4株苯磺隆降解菌的分离鉴定及其生长特性

从长期受农药苯磺隆污染的土壤中通过采用富集培养分离技术得到4株以苯磺隆为唯一碳源生长的细菌,分别将其命名为B1、B2、B3和B4。通过观察这4种菌株的形态学特征,研究其生理生化特性以及分析其16S rDNA序列,初步鉴定菌株B1为铜绿假单胞菌(Pseudomonas aeruginosa),B2为戴尔福特菌(Delftia sp.),B3为微杆菌(Microbacterium sp.),B4为产碱杆菌(Alcaligenes sp.)。并通过研究温度、初始pH值、接种量、苯磺隆初始浓度、培养基体积、氮源、碳源、Mg2+浓度等因素对4种菌株生长情况的影响,确定了菌株的最佳生长条件。结果显示,B1菌株的最适温度为35℃,其他3株菌株均为30℃。菌株B3最适pH为8.0,其余3株菌株均为pH7.0。B1和B3菌株最适接种量为15%,B2和B4最适接种量为10%。菌株B3最适苯磺隆初始浓度为100mg·L-1,其余菌株最适苯磺隆初始浓度均为200mg·L-1。4株菌株最适培养基体积均为75mL,最适氮源均为硝酸铵,最适碳源均为葡萄糖。B2菌株最适Mg2+浓度为100mg·L-1,其余3株菌株均为200mg·L-1。B1和B4菌株最适NaCl浓度为20g·L-1,B2菌株NaCl浓度为5~30g·L-1,B3菌株最适NaCl浓度为50g·L-1。该结果为利用微生物对农药苯磺隆污染的土壤进行原位生物修复提供理论依据。     

结晶纤维素降解细菌的筛选、分离与鉴定

采用以结晶纤维素（Avicel）为唯一碳源的平板活性筛选法,从60份森林腐殖土、腐术样品中分离得到2株在pH5．0、37℃条件下高效降解结晶纤维素的细菌菌株GXN152和GXN153。以菌株的总DNA基因为模板,用细菌的16S rRNA基因的通用引物扩增得到菌株GXN152和GXN153的16S rRNA基因序列。测序分析表明菌株GXN152的16S rRNA基因序列和洋葱假单胞菌（Burkholderia cepacia）菌株CNR22的16S rRNA基因序列的同源性最高,具有98％的同源性,生理生化特性检测表明菌株GXN152具有洋葱假单胞菌的鉴别特征,将结晶纤维素降解菌GXN152鉴定为洋葱假单胞菌。菌株GXN153的16S rRNA基因序列和类芽孢杆菌（Paenibacillus favisporus）菌株GMP01的16S rRNA基因序列的同源性最高,具有99％的同源性,生理生化特性检测表明菌株GXN153具有类芽孢杆菌的鉴别特征,将纤维素降解菌GXN153鉴定为类芽孢杆菌。     

水稻根际耐镉细菌的筛选与鉴定

为获得稻田土壤镉污染修复的微生物,采用培养基加镉平板法,从水稻根际土壤分离耐镉细菌。本研究分离得到3株耐镉菌株,经16S rDNA测序、比对,鉴定为假单胞菌属细菌。对菌株细胞形态的电镜观察、菌株生长曲线的测定结果表明,在镉处理浓度为100 mg L 1条件下,菌株生长未受明显抑制,但菌株形态发生了适应性的变化;在镉处理浓度超过300 mg L 1时,菌株生长受到明显抑制;在镉处理浓度700 mg L 1条件下,菌株仍能生长,但生长延滞期延长,对数期后的生长量亦低;对菌体镉含量分析表明,在100 mg L 1镉处理下,3株菌株菌体中的镉含量分别为9.04 mg g 1、4.96 mg g 1和28.58 mg g 1,富集系数依次为90.4、49.6和285.8。可见,分离得到的3株菌株具有较强镉耐性和镉吸收能力,将为稻田土壤重金属镉污染的修复提供重要微生物资源。     

车前草根际土壤细菌的筛选鉴定与功能初探

从车前草(Plantago asiatica L.)根际土壤中筛选发现潜在的具有活硒能力、抑镉能力或二者能力兼之的细菌菌株。首先,采用稀释涂布平板法从车前草根际土壤中分离纯化出耐硒耐镉细菌;将分离得到的耐硒耐镉细菌进行16S rRNA基因鉴定;然后,利用土壤固体培养基对细菌进行培养,并用氢化物发生原子荧光光谱法和电感耦合等离子体发射光谱法分别测定不同细菌对应土壤的有效硒和有效镉,计算出菌株活硒率和抑镉率;最后,初步评价菌株对土壤硒或镉活化或抑制的能力。试验结果从车前草根际土壤中分离鉴定二级细菌8株,其中活硒率超过100%的菌株有CQC-3、CQC-8、CQC-11、CQC-12,抑镉率超过20%的菌株有CQC-7、CQC-8、CQC-12、CQC-13,活硒抑镉能力兼具的菌株有CQC-8和CQC-12,此研究结果为后续解决土壤和植物硒镉伴生问题、活硒抑镉机理研究和菌剂的开发提供基础资料。     

双歧杆菌的分离鉴定及活菌胶囊的研制

从母乳哺育的３～５月龄健康婴儿的粪便中分离到可疑双歧杆菌，通过驯化，获得一株较耐氧的菌株。经属和种的系统生理生化鉴定，确认为两叉双歧杆菌。将它制成活菌胶囊，在本校小范围服用结果表明，该菌对急慢性肠炎、腹泻、便秘等症有显著疗效。     

阿特拉津降解菌FM326（Arthrobacter sp.）的分离筛选、鉴定和生物学特性

采集除草剂阿特拉津污染的土壤,通过直接涂布法和富集驯化培养分离法,分别获得6株和5株能够降解阿特拉津的细菌。通过降解效率和降解动态试验,筛选到1株高效降解阿特拉津的菌株FM326,该菌株能以阿特拉津为唯一的碳源和氮源生长,培养96h后对1000mg·L-1阿特拉津降解效率达到97%。通过生理生化鉴定和16SrDNA序列分析,菌株FM326鉴定为节杆菌属（Arthrobacter sp.）细菌。该菌株表现出最适生长温度30～35℃,最适生长pH值5～9,好氧生长的生长特性。     

紫外诱变与羧甲基半胱氨酸复合筛选高产L-精氨酸菌株

为获得L-精氨酸高产菌株,以实验室初步鉴定为格兰氏阳性细菌,菌落特征为金黄色的产全氨基酸菌株为出发菌株,利用紫外线(UV)诱变和精氨酸结构类似物羧甲基半胱氨酸(羧甲司坦)进行复合筛选,得到1株L-精氨酸高产诱变菌株。对所筛选到的高产诱变菌株进行160r/min,30℃摇瓶发酵90h,测得该菌株L-精氨酸的产量为78.79μg/3ml,与出发菌株相比,L-精氨酸的产量提高约3.71倍。经多批次发酵试验进一步验证,所筛选的高产L-精氨酸菌株具有较好的遗传稳定性,此研究不仅对筛选L-精氨酸高产菌株具有一定参考和借鉴意义,对筛选其他类型氨基酸高产菌株也可提供一定参考。     

木糖氧化无色杆菌反硝化亚种细菌的分离鉴定及其菲降解特性研究

利用选择性富集培养及升华法,从石油污染的土壤中分离到2株菲降解细菌,它们在以菲为唯一碳源的培养基上生长良好。应用BIOLOG细菌鉴定系统和分子生物学方法对两株细菌进行鉴定,两株菌分别为坚强芽孢杆菌(Bacillus.firmus)和木糖氧化无色杆菌反硝化亚种(Achromobacter.xylosoxidanssub.sp.denitrificans),两株菌均具有邻苯二酚氧化酶活性。两株细菌在液体培养条件下都表现较强降解菲的能力,液体培养60.h约90%的加入菲被降解。通过测定液体培养基中菲浓度和菌体密度变化发现,菌株降解菲的量与其生长密度相关;随着菌体浓度(吸光度)的增加,代谢底物菲的浓度明显降低,两株菌混合使用能够大幅度提高降解菲的能力。     

四环素类抗生素降解菌的分离与鉴定

为了获得四环素类抗生素的高效降解菌,从长期受四环素类抗生素污染的土壤中分离、重复驯化筛选得到6株对四环素类抗生素具有降解能力的菌株;采用高效液相色谱法研究了6株降解菌的降解效果,并通过形态、生理生化特征及16S rDNA序列分析对其进行鉴定。结果表明,菌株TJ-2#对土霉素、四环素和金霉素3种四环素类抗生素的降解效果最好,降解率分别为58.3%、63.9%和65.5%,TJ-6#其次;6株降解菌均能以四环素类抗生素作为唯一碳源生长。经形态、生理生化特征及16S rDNA鉴定,菌株TJ-2#为木糖氧化无色杆菌(Achromobacter sp.),TJ-6#为枯草芽胞杆菌(Bacillus sp.)。本研究结果对四环素类抗生素的生物降解具有一定的现实意义。     

一株烟草青枯拮抗细菌的筛选、鉴定和培养特性研究

为烟草生物肥料的研制和生产提供优良菌株资源,筛选可有效拮抗烟草青枯病原菌的菌株,优化其培养条件,确定其分类地位。采用抑菌圈测定法从高粱根际土中筛选烟草青枯拮抗菌株,并对其单因素液体拮抗条件进行优化,通过其表型特征、生理生化特征和遗传特征等多相分析来确定其分类地位。获得一株对烟草青枯病原菌具有较好拮抗效果的细菌 MT-002-B-7,抑菌圈直径可以达到 28 .67 mm,该菌的液体最佳发酵条件为以葡萄糖为碳源,以谷氨酸为氮源,装液量 10%,培养基初始 pH值为 7 .0,接种量3%,温度 35℃,转速 150r/min,培养25 h。鉴定结果表明该菌为铜绿假单胞菌。该菌株对烟草青枯病原菌拮抗效果良好,可作为研制烟草生物肥料的菌株资源。     

生物有机肥对土壤微生物活性的影响

通过两次连续温室玉米盆栽试验,研究了施用具有调节微生物功能的生物有机肥对土壤微生物数量与活性的影响,并利用传统平板计数法与BIOLOGECO方法相结合研究生物有机肥对土壤微生物生态的影响。结果表明,与化肥相比,施用生物有机肥可显著提高土壤微生物中3大菌群的数量;AWCD值及微生物对不同碳底物利用水平的测定结果表明,施用生物有机肥可明显提高土壤微生物对碳源的利用率,尤其土壤中的羧酸、胺类和其他类碳源等。表明生物有机肥的施用能增加土壤微生物利用碳源能力,改善微生物营养条件,使微生物保持较高活性,提高土壤微生物多样性。     

生物复混肥对土壤微生物群落功能多样性和微生物量的影响

在温室盆栽条件下,采用Biolog微平板法和氯仿熏蒸浸提法,研究了玉米施用等养分量的无机肥、有机无机复混肥和生物复混肥后土壤微生物群落功能多样性及土壤微生物量的变化。结果表明:生物复混肥处理的土壤微生物平均颜色变化率(AWCD)、微生物群落Shannon指数(H)和微生物群落丰富度指数(S)均最高;施用生物复混肥可明显提高土壤微生物对碳源的利用率,尤其是多酚化合物类和糖类;不同处理土壤微生物碳源利用特征有一定差异,生物复混肥在第1主成分上的得分值为正值,其他各处理在第1主成分上的得分值基本上为负值,起分异作用的主要碳源是糖类和羧酸类。在玉米生长期间各处理土壤微生物量大致呈先升高后逐渐平稳的趋势,且土壤微生物量碳、氮、磷的含量均以生物复混肥处理最高,最高值分别为333.21mg.kg 1、53.02 mg.kg 1和22.20 mg.kg 1。研究表明,生物复混肥的施用比等养分量的有机无机复混肥处理能显著提高土壤微生物群落碳源利用率、微生物群落丰富度和功能多样性,显著增加土壤微生物量碳、氮、磷的含量,有利于维持良好的土壤微生态环境。     

Diversity of rhizobia isolated from an agricultural soil in Argentina based on carbon utilization and effects of herbicides on growth

Seventy-six rhizobial isolates belonging to four different genera were obtained from the root nodules of several legumes (Vicia sativa, Vicia faba, Medicago sativa, Melilotus sp., Glycine max and Lotus corniculatus). The action of five commonly used herbicides [2,4-dichlorophenoxyacetic acid (2,4-D), glyphosate (GF), dicamba, atrazine and metsulfuron-methyl] on the growth of rhizobial strains was assessed. Subsequently, GF and 2,4-D were tested in a minimum broth as C and energy sources for 20 tolerant strains. The ability of these strains to metabolize different carbon sources was studied in order to detect further differences among them. Tolerance of the bacteria to agrochemicals varied; 2,4-D and GF in solid medium inhibited and diminished growth, respectively, in slow-growing rhizobial strains. Among slow-growing strains we detected Bradyrhizobium sp. SJ140 that grew well in broth + GF as the sole C and energy source. No strain was found which could use 2,4-D as sole C source. The 20 strains studied exhibited different patterns of C sources utilization. Cluster analysis revealed three groups, corresponding to four genera of rhizobia: Rhizobium (group I), Sinorhizobium (group II) and Mesorhizobium–Bradyrhizobium (group III). On the basis of the results obtained on responses to herbicides and C sources utilization by the isolates investigated, it was possible to differentiate them at the level of strains. These results evidenced a considerable diversity in rhizobial populations that had not been previously described for Argentinean soils, and suggested a physiological potential to use natural and xenobiotic C sources.     

Misidentification of soil bacteria by fatty acid methyl ester (FAME) and BIOLOG analyses

 Fatty acid methyl ester (FAME) analysis is commonly used by soil scientists as a sole method for identifying soil bacteria. We observed discrepancies with this method for identifying certain species of bacteria. Therefore, we used carbon substrate oxidation patterns (BIOLOG) and some simple physical and chemical tests to determine the extent of these discrepancies. Identification with FAME profiles gave false positives for Arthrobacter globiformis, Micrococcus kristinae, and M. luteus, and identification with BIOLOG patterns gave a false positive identification for A. globiformis. A visual check and Gram stain are recommended when FAME analysis identifies soil isolates as M. kristinae or M. luteus, and an additional spore formation test is recommended when FAME and BIOLOG analyses identify isolates as A. globiformis. Received: 14 January 2000     

两株解磷细菌的解磷活性及作用机制研究

解磷细菌在增加土壤可溶性磷含量、提高磷肥利用效率方面具有重要作用。为选筛高效解磷菌、探讨其解磷机制,本文利用平板溶磷圈法筛选解磷细菌,采用钼锑抗比色法研究其解磷活性,苯磷酸二钠法研究其磷酸酶活性,利用薄层层析分析其产生的有机酸,根据生理生化特征和16S r RNA基因序列系统发育分析,确定其分类学地位。结果表明,菌株JXJ-11和JXJ-15对植酸钙的降解活性很强,3 d后培养液中可溶性磷浓度分别增加219 mg·L~(-1)和216 mg·L~(-1);对磷酸钙降解活性较弱,最高可溶性磷浓度仅为植酸钙的21.79%~30.37%;解磷细菌可分泌酸性、中性和碱性磷酸酶,降解不溶性磷,可能产生丙酸和琥珀酸等有机酸,降低培养液p H,增加可溶性磷浓度。两株细菌均为革兰氏阴性杆菌,无芽孢,产生硫化氢,其中菌株JXJ-11的16S rRNA基因序列与Sphingomonas melonis DAPP-PG 224T和S.aquatilis JSS7T相似性最高(99.79%),菌株JXJ-15的16S rRNA基因序列与Klebsiella pneumoniae subsp.pneumoniae DSM 30104T相似性最高(99.73%),根据以上信息,确定菌株JXJ-11和JXJ-15分别是鞘氨醇单胞菌属和克雷白氏杆菌属的成员。菌株JXJ-11和JXJ-15的解磷机制包括分泌有机酸和磷酸酶,其中JXJ-11在微生物磷肥研制方面具有潜在应用价值。     

Soil Microbial Community Diversity and Its Relationships with Geochemical Elements under different Farmlands in Shouguang,China

The aims of this study were to investigate soil microbial community characteristics and their interrelationships with soil geochemistry under different farmlands in Shouguang, China. The traditional dilution plate counts, BIOLOG system, and fatty acid methyl ester (FAME) analyses were used to assess microbial populations, substrate utilization, and fatty acid profiles. The number of aerobic heterotrophic bacteria varied significantly among untilled land, maize, and mungbean fields. The amounts of actinomycetes, fungal fatty acids, and Gram-positive/Gram-negative bacteria ratios varied greatly among celery, tomato, and aubergine fields. In the tomato field, the soil microbial community characteristics were significantly different from other fields. Principal component analysis of BIOLOG and FAME data revealed differences in the catabolic capability and fatty acid profiles of soil microbial communities among different farmlands. Spearman correlation analyses showed that in these sand clay soils of Shouguang, microbial communities in different farmlands were closely correlated with soil geochemical elements, moisture, and organic matter.     

间套作玉米对线辣椒根际土壤微生物生态特征的影响

采用常规稀释平板法、氯仿熏蒸法、BIOLOG GN微平板反应系统及种间根系分隔技术, 以线辣椒单作(SC)为试验对照, 研究了间套作处理[玉米/线辣椒套作+根部塑料膜分隔(ICP)、玉米/线辣椒套作+根部尼龙网分隔(ICM)、玉米/线辣椒套作根部无分隔(ICN)]对线辣椒根际土壤微生物生态特征的影响。结果表明: 整个线辣椒生育期内, 各套作处理线辣椒根际土壤微生物总数与细菌总数具有相同的变化趋势, ICN和ICM处理的真菌、细菌、放线菌数量和细菌/真菌(B/F)、放线菌/真菌(A/F)比值均大于ICP与SC处理。盛果期, ICN处理根际土壤微生物量碳和微生物量氮比同处理其他生育期增加14.2%~54.0%和10.6%~54.7%。各处理土壤微生物群落AWCD的变化随培养时间呈现明显的"S"型曲线。间套作玉米显著提高了线辣椒根际土壤微生物群落的Shannon-Wiener指数(P<0.05)、Simpson指数、种间相遇几率和McIntosh指数(P<0.05), 并改变了土壤微生物对单一碳源的利用能力。线辣椒根际土壤微生物的不同多样性指数分别与其生物学产量之间存在显著或极显著正相关。说明间套作改善了土壤微生态环境。     

土壤微生物群落结构与多样性对农田集约化管理程度降低的响应

Using a scheme of agricultural fields with progressively less intensive management （deintensification）, different management practices in six agroecosystems located near Goldsboro, NC, USA were tested in a large-scale experiment, including two cash-grain cropping systems employing either tillage （CT） or no-tillage （NT）, an organic farming system （OR）, an integrated cropping system with animals （IN）, a successional field （SU）, and a plantation woodlot （WO）. Microbial phospholipid fatty acid （PLFA） profiles and substrate utilization patterns （BIOLOG ECO plates） were measured to examine the effects of deintensification on the structure and diversity of soil microbial communities. Principle component analyses of PLFA and BIOLOG data showed that the microbial community structure diverged among the soils of the six systems.Lower microbial diversity was found in lowly managed ecosystem than that in intensive and moderately managed agroecosystems, and both fungal contribution to the total identified PLFAs and the ratio of microbial biomass C/N increased along with agricultural deintensification. Significantly higher ratios of C/N （P 〈 0.05） were found in the WO and SU systems, and for fungal/bacterial PLFAs in the WO system （P 〈 0.05）. There were also significant decreases （P 〈 0.05） along with agricultural deintensification for contributions of total bacterial and gram positive （G＋） bacterial PLFAs.Agricultural deintensification could facilitate the development of microbial communities that favor soil fungi over bacteria.     

BIOLOG法测定土壤微生物群的一些局限性

Reduction of Cr(VI)to Cr(Ⅲ）were studied in a fresh wheat rhizosphere soil(Kuroboku,high humicandosol) pretreated with a basal fertilizer consisting of (NH4)2SO4,P2O5 and KH2PO4 and with K2Cr2O7 by using a rhizobox system.It was found that rhizosphere exerted a positive effect on Cr(VI) reduction.Part of the reason was the decrwease of pH in the rhizosphere due to application of (NH4)2SO4,implying that application of physiologically acid fertilizers would reduce Cr(VI) toxicity to plants.