华北平原农田厚包气带及含水层反硝化细菌的分离鉴定及作用机制研究

华北平原农田由于长期过量施用氮肥,造成了土壤硝酸盐累积,导致地下水硝酸盐污染日趋严重。微生物的反硝化作用可将土壤中累积的硝酸盐或亚硝酸盐还原为气态产物,是消减厚包气带土壤累积的硝酸盐的重要途径。因此筛选高效反硝化微生物资源,对人工强化厚包气带土壤反硝化脱氮,阻控地下水硝酸盐污染具有重要作用。基于此,本研究采集位于华北平原的中国科学院栾城农业生态系统试验站长期施氮[施氮量为600kg(N)·hm~(-2)·a~(-1)]定位试验0～150m农田厚包气带及含水层土壤样品,从中筛选到62株细菌。16SrRNA基因序列分析表明这62株菌株与变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、厚壁菌门(Firmicutes)中的9个属具有较高的同源性。根据系统发育树的结果,挑选7株亲缘关系较远的菌株进行反硝化潜势试验,结果表明,菌株L71、L13和L103具备反硝化产气能力。电镜观察结果表明,这3株菌均为无鞭毛的杆状细菌,其长度分别为1.0μm、1.5μm和1.5μm,只有L103具有运动能力。此外,菌株L103具有完全反硝化能力,且脱氮能力受到pH的影响,在本试验条件下,菌株L103的反硝化速率高达1.62～2.36g(KNO_3)·d~(-1)·L~(-1),具备实际应用潜力。本研究表明华北平原厚包气带土壤中存在完全反硝化微生物,并可为人工强化治理厚包气带土壤硝酸盐污染提供菌种资源和理论依据。     

蜡样芽孢杆菌及其生物被膜对有机酸及乙醇的抗性比较

为比较蜡样芽孢杆菌及其生物被膜对环境压力的抗性,该文主要研究了4种有机酸（乙酸、柠檬酸、乳酸和苹果酸）和乙醇对蜡样芽孢杆菌及其生物被膜存活率的影响。结果表明：生物被膜态蜡样芽孢杆菌对乙酸的抗性高于浮游态菌。扫描电镜结果显示,经乙酸处理后,浮游态蜡样芽孢杆菌的细胞表面严重受损,而生物被膜态菌的表面形态未发生明显变化。在柠檬酸、乳酸、苹果酸和乙醇存在的条件下,生物被膜态蜡样芽孢杆菌比浮游态菌表现出更强的压力抗性,特别是在高浓度（有机酸16%～20%,乙醇50%～60%）的情况下,该现象尤为显著。因此,在食品工业中控制被膜态蜡样芽孢杆菌对预防和阻止食品腐败非常重要。该研究结果为实际生产中有效控制蜡样芽孢杆菌及其生物被膜的形成提供参考。     

生物表面活性剂产生菌的筛选及其发酵条件的初步优化

以石油为 C 源,经过富集培养从石油污染土壤中筛选出 15 株具有较强产生物表面活性剂能力的菌株.其中菌株 BS-5 产生物表面活性剂的能力最强,该菌发酵液的表面张力可达 27.3 mN/m(空白发酵液表面张力为 54.5 mN/m).通过红外光谱分析发现该菌产生的生物表面活性剂为糖脂类物质.另外,通过正交试验对该菌的培养基条件进行初步优化,结果以植物油 10 g/L、MgSO4 0.2 g/L、K2HPO4 1.0 g/L、KH2PO4 1.0 g/L、蛋白胨 1.0 g/L、FeSO4 0.05 g/L、CaCl2 0.02 g/L、初始 pH 值 7.5 为最佳.通过16S rDNA 测序结果表明该菌为铜绿假单胞菌 (Pseudomonas aeruginosa).     

产共轭亚油酸菌株的筛选和鉴定

通过测定乳酸菌发酵液中共轭亚油酸(CLA)的含量筛选出具有高共轭亚油酸(CLA)转化能力的非厌氧菌株.本研究测定了加株乳酸菌液中的CLA含量,筛选出A6-1和SL-3两株具有较高CLA转化能力的菌株;将A6-1和SL-3分别接种于含1.0%LA的MRS培养基中,37℃培养24h,采用气相色谱(GC)分析发酵液中CLA的组成,结果表明,A6-1的发酵液中两种活性异构体c-9,t11CLA和t-10,c12CLA的含量分别为61.554μg/ml和14.235μg/ml;SL-3发酵液中c-9,t11和t-10,c12 CLA含量分别为46.161μg.ml和8.7399μg/ml.经API 50鉴定系统确定,A6-1和SL3均为植物乳杆菌.     

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。     

重组H-NS蛋白对胸膜肺炎放线杆菌生物被膜形成的影响

摘要：胸膜肺炎放线杆菌（APP）是重要的猪呼吸道病原菌,给世界养猪业造成严重的经济损失。生物被膜在许多细菌的感染与致病过程中起重要作用。我们在筛选APP转座突变体库时,发现hns基因缺失突变可显著增强APP血清1型生物被膜的形成,该基因编码一种DNA结合蛋白（组蛋白样类核结构蛋白）。为了进一步研究H-NS蛋白对APP生物被膜形成的影响,本文以APP血清1型4074株基因组DNA为模板,PCR扩增了408bp的hns基因编码区,克隆到原核表达载体pET-28c中获得重组质粒pET-hns,转化大肠杆菌BL21(DE3),经IPTG诱导表达和组氨酸亲和层析柱纯化获得大小约19KDa的重组蛋白rH-NS。将不同浓度的rH-NS添加到hns突变株1-21及其亲本菌株4074的培养基中,用微孔板法测定生物被膜的形成。结果显示,在不添加rH-NS时,4074株不能形成可见的生物被膜,而1-21株形成明显的生物被膜;在添加0.1-0.3μM的rH-NS的情况下,1-21株生物被膜的形成量随着rH-NS浓度的升高而降低,而4074株随着rH-NS浓度的升高而升高;rH-NS添加量超过0.4μM对两个菌株生物被膜形成未见明显影响。结果表明H-NS蛋白负调控APP生物被膜的形成,并呈现一定的剂量效应。     

紫外诱变筛选海洋红酵母S8的尿嘧啶缺陷型菌株

本课题组从海南天然海域筛选到一株高产类胡萝卜素的海洋红酵母菌株S8,该菌株对鱼无毒害,并与鱼共生,欲将其应用于盐诱导表达外源蛋白的海洋红酵母工程菌的构建。本研究利用紫外诱变筛选的方法处理S8菌株,通过统计其UV致死率、5-氟乳清酸致死率等筛选S8的尿嘧啶营养缺陷型突变株。研究结果表明,供试菌株通过紫外线诱变、5-氟乳清酸致死和回复突变率的实验筛选,共获得16株稳定的尿嘧啶缺陷型突变株,突变菌株在基本培养基中培养了8d仍不能生长。选择了其中的一株ST5进行了产胡萝卜素能力的测定,结果表明,在同样的培养条件下,野生型S8菌株细胞生物产量可达87.55g/L,类胡萝卜素含量可达520μg/g,突变株ST5的细胞生物产量为85.45g/L,类胡萝卜素含量为512μg/g;ST5的产胡萝卜素能力方面与野生型S8无明显差异。因此,尿嘧啶缺陷型菌株ST5可为下一步海洋红酵母工程菌的构建提供受体菌。     

黄化英红九号红茶体外抗氧化活性分析

以黄化英红九号红茶为材料,分析其茶多酚及儿茶素含量,测定其FRAP铁离子还原能力、 DPPH和ABTS自由基清除能力,以揭示黄化英红九号的抗氧化机理。结果表明,黄化英红九号红茶中茶多酚、 EGC、 C、 EC、 EGCG、 GCG和ECG与英红九号有显著差异,其多酚提取物抗氧化活性随质量浓度增大,活性增强。黄化英红九号多酚提取物平均EC50分别为6.01μg/m L(FRAP)和4.63μg/m L(ABTS)极显著低于英红九号(7.13μg/m L和6.03μg/m L), DPPH EC50为3.59μg/m L,与英红九号(3.90μg/m L)没有显著差异。相关性分析表明, FRAP、 DPPH EC50值与茶多酚、 EGC、 C、 EC、 GCG和ECG呈极显著负相关; ABTS EC50值与茶多酚、 EGC相关性达到极显著水平,相关系数分别为-0.621 0和-0.535 3。因此,黄化英红九号红茶多酚提取物具有优良的抗氧化活性。     

重组蛋白H-NS对胸膜肺炎放线杆菌生物被膜形成的影响

在筛选胸膜肺炎放线杆菌(Actinobacills pleuropneumoniae,APP)转座突变体库时,发现hns基因缺失突变可显著增强APP血清l型生物被膜的形成,该基因编码一种DNA结合蛋白(组蛋白样类核结构蛋白,H-NS).为了进一步研究H-NS对APP生物被膜形成的影响,实验以APP血清l型4074株基因组DNA为模板,PCR扩增了408 bp的hns基因编码区,并克隆到原核表达载体pET-28c中获得重组质粒pET28c-hns,转化大肠杆菌(Escherichia coli)BL21(DE3),经IPTG诱导表达和组氨酸亲和层析柱纯化获得大小约19 kD的重组蛋白rH-NS.将不同浓度的rH-NS添加到hns突变株1-21及其亲本菌株4074的培养基中,用微孔板法测定生物被膜的形成.结果显示,在不添加rH-NS时,4074株不能形成可见的生物被膜,而1-21株形成明显的生物被膜;在添加0.1～0.3μmol/L rH-NS的情况下,1-21株生物被膜的形成昔随着rH-NS浓度的升高而降低.而4074株随着rH-NS浓度的升高而升高;rH-NS添加量超过0.4 μmol/L对两个菌株生物被膜形成未见明显影响.结果表明H-NS蛋白负调控APP生物被膜的形成,并呈现一定的剂量效应.     

螺旋藻的分离筛选及培养条件的选择

采用选择性Zarrouk无机培养基从北海螺旋藻养殖场水样中富集和稀释平板分离出9株螺旋藻,经对其生长测定,从中筛选出一株生长较快、藻体粗壮的螺旋藻藻种（暂定名SP06）,通过对其形态学观察、培养基优化和最佳生长条件的选择试验,结果表明：该藻螺旋状,细胞为短圆柱形,藻体长50-600μm,径宽30-70μm,螺旋数3-10,在配方为NaHCO3 16.8g／L、KH2PO4 0.4g／L、NaNO3 2.7g／L、NaCl 1.0g／L、FeSO4 0.005g／L、MgSO4 0.1g／L、K2SO4 1.0g／L、CaCl2 0.04g／L液体培养基中,在起始pH8-10,光照强度4000Lx,30℃／25℃光／暗变温条件下生长良好,培养8d每升养殖水可收获湿藻46-48g。     

有机生活垃圾半干发酵菌群的分布变化特征

为了研究有机生活垃圾厌氧发酵中菌群的分布变化特征,以有机生活垃圾为生物质资源,进行半干式厌氧发酵试验。采用最大或然数法(most probable number,MPN)分析发酵过程中厌氧菌群时间和空间上的数量变化。结果表明:厌氧菌中产酸菌先于氨化菌达到最大值并占据优势地位,产甲烷菌在启动阶段初期基本没有增殖,第25天左右达到最大值3.2×109个/m L,随后产甲烷菌在整个盛产期数量维持在这一数量级上。厌氧纤维素降解菌菌数呈现缓慢增长的趋势,直到投料的第45天才增加到106个/m L。空间上厌氧产酸菌和甲烷菌的数量均是在基质条件稳定的中部位置和流动性较好的底部位置较多;厌氧氨化菌数量较多的为中部边缘和中部中心位置;厌氧纤维素降解菌主要在底部增殖。初步构建了产酸菌与产甲烷菌时间和空间的动力学模型,模型拟合效果良好,可为厌氧发酵工艺提供参考。该文对有机垃圾制取生物燃气的工艺过程具有理论指导意义。     

Low soil C:N ratio results in accumulation and leaching of nitrite and nitrate in agricultural soils under heavy rainfall

Nitrate (NO^-₃) and nitrite (NO₂^-) leaching threatens groundwater quality.Soil C:N ratio,i.e.,the ratio of soil organic carbon to total nitrogen,affects mineralization,nitrification,and denitrification;however,its mechanism for driving soil NO^-₃ and NO^-₂ accumulation and leaching remains unclear.Here,a field investigation in a fluvo-aquic soil and a soil column experiment were performed to explore the relation...     

Plant growth-promoting bacteria in the rhizo- and endosphere of plants: Their role, colonization, mechanisms involved and prospects for utilization

In both managed and natural ecosystems, beneficial plant-associated bacteria play a key role in supporting and/or increasing plant health and growth. Plant growth-promoting bacteria (PGPB) can be applied in agricultural production or for the phytoremediation of pollutants. However, because of their capacity to confer plant beneficial effects, efficient colonization of the plant environment is of utmost importance. The majority of plant-associated bacteria derives from the soil environment. They may migrate to the rhizosphere and subsequently the rhizoplane of their hosts before they are able to show beneficial effects. Some rhizoplane colonizing bacteria can also penetrate plant roots, and some strains may move to aerial plant parts, with a decreasing bacterial density in comparison to rhizosphere or root colonizing populations. A better understanding on colonization processes has been obtained mostly by microscopic visualisation as well as by analysing the characteristics of mutants carrying disfunctional genes potentially involved in colonization. In this review we describe the individual steps of plant colonization and survey the known mechanisms responsible for rhizosphere and endophytic competence. The understanding of colonization processes is important to better predict how bacteria interact with plants and whether they are likely to establish themselves in the plant environment after field application as biofertilisers or biocontrol agents.     

冷鲜鸡肉表面四环素和磺胺耐药菌的菌群多样性分析

为探索冷鲜鸡肉产品表面的抗生素耐药菌的菌群结构,利用IonS5TMXL测序平台对18个市售冷鲜鸡肉样品表面的可培养四环素耐药菌和磺胺耐药菌进行了研究。结果表明,2类耐药菌中分别注释出59个和58个已明确属名的属,相对丰度最大的3个门均为变形菌门、拟杆菌门和厚壁菌门。共享菌属中不动杆菌属、假单胞菌属、变形杆菌属、柠檬酸杆菌属、香味菌属和漫游球菌属的禽源和人源分离株的多重耐药性已被大量研究所证实;而禽源肉杆菌属等16个菌属的耐药特性还未见报道。各采样点分别鉴定出5～39个特有操作分类单元,分属3～32个属。该研究反映了冷鲜鸡肉表面2类耐药菌的污染状况,为后期冷鲜鸡肉产品表面耐药菌的迁移风险评估和控制技术研究提供参考。     

稻田固氮解磷解钾菌筛选及其复合菌剂对土壤培肥作用

用固氮、解磷、解钾培养基,分别从稻田土壤中分离获得9个固氮菌株(A1-A9)、7个解磷菌株(B1-B7)和6个解钾菌株(C1-C6)。对各菌株固氮、解磷、解钾能力及水稻促生效果分析表明,最突出菌株分别为A5、B2、C5;各菌株等比例混合组成复合菌剂接入灭菌稻田土壤,30 d后可使土壤总氮、有效磷、速效钾分别增加21.32%、32.17%、45.57%;初步鉴定各菌株分别为固氮菌属(Azotobacter sp.)、假单胞菌属(Pseudomonas sp.)和芽孢杆菌属(Bacillus sp.)。复合菌剂处理稻田土壤,水稻苗高、鲜重、干重及植株全氮、全磷、全钾显著优于对照组,说明筛选菌株可在稻田土壤氮、磷、钾转换及培肥过程中起重要作用。     

兼具固氮、解磷功能菌株固氮特性的研究

试验研究 9株兼具固N、解P功能菌株在无N、N源充足不同P源条件和混合培养时菌株固N特性结果表明 ,供试菌株在无N培养基上均有固N活性 ,但固N量差异较大 ,菌株“N5 1”固N量最高达 5 5 .4mg/kg ,为固N量最低菌株“K10 2”的 3.1倍。在以Ca3 (PO4) 2 作P源、培养液N素供应充足时“K10 2”和“N17”菌株仍有较高固N能力 ,培养 3d后培养液全N含量增幅达 5 5 .2 % ;“N11”、“N12”和“P14 2”菌株固N量也维持在较高水平 ,全N含量增加30 %左右 ;“K16”菌株培养液全N含量略有增加 ,“N5 1”、“N13 1”和“K3”菌株则导致N的挥发损失。以磷矿粉作P源可使该过程逆转。在Ca3 (PO4) 2 P源培养基上混合培养能有效降低“N5 1”、“N13 1”和“K3”菌株单独培养时所造成的N损失。     

Copper and temperature modify microbial communities,ammonium and sulfate release in soil

Recent studies suggest an important role of thermophilic bacterial communities of the Phylum Firmicutes on soil C, N and S cycling, and a positive effect on crop productivity through the production of sulfate (SO $ _4^{2 - } $ ) and ammonium (NH $ _4^+ $ ), essential plant nutrients. Copper (Cu) is commonly supplemented to soils as a fungicide in phytosanitary treatments although its consequences to the bacterial communities is frequently overlooked. Herein, we report on the influence of temperature and Cu on the microbial communities, namely those of the Phylum Firmicutes, from a soil collected at an olive orchard in S Portugal. Community fingerprints and band identification through sequencing was combined with measurement of SO $ _4^{2 - } $ and NH $ _4^+ $ production at different supplemented amounts of Cu and at moderate and high temperatures (30°C and 50°C, respectively). Both temperature and Cu induced changes in these communities, selecting for specific bacteria. Temperature induced the dominance of Brevibacillus, and Cu addition to soil caused a reduction of SO $ _4^{2 - } $ release by soil bacteria. Ammonium production during bacterial growth at moderate and high temperatures was not affected by Cu addition. A Cu‐tolerant thermophilic isolate, belonging to the Bacillus genus, showed significant inhibition by high Cu concentrations and a reduction of NH $ _4^+ $ release during growth; genera Brevibacillus and Bacillus have been previously reported as high NH $ _4^+ $ and SO $ _4^{2 - } $ producers of the Firmicutes phylum. Results indicate that Cu treatments select specific tolerant bacterial strains which could influence natural soil fertilization in Cu‐treated orchards.     

沼泽红假单胞菌与枯草芽孢杆菌混施对水稻根域细菌多样性与功能的影响

[目的]微生物菌肥作为一种绿色环保的生物肥料,可以改善土壤质量,促进作物生长.以水稻根际和根表微生物为对象,探究不同微生物菌肥单施和混施对其群落多样性和功能的影响.[方法]采用盆栽试验,设单施沼泽红假单胞菌(Rhodopseudomonas palustris)、单施枯草芽孢杆菌(Bacillus subtilis)及...     

Characterization of nitrifying bacteria communities of soils from different ecological regions of China by molecular and conventional methods

Soil nitrification rate is very different among soil types, as a result of differences in physical and chemical properties. Little is known about the composition of the nitrifying bacteria community. In this investigation, three soils (fluvo-aquic soil, permeable paddy soil and red earth) from different geo-ecological regions in China were characterized for their nitrification activities and their nitrifying bacteria communities determined either by molecular approaches or by conventional culture methods. A 28-day long-term soil incubation showed that the maximum nitrification potential was found in the fluvo-aquic soil with almost 100% of inorganic N present as NO₃^–-N, while the minimum nitrification potential was in red earth with only a 4.9% conversion rate from ammonium into nitrate. There was no relationship between nitrification potential and numbers of nitrifiers in the soil. The conventional most probable number (MPN) method could enumerate ammonia oxidizers, but failed in enumerating nitrite oxidizers. Therefore, we used an MPN-PCR procedure which gave a convincing nitrite oxidizer count result, instead of MPN-diphylamine. Soils were characterized by denaturing gradient gel electrophoresis (DGGE) of DNA extracted from soils and amplified using a primer specific for the 16S rRNA gene and/or for the amoA gene. The DGGE columns of the three soils differed from each other. There were two similar bands present in DGGE columns of the fluvo-aquic and permeable paddy soils, but no similar band was found in DGGE columns of the red earth. The sequence of amoA indicated that all ammonia oxidizers in these soils were grouped into Nitrosospira clusters 1 and 3, and each soil had a common band similar to the other soils and a special band which differed from the other soils.     

Interactions of Azospirillum spp. in soils: a review

 This review summarizes and discusses the current knowledge and the, as yet, unanswered questions on the interactions of Azospirillum spp. in bulk soil (but not in the rhizosphere). It contains sections on the isolation of these bacteria from tropical to temperate soils, and on their short- and long-term persistence in bulk soil. The interactions of these bacteria with soil particles and minerals such as clay, sand and Ca, and the effect of soil pH, soil redox potential, and the cation exchange capacity of the soil on them is demonstrated. Data is presented on the distribution of Azospirillum spp. in soils, on their production of fibrillar material essential for anchoring the cells to soil particles, on the effects of soil irrigation, and of external soil treatments, and on the effect of soil C and C used in bacterial inoculants on the cells. It shows that root exudates possibly govern bacterial motility in the soil. Finally, the effect of pesticide applications, the relationships with other soil microorganisms such as Bdelovibrio spp., Bradyrhizobium spp., and phages, and the potential use of a community-control model of Azospirillum spp. in soil and in the rhizosphere is suggested. Received: 11 November 1998