中国科学院沈阳应用生态研究所

本研究通过选择性富集培养，从辽河油田稠油污染土壤中获得了能以2000mg·L~(-1)菲为唯一碳源和能源快速生长的优势降解菌株ZL5，经165 roNA序列分析认其属变形细菌a亚类中的鞘氨醇单胞菌属。ZL5菌株具有较强的降解菲、花能力，但是不能以蔡为唯一碳源和能源生一氏。外加碳源葡萄糖能有效促进PAHs降解，降解率提高达18.21-23.5％，但糖过量会表现出抑制效应。菌株ZL5含有一个大小约为60 kb的内生大质粒。丝裂霉素C消除试验表明，随着质粒丢失，菌株利用菲和花的能力也丧失。应用电转化和氯化铆转化将质粒导入大肠杆菌中，转化子获得了相应的降解菲、花能力，表明菌株ZLS的内生大质粒是与代谢PAHs功能有关的降解质粒。菌株ZL5具有PAHs诱导的邻苯二酚2，3-双加氧酶酶活（C230），指明其能以菲为唯一碳源和能源生长但不能降解蔡的原因可能为一未见报道的菲代谢新途径-水杨酸和邻苯二酚途径。采用特异性引物，扩增菌株ZLS的C230编码基因全序列，再与表达载体pET-30a（+）连接，实现了在E.coli JM109（OE3）中的高效表达。SDS-PAGE结果表明：C230蛋白不仅在细胞内存在，也能被大量地分泌到胞外，属国内首次报道。经Southern杂交，将菌株ZLS的C230基因定位在内生大质粒的不同酶切片段上。

Gene cloning and functional analysis of glycosaminoglycan-degrading enzyme chondroitin AC lyase from Flavobacterium columnare G(4)

The chondroitin AC lyase gene, cslA, was cloned for the first time from the fish bacterial pathogen F. columnare G(4). From the first transcription initiation site, the cslA extends 2620 nucleotides to the end of the 3' region. The open reading frame of cslA transcript has 2286 nucleotides encoding 762 amino acids with a 16 residues long signal peptide at the N-terminus. The gene, cslA was then successfully expressed in Escherichia coli and recombinant chondroitin AC lyase, rChonAC was purified, with its lytic activity analyzed. Zymography analysis copolymerized with chondroitin sulphate revealed the lytic activity of rChonAC and also the crude native ChonAC isolated from periplamic space of cultured F. columnare G(4). The low level of lytic activity observed in crude native ChonAC may be due possibly to the low level of expression of this gene in the cultured condition. The expression and the role of this virulence factor is of interest for further research on the pathogenesis of F. columnare.

Roles of substrate microorganisms and urease activities in wastewater purification in a constructed wetland system

A vertical/reverse-vertical flow constructed wetland system was set up in Wuhan, China, to study its treatment efficiency of polluted lake water. The numbers of substrate microorganisms and urease activities in the substrate of the constructed wetland were determined by plate counts and colorimetric analysis, respectively. The removal efficiencies of biochemical oxygen demands (BOD5). chemical oxygen demands (COD), total phosphorus (TP), total Kjeldahl nitrogen (TKN), and total suspended solids (TSS) were measured by EPA approved methodology. The results showed there were significant positive correlations (P < 0.05) between numbers of microorganism in the substrate and removal rates of TKN and CODCr. Meanwhile, there was significant positive correlation (P < 0.05) between urease activities and removal efficiencies of TKN and negative correlation between urease activities and removal efficiencies of BOD5. Substrate microorganisms and urease activities played key factors during purification processes and they could be utilized as indicator of wastewater treatment performances in the constructed wetland system. (C) 2004 Elsevier B.V. All rights reserved.

Microorganisms linked to Neoproterozoic microspar carbonate sedimentation in the Jilin-Liaoning area

Molar-tooth carbonate refers to a sort of rock that has ptygmatical folded structure comparable to the ivory. This kind of carbonate exists in a special time range (from Middle to Neoproterozoic). Its origin and the possibility to use it in stratigraphic correlation of the paleocontinent is the key task of the IGCP447, a project on Proterozoic molar tooth carbonates and the evolution of the earth (2001-2005). The importance lies in that the molar-tooth structure is the key to solving problems related to Precambrian biological and global geochemical events. The molar-tooth structure is associated with microorganisms. Development and recession of such carbonates have relations with the evolution process of early lives and abrupt changes in sea carbonate geochemistry. In recent years, based on researches on petrology, geochemistry and Sr isotope of molar-tooth carbonate in the Jilin-Liaoning and Xuzhou-Huaiyang area, the authors hold that it can be used as a marker for stratigraphic sequence and sedimentary facies analyses.

Isolation and Characterization of a Methomyl-Degrading Paracoccus sp mdw-1

Methomyl, an extremely toxic pesticide, is widely used in agriculture. A strain named mdw-1 capable of degrading methomyl rapidly was successfully isolated from activated sludge in this study. It could utilize methomyl as the sole carbon or nitrogen source. The optimal temperature and medium pH for its growth and methomyl biodegradation were 30 degrees C and 7.0, respectively. It was identified as a Paracoccus sp. according to its morphological features, physiological and biochemical characteristics, and phylogenetic analysis based on the sequence of 16S rDNA. Gas chromatography-mass spectrometry (GC-MS) analysis showed that methomyl could be completely transformed to S-methyl-N-hydroxythioacetamidate in 10 h of incubation with the isolate mdw-1.

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.

Rhodococcus qingshengii sp nov., a carbendazim-degrading bacterium

A Gram-positive, aerobic, non-motile, mesophilic strain, djl-6(T), able to degrade carbendazim, was isolated from a carbendazim-contaminated soil sample from Jiangsu province, China. The taxonomic position of this isolate was analysed by using a polyphasic approach. Chemotaxonomic analysis including peptidoglycan type, diagnostic sugar composition, fatty acid profile, menaquinones, polar lipids and mycolic acids showed that the characteristics of strain djl-6(T) were in good agreement with those of the genus Rhodococcus. DNA-DNA hybridization showed that it had low genomic relatedness with Rhodococcus baikonurensis DSM 44587(T) (31.8%), Rhodococcus erythropolis DSM 43066(T) (23.8%) and Rhodococcus globerulus DSM 43954(T) (117.7%), the three type strains to which strain djl-6(T) was most closely related based on 16S rRNA gene sequence analysis (99.78, 99.25 and 98.91% similarity, respectively). Based on the phenotypic properties and DNA-DNA hybridization data, strain djl-6(T) (=CGMCC 1.6580(T) =KCTC 19205(T)) is proposed as the type strain of a novel Rhodococcus species, Rhodococcus qingshengii sp. nov.

Microorganisms linked to Neoproterozoic microspar carbonate sedimentation in the Jilin-Liaoning area

Molar-tooth carbonate refers to a sort of rock that has ptygmatical folded structure comparable to the ivory. This kind of carbonate exists in a special time range (from Middle to Neoproterozoic). Its origin and the possibility to use it in stratigraphic correlation of the paleocontinent is the key task of the IGCP447, a project on Proterozoic molar tooth carbonates and the evolution of the earth (2001-2005). The importance lies in that the molar-tooth structure is the key to solving problems related to Precambrian biological and global geochemical events. The molar-tooth structure is associated with microorganisms. Development and recession of such carbonates have relations with the evolution process of early lives and abrupt changes in sea carbonate geochemistry. In recent years, based on researches on petrology, geochemistry and Sr isotope of molar-tooth carbonate in the Jilin-Liaoning and Xuzhou-Huaiyang area, the authors hold that it can be used as a marker for stratigraphic sequence and sedimentary facies analyses.

植物细胞色素P450对土壤PAHs污染早期诊断研究

以高等植物为试验材料，建立了细胞色素P450含量的测定方法。在此基础上通过对5种植物（小麦、玉米、白菜、萝卜、大豆）的筛选试验，最终选择小麦绿苗和玉米黄化苗作为供试材料，进行土壤菲、芘单一与复合污染条件下，细胞色素P450含量与多环芳烃污染的量-效关系生态毒理诊断研究。同时为比较不同指标对污染物生态毒性敏感性，进行了相同剂量条件下，SOD、POD、CAT活性,高等植物种子发芽与根伸长抑制率与菲、芘单一与复合污染诱导的剂量-效应关系试验研究。 细胞色素P450含量污染诱导研究结果表明，在较低的浓度下，土壤菲、芘单一暴露与小麦和玉米幼苗P450含量具有显著的污染诱导剂量-效应关系，菲、芘复合污染对小麦和玉米的P450产生协同毒性作用。两种植物P450含量对菲、芘污染指示较为敏感。 SOD、POD、CAT活性比较试验结果表明，SOD、POD、CAT活性对菲、芘污染具有响应，但响应敏感性不及植物色素P450，有些不能进行定量诊断，但可用于定性诊断。 发芽率和根伸长抑制率试验比较结果表明，在同样的供试浓度范围内，小麦发芽率和根伸长与对照相当，未出现明显诱导或抑制的响应。植物色素P450含量对菲、芘单一与复合污染的指示作用比植物种子发芽率和根伸长抑制率更为敏感。 植物细胞色素P450这一敏感指标可以作为生物标记物，与化学分析及抗氧化酶系指标结合起来进行土壤PAHs早期诊断。既有助于提高指标的准确性和敏感性，推动形成完善的土壤污染生态毒理诊断方法，还可从生理和分子水平使污染诱发障碍的机理等得到破译。

多环芳烃胁迫下农田土壤微生物特征及高效芘降解菌筛选

本文通过传统和现代微生物生态学方法，从土壤微生物数量、土壤底物诱导呼吸强度和微生物群落多样性角度，评价了长期污灌所致PAHs污染对土壤微生物特征的影响。结果发现沈抚灌区农田PAHs总量在表层为612.3-6362.81μg•kg-1干土，在亚表层为319.5-4318.51μg•kg-1干土。土壤微生物主要类群、功能群数量、微生物生物量碳和代谢商与土壤PAHs污染程度无明显相关性，土壤底物诱导呼吸强度和所试土壤酶与PAHs含量呈显著正相关，微生物商与PAHs含量呈显著负相关，可以作为土壤PAHs污染评价的敏感生化指标之一。污染稻田土壤细菌群落中优势菌群为β-和γ-变形细菌亚纲的成员，中度PAHs土壤的分支杆菌多样性指数较重度和轻度的略高，PAHs污染使一种或几种分支杆菌得到富集。长期污水灌溉造成土壤固氮细菌种群多样性降低，清水灌溉一段时间后，固氮细菌种群结构得到不同程度的恢复，但是，即使清灌不能使其种群结构得到完全恢复。 通过富集得到一株高效降解芘的细菌N12，经鉴定确认为分支杆菌。经10天培养菌株可将100mg•l-1芘降解97.84%。还可降解菲、苊、芴，不能降解萘、蒽和苯并[a]芘。污染土壤修复实验表明，单一接种菌剂对芘的降解率为57.42%，含N12的混合接种菌剂对芘的降解率为61.11%。

土壤低浓度PAHs胁迫下赤子爱胜蚓差异表达基因初步筛选

为探索土壤低浓度多环芳烃污染的生态毒性及其对土壤生物的致毒机理，本论文初步研究了菲、芘、荧蒽和苯并[a]芘等四种多环芳烃人工土壤污染在0.1mg.kg-1~10.0mg.kg-1浓度水平对赤子爱胜蚓（Eisenia fetida）产卵量、体重变化、排卵激素annetocin基因和翻译控制肿瘤蛋白（translationally controlled tumor protein, TCTP）转录水平的影响，发现在相同的低浓度水平下，只有苯并[a]芘对蚯蚓annetocin前体基因和TCTP基因的表达有显著影响，故其对生物体的生殖风险和致癌风险可能最大。另一方面，低浓度苯并[a]芘对蚯蚓体重和产茧量并无显著影响，这表明基因表达水平作为污染生态监测指标比宏观观测指标更灵敏。 为进一步研究土壤PAHs污染的生态毒性效应，在上述研究基础上，我们采用SSH-PCR的方法构建了人工土壤1.0mg.kg-1苯并[a]芘胁迫下的蚯蚓与对照组蚯蚓之间的消减cDNA文库，随机挑取上调文库301个克隆及下调文库283个克隆进行测序，与NCBI蛋白数据库比对结果表明，其中有391个克隆与已知的75种蛋白质基因显著匹配（期望值< 10-5），其余克隆匹配不显著（期望值> 10-5）或找不到匹配蛋白。显著匹配的基因序列包括：一相解毒酶细胞色素P450，二相解毒酶谷胱甘肽硫转移酶，蛋白质合成所需的核糖体蛋白亚基，参与新合成肽链折叠的热休克蛋白，线粒体基因组编码的呼吸链复合酶体亚基，过氧化物还原蛋白，铁蛋白，钙结合蛋白，半胱氨酸蛋白酶等。表明低浓度苯并[a]芘胁迫引起蚯蚓的生理变化是非常复杂的，涉及污染物降解与解毒、抗氧化保护、能量代谢、蛋白质合成、金属离子调节与蛋白质降解等过程。 Real-time PCR检测验证消减文库中部分差异基因对不同剂量BaP胁迫响应结果表明，各检验基因序列受1.0 mg∙kg-1 BaP 胁迫影响均与消减结果一致，且影响程度均高于0.1 mg∙kg-1浓度水平的BaP；其中，在0.1 mg∙kg-1 BaP胁迫下，过氧化物还原酶PRDX和类似Cyp2R1的P450基因表达未见明显变化。其余的HSP70、HSP90、rpL10、COXⅡ、SCBP、Ferritin等基因在0.1 mg∙kg-1 BaP胁迫组蚯蚓中均有检测到预期表达变化，说明虽然从消减文库中获得的基因在一定的污染物浓度范围内均表现浓度效应，但各个基因对污染物的响应浓度不尽相同。 Real-time PCR检测消减文库中部分差异基因对不同PAHs胁迫响应结果表明，1.0 mg∙kg-1 浓度水平的荧蒽、菲、芘和苯并[a]芘对差异表达基因的影响不尽相同，主要有以下三种情况：(1)广谱响应型：蚯蚓线粒体编码的亚基COXⅡ、可溶性钙结合蛋白、铁蛋白等基因对荧蒽、菲、芘及苯并[a]芘的胁迫均有相似的响应；(2)随芳烃环数而变化型：热休克蛋白HSP70和过氧化物还原酶PRDX表现出响应程度随胁迫多环芳烃的环数增加而提高的现象；(3)仅在苯并[a]芘中有响应型：核糖体蛋白亚基L10和细胞色素P450（类似Cyp2R1）基因，在1.0 mg∙kg-1浓度条件下，它们仅受BaP诱导表达，而芘、菲和荧蒽却没有显示诱导作用。 上述结果表明，在土壤中的低浓度的多环芳烃污染胁迫对蚯蚓的影响是多方面的，这些影响至少涉及能量代谢、污染物降解与解毒、蛋白质合成与修复、信号转导、细胞凋亡、排卵生殖、个体发育等多方面的生理功能。目前蚯蚓基因组还未有完整测序，本文论述的多个差异表达基因是首次在蚯蚓中发现的，这些新发现的基因序列在为低浓度PAHs的生态毒性机理研究提供依据的同时，也为以蚯蚓为模式生物的土壤污染生物监测提供了备选的生物分子标记。另一方面，由于蚯蚓基因组未完整测序，本研究构建的消减文库中仍不少未知功能基因，其功能与调控有待进一步研究。

土壤低剂量PAHs污染生态毒理诊断指标研究

本文探讨了蚯蚓细胞色素P450作为生物标记物诊断土壤低剂量PAHs污染的可行性，主要以蚯蚓P450含量和AHH活性二项指标为主，开展“土壤低剂量PAHs污染敏感生态毒理诊断指标研究”，并结合其它分子毒理诊断指标，通过指示敏感性比较，最终建立土壤低剂量PAHs污染早期诊断的敏感生物标记指标或指标组合，为土壤污染早期诊断奠定基础。 蚯蚓P450含量测定方法研究表明，在强化预处理的基础上，通过微粒体增溶、物理分离和解剖后内脏直接测定等去干扰手段均可实现P450含量的测定。通过三种方法的比较，最终确定内脏直接测定法为最佳方法。在此基础上，对CO通气量进一步优化，确保P450含量的准确定性和定量。 低剂量PAHs胁迫下蚯蚓细胞色素P450响应研究表明，P450含量和AHH活性两指标随暴露时间的增加总体上表现为“先诱导后抑制”的响应规律，且响应因PAHs的不同而存在差异，对芘的指示效应最强，荧蒽次之，菲最弱，与其毒性大小相关。其中，AHH对PAHs的指示作用优于P450含量，两指标的响应情况显示了它们作为生物标记物的可行性。为增强污染诊断的灵敏性和有效性，可将蚯蚓P450含量和特异性同工酶活性联合，彼此互为补充，共同应用于土壤污染生态毒理诊断中。 低剂量PAHs胁迫下，参比指标的响应情况与P450相比结果显示，P450含量和AHH活性的指示效应最明显；SOD和POD活性的指示次之；GST和CAT活性虽表现出指示效应，但并不是对三种PAHs都具有指示效果；MDA含量对低剂量PAHs的指示性最差。不同指标对污染物的指示敏感性存在差异，因而在进行土壤污染生态毒理诊断时，应选用多指标联合，彼此相互补充，以适应不同污染状况下的污染诊断。酶活性对毒物暴露的响应在时间上是一个动态变化过程，单一时段的暴露结果可能不足以揭示污染物的毒性效应，选择多时段检测对污染暴露的灵敏指示尤为重要。 应用蚯蚓生化指标对沈阳污灌区土壤进行毒性诊断，结果表明P450、AHH、GST、POD和CAT五个指标通过不同时段的检测均可对供试土壤显示出指示效应；而SOD和MDA两指标均出现“漏诊”现象。同实验室条件下低剂量污染情况相比，P450含量和POD的指示效果在两种情况下均较好；AHH、SOD在实验室条件下的指示更为优越；而GST、CAT和MDA更适合于污染物复杂的实际土壤诊断。研究结果从实际应用的角度再一次证实，任何一种指标若采用单时段的检测均存在其污染诊断的“盲区”。在进行实际环境污染诊断时，采用多指标和多时段检测是不可或缺的。