，引入功能菌剂的SBR系统明显能够改善和提高生物降解的效率。与仅有活性污泥的系统相比，系统对COD的平均去除率可以提高27.1%，同时，系统的耐负荷冲击以及耐毒害冲击的性能比仅有活性污泥的SBR系统强，特别是负荷冲击对引入功能菌剂的SBR系统影响很小。仅有活性污泥的SBR系统经过负荷冲击和毒害冲击之后，不能恢复到冲击之前的水平，而且系统有效作用时间的周期比引入功能菌剂的SBR系统相比大大缩短，而引入功能菌剂的SBR系统处理效果较为稳定，恢复能力很强。 Along with the development of industries, many recalcitrant organic chemicals have been discharged into natural environments together with wastewaters and can exist in waters, soil and sediments for a long time without degradation. These haz-ardous substances, their byporducts and metabolizabilities can be highly toxic, mu-tagenic and carcinogenic, thereby threatening animals, plants and human health through food chain. Consequently the removal of these compounds is of significant interest in the area of wastewater treatment. In this dissertation, the phenol, hydro-quinone, chlorobenzene and hexadecane treated as the model pollutants, the func-tional microorganism agent was used as the starting strains, they treated with ultra-violet light, and then the mutant strains with high degradation ability were screened out and identified primarily, the relationship between these stains were studied, the medium composition and fermentation conditions were optimized, the degradation ability of the fermented production was tested. The literature survey indicates that the study of the microorganism agent is far from complete and more information is re-quired on following problems. 1, Because of the complexity of relationship in mixed fermentation and the complicated factors, the study is hardly to process.2, There is a lack of information on the quality control of the producing process .3, And there is a lack of information on the stability about the microorganism agent. In this dissertation, the main results of the present study could be summarized as follows: (1)The degenerate starting strains were treated with the ultraviolet light, and six mutant strains with high biodegradation ability were screened out by using the me-dium with selective pressure of model pollutants. The mutant strains had great changes in colonialmorphology and ERIC-PCR fingerprinting. And the mutant strains got obvious advantages over the starting strains in degradation ability and over 20% improvement of removal rates was achieved for FPN、FCB、F14 and FEm. The de-gradation ability of the mutant strains was stable after seven generations. After that, the mutant strains were primarily identified as bacillus respectively. (2) The relationship between these mutant strains was studied. By the compari-son of antibiosis effect, biomass and consumption of substrate, the relationships were neutralism and they could be mixed fermented. (3) The optimized cultivation conditions were as follows: glucose 31.0 g/L, corn power 10 g/L, K2HPO4 1.0 g/L, (NH4)2SO4 1.1 g/L, MgSO4 0.55 g/L, initial pH7.5, temperature 32℃, working volume 125 mL/250 mL, and cultivation time 20h (con-sidering the time effect on degradation ability), adding pollutants phenol (1.125 g/L) and hydroquinone (0.1 g/L) into the broth at 4 h after cultivation. (4) Based on the above optimum condition, the batch fermentation was per-formed with strains FPN, F10, FCB, FNa, F14 and FEm in shake flask. The batch fermentation kinetics was studied based on the experimental data. Two kinetic models were constructed which could reflect the regularity of growth and substrate consump-tion in the process of batch fermentation. (5) The co-operation of functional microorganism agent and activated sludge could raise biodegradation of system by adding some microorganism agent and 3500 mg/L fresh activated sludge. Bioaugumentation by the addition of high effective deg-radation culture enhanced the treatment effect of SBR system and the COD removal rate was increased by 20%-36.8%. Its biodegradation matched first-order dynamical reaction equation, and the reaction equation was ln0.2327.391ct=−+. The micro-organism agent had the effect of optimization to activated sludge micro-ecosystem. The SBR system adding 2% microorganism agent, the average COD removal rate of that was increased by 27.1% and stronger anti-shock ability to load and toxicant were achieved (compared with SBR system just adding activated sludge). Especially the load-shock has barely effect to the SBR system adding microorganism agent. After the load and toxicant shock, the SBR system just adding activated sludge couldn’t come back to original level and the activated sludge micro-ecosystem was frustrated. The applying of microorganism agent increased biological activity and system’s re-sistance ability to load shock and toxicant shock.

制革废水生物强化处理及中水回用试验研究

制革行业是轻工行业中仅次于造纸业的高耗水、重污染行业，作为劳动密集型行业，在解决大量人口就业问题的同时，也对所在地区环境造成了严重污染。目前我国制革行业每年排放废水8,000~12,000万吨，废水中含铬约3,500 t，SS为1.2×105 t，COD为1.8×105 t，BOD为7×104 t，对水体污染严重。 本研究在对厌氧酸化工艺进行研究、一级好氧处理段进行工艺比选研究的基础上，获得了匀质调节—SBBR—BAF的生物处理工艺，并依托该工艺进行了生物强化处理的研究，考察了菌剂的强化运行效果及其处理水回用的可行性。 研究表明，在进水COD＞3,000 mg/L，厌氧酸化具有很好的抗冲击作用，保证了好氧工艺出水COD＜200 mg/L；在进水COD＜3,000 mg/L，可只通过好氧处理实现出水COD＜200 mg/L。厌氧酸化停留时间选择不当，会导致厌氧出水硫化物浓度升高，严重影响好氧系统，会使好氧活性污泥因中毒而解絮。 研究表明，当进水COD为2,000~2,500 mg/L，NH4+-N为130~146 mg/L时，COD、NH4+-N去除率SBBR分别为93.8%~96.6%和14.5%~55.9%，SBR分别为88.8%~94.9%和13%~50.7%，表明SBBR优于SBR。同时，研究发现SBBR污泥增长率为0.05 kgVSS/kgCOD，仅为SBR0.57 kgVSS/kgCOD的8.8%。此外，研究发现SBBR在停止运行后经3个运行周期可回复原油能力，而SBR池经9个周期培养也不能恢复，说明SBBR恢复能力明显优于SBR。 研究表明，以匀质调节—SBBR—BAF为主的制革废水处理工艺，出水水质稳定，进水COD 801~2,834 mg/L、NH4+-N 87~203 mg/L，出水COD＜80 mg/L、NH4+-N＜10 mg/L，基本达到中水回用标准；操作简单灵活，没有污泥回流系统，污泥产率低，污泥处理费用低；工艺基本不需要添加化学药剂，既节约成本、又避免了二次污染；两级生物膜使得该工艺具有很强的耐冲击负荷能力，特别适合制革废水水质水量波动大的特点。 研究表明，高效菌对系统的启动具有一定的促进作用，强化系统生物膜6天可以成熟，对照系统生物膜9天可以成熟。同时高效菌能加速COD降解，缩短停留时间，强化系统6~8 h可使COD＜200 mg/L，对照系统8~10 h可使COD＜200 mg/L。长期运行表明，强化系统的SBBR在COD和NH4+-N的去除率都优于对照系统的SBBR。最终出水COD强化系统平均为53 mg/L、对照系统为74 mg/L。在模拟循环过程中，强化系统均有更高的稳定性。可实现8次理论循环，而对照系统只能实现4次理论循环。 研究表明，通过合理的工艺设计，可以实现猪皮制革废水达到《污水综合排放标准GB8976-1996》一级标准，同时满足工厂部分用水要求。通过添加高效微生物，可提高生物处理系统处理能力，使处理水能够满足工厂的多次回用。 As a labour-intensive industry, tanning has created large amount of working opportunities as well as caused severe contamination to environment. And it is one of the highest water-consuming and polluting industry, only second to manufacturing. At present time, Chinese leather industry emits wastewater about 80,000,000~120,000,000 t annually, which contains chromium about 3,500 t, SS 1.2×105 t, COD 1.8×105 t, BOD 7×104 t and ambient riverhead has been polluted greatly. Based on the research of anaerobic acidification and comparison of SBBR and SBR, biotreatment process (Homogenization—SBBR—BAF) had been established to amend the disadvantages of traditional sewage treatment such as too much sludge, high cost of advanced treatment and NH4+-N can not reach the emission standard. Research on the bioaugmentation was also been carried out. Researches showed, when COD of influent was beyond 3,000 mg/L, anaerobic acidification could resist strong impact, thus COD of effluent was less than 200 mg/L; when COD of influent was less than 3,000 mg/L, only throughout aerobic sewage treatment could COD of effluent beless than 200 mg/L. False residence tiome of anaerobic acidification would lead to the higher effluent concentration of sulfide and disintegration of aerobic activated sludge. Researches showed SBBR worked a better than SBR: when influent between 2,000 and 2,500 mg/L, NH4+-N between 130 mg/L and 146 mg/L, COD, NH4+-N removal rate of SBBR was 93.3%~96.6%, 14.5%~55.9% respectively while COD, NH4+-N removal rate of SBR was 88.8%~94.9%, 13%~50.7% respectively. Sludge growth rate of SBBR was 8.8% of that of 0.05 kgVSS/kgCOD. Besides, SBBR could recovered after 3 operating periods while SBR worked no better after 9 operating periods.Therefore, SBBR excelled SBR. Researches showed, effluent quantity of tannery wastewater treatment process (Homogenization—SBBR—BAF) was stable. When COD of influent was between 801 and 2,834 mg/L, NH4+-N was between 87 mg/L and 203 mg/L, COD of effluent was less than 80 mg/L, NH4+-N was less than 10 mg/L, which achieved the standard of reuse. This biotreatment was featured in low cost, easy and flexible management, less sludge, no inverse sludge system. Besides, this technique required no chemical, which could lower the cost and avoid secondary pollution. Great resistant of impact due to two membranes and was suitable for tannery wastewater which was featured by fluctuation of influent quality and quantity. Researches showed effective microorganisms promotes the startup of the process.Biofilm in the bioaugmentation process matured with 6 days while biofilm in normal process matured with 9 days. Effective microorganisms could accelerate the degradation of COD and shorten the residence time. Aggrandizement system could make COD<200 mg/L with 6 to8 hours while cntrolling system could make COD<200 mg/L with 8 to 10 hours. Long-term operating shows that SBBR in the bioaugmentation system worked better than the normal system in the treatment of COD and NH4+-N. The average COC of effluent in bioaugmentation system was 53 mg/L, normal system was 74 mg/L. In the simulative circulation process,aggrandizement process, which could fulfill 8 times theoretical circulation, works more stably than controlling process which could only fulfill 4 times theoretical circulation. Researches showed that reasonable design could make the wastewater meet the first grade of discharging standard of National Integrated Wastewater Discharge Standard (GB8976-1996), and partially meet the demand of water using of the factory. Adding effective microorganisms could enhance the biotreatment and make the effluents reuse many times.

功能菌强化应急处理苯胺突发污染事故研究

近年来各种环境污染事故频发，据统计仅2001~2003年间，发生的各类环境污染事故就高达5606次，其中水污染事故3235次，占全部的57.7%。这些事故不仅给人民生命财产造成巨大损失，也给生态环境造成严重的破坏。因此开发安全高效的应急处理技术迫在眉睫。本研究以筛选高效苯胺降解菌为基础，通过对高效菌降解性能的研究指导将高效菌作为功能郡主投加到已有生物处理系统强化应急处理苯胺突发污染事故废液，取得了良好的效果。 苯胺高效降解菌AN-P1为红球菌（Rhodococcus sp.），其通过间位途径降解苯胺，AN-P1利用苯胺生长和降解的最佳pH为6，最适浓度为2000 mg/L，最适温度为30 ℃，最佳接种量为0.3‰。AN-P1降解含500 mg/L、1000 mg/L、2000 mg/L苯胺的培养物分别经过28 h、24 h、32 h降解，出水苯胺含量能达到《污水综合排放标准》（GB8978-1996）一级标准。但由于苯胺降解过程中释放了大量氨氮，出水氨氮仍较高未能达标排放。而常规SBR系统应急处理效果较差，苯胺和COD去除率均低于10%，出水未能达标排放。活性碳吸附后的回收和后续处理也会带来操作不变和二次污染问题，且处理后出水往往难于达标排放，尚需进行进一步处理。 生物处理系统应急处理后恢复运行处理效果监测和PCR-DGGE图谱分析显示，用AN-P1菌强化应急处理系统后不仅能快速高效的去除苯胺，而且可以有效保障处理系统对污染物的净化性能，有效的保护系统中的功能微生物免受苯胺毒害。 研究结果表明，从实际处理效果、对原有生物系统性能保护及实际应用操作等多方面考虑，用AN-P1菌强化应急处理苯胺突发污染事故在技术上都是可行的。本研究为应急处理苯胺突然污染事故废液提供了新的方法。 Recent years, environment pollution accidents happened frequently, the data showed that there are 5606 accidents between 2001 and 2003, including 3235 water environment accidents, which is 57.7% of all. These accedents not only caused money lost and life lost but also caused serious damage to the ecologicl environment. So exploring highly-effective and secure methods to solve these accidents is an urgent mission. We screened a highly-effective aniline-degrading bacterium and did some researches on its ability to degrade aniline, in order to guide the emergency treatment of aniline containing wastewater that caused by sudden accident pollution with bioaugmentation. A highly-effective aniline-degrading bacterium AN-P1 was isolate and characterized as Rhodococcus sp. It degrades aniline through meta-cleavage pathway. The optimal pH and temperature for cell growth and aniline degradation were 6 and 30 ℃, respectively, and the opitimal concentration of aniline was 2000 mg/L, the optimal inoculation amount was 0.3‰.It took bacterium AN-P1 only 18 h, 24 h and 32 h, respectively, for the treatment of MSB containing 500 mg/L, 1000 mg/L, 2000 mg/L aniline to meet the first grade of national some of the NH4+-N which caused by aniline degradation. It took bacterium AN-P1 only 10 h, 20 h and 32 h, respectively, for the treatment of wastewater containing 500 mg/L, 1000 mg/L, 2000 mg/L aniline to meet the first grade of national integrated wastewater discharge standard. The bacterium AN-P1 can also remove some of the NH4+-N which caused by aniline degradation. It took bacterium AN-P1 only 10 h, 20 h and 32 h, respectively, for the treatment of wastewater containing 500 mg/L, 1000 mg/L, 2000 mg/L aniline to meet the first grade of national integrated wastewater discharge standard. By combing AN-P1 with regular SBR system, it took only 36 h for the emergency treatment of wastewater containing 2000 mg/L aniline under simulating engineering conditions to meet the discharge standard. While the NH4+-N of effluent can not meet the standard because of the high amount NH4+-N caused by aniline degradation. The regular SBR system was not good at aniline and COD removal. The removal efficiency of which are less than 10%. It cost 67.8 g activated carbon to absorbed 1000 mg aniline. It is inconvenient to transport and use it for the emergency treatment of aniline when the sudden pollution accident happened. Meanwhile, it was complex ad hard to recycle the activated carbon and treat the aniline wastewater get from activated carbon recycling too. Hard to meet the effluent standard was also a problem of activated carbon absorption method. According to the PCR-DGGE profile and removal efficiency of pollutants and COD when the systerm recover from emergency treatment, AN-P1 can efficiently protect the microbial community of regular activated sludge system against the aniline. It proved that combing AN-P1 with regular biological system is a feasible strategy for emergency treatment of aniline sudden pollution accident. The research offered a new way for emergency treatment of aniline sudden pollution accident.

Immobilization of nitrifying bacterial consortia on wood particles for bioaugmenting nitrification in shrimp culture systems

Shrimp grow out systems under zero water exchange mode demand constant remediation of total ammonia nitrogen (TAN) andNO2 −–Nto protect the crop. To address this issue, aninexpensive and user-friendly technology using immobilized nitrifying bacterial consortia (NBC) as bioaugmentors has been developed and proposed for adoption in shrimp culture systems. Indigenous NBC stored at 4 °C were activated at room temperature (28 °C) and cultured in a 2 L bench top fermentor. The consortia, after enumeration by epifluorescence microscopy,were immobilized on delignifiedwood particles of a soft wood tree Ailantus altissima (300–1500 μm) having a surface area of 1.87m2 g−1. Selection of wood particle as substratumwas based on adsorption of NBC on to the particles, biofilm formation, and their subsequent nitrification potential. The immobilization could be achievedwithin 72 h with an initial cell density of 1×105 cells mL−1. On experimenting with the lowest dosage of 0.2 g (wet weight) immobilized NBC in 20 L seawater, a TAN removal rate of 2.4 mg L−1 within three days was observed. An NBC immobilization device could be developed for on site generation of the bioaugmentor preparation as per requirement. The product of immobilization never exhibited lag phase when transferred to fresh medium. The extent of nitrification in a simulated systemwas two times the rate observed in the control systems suggesting the efficacy in real life situations. The products of nitrification in all experiments were undetectable due to denitrifying potency, whichmade the NBC an ideal option for biological nitrogen removal. The immobilized NBC thus generated has been named TANOX (Total Ammonia Nitrogen Oxidizer)

Biodegradability of commercial and weathered diesel oils

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Biodegradation of biodiesel/diesel blends by Candida viswanathii

This work is aimed to assess the aerobic biodegradation of biodiesel/diesel blends (0, 2, 5, 20 and 100%, v/v) by Candida viswanathii. The biodegradation potential of the inoculum was assessed with the redox indicator 2,6-dichlorophenol indophenol (DCPIP) test and with respirometric experiment in biometer flasks (250 mL) used to measure the microbial CO(2) production. In the latter, the inoculum was added to a contaminated soil with the blends (addition of 50 mL of fuel/Kg of soil from a non-contaminated site). C. viswanathii was able to increase significantly (approximately 50% in terms of CO(2) production) the biodegradation in soil of biodiesel/diesel blends and neat biodiesel since it preferable biodegrades biodiesel. Without inoculum the biodegradation of diesel oil was higher than biodiesel and blends (47.3, 51.1, 5.7 and 22.1% in terms of CO(2) production by B2, B5, B20 and B100, respectively) presumably due to the presence of the antioxidant terc-butyl-hydroquinone (TBHQ) in the biodiesel.

Laboratory study on the bioremediation of diesel oil contaminated soil from a petrol station

O objetivo do presente estudo foi investigar possíveis métodos para aumentar a taxa de biodegradação aeróbia de hidrocarbonetos (tratamentos ex-situ). Neste trabalho, processos de biorremediação foram aplicados a um solo arenoso com alto nível de contaminação ocasionada por um vazamento de um tanque de armazenamento de óleo diesel subterrâneo em um posto de combustíveis. Experimentos em escala laboratorial (respirômetros de Bartha) foram utilizados para avaliar a biodegradação do óleo diesel. Estímulo da biodegradação foi realizado utilizando-se as técnicas de bioestímulo (adição de soluções de nitrogênio e fósforo ou surfactante Tween 80) e de bioaumento (consórcio bacteriano isolado de um sistema de landfarming). Para investigar as interações entre os fatores otimizadores, e encontrar a melhor combinação entre esses agentes, o estudo foi baseado em um delineamento experimental fatorial completo. A eficiência de biodegradação foi simultaneamente medida com dois métodos: respirométrico (produção de CO2 microbiano) e cromatografia gasosa. Testes de toxicidade aguda com Daphnia similis foram aplicados para examinar a eficiência dos processos em termos de geração de produtos menos tóxicos. Resultados mostraram que todas as estratégias de biorremediação aceleraram a biorremediação natural do solo contaminado e os melhores resultados foram obtidos quando os tratamentos tinham adição de nutrientes. Dados respirométricos indicaram uma máxima mineralização de hidrocarbonetos de 19,8%, obtida com a combinação dos três agentes, com uma remoção de hidrocarbonetos totais de petróleo (TPH) de 45,5% em 55 dias de tratamento. No final dos experimentos, duas espécies predominantes de bactéria foram isoladas e identificadas como Staphylococcus hominis e Kocuria palustris.

Avaliação do potencial de biorremediação de solos e de águas subterrâneas contaminados com óleo diesel

Pós-graduação em Geociências e Meio Ambiente - IGCE