二种淡水微囊藻rDNA16S-235基因间隔区的序列测定与分析

本研究采用PCR及序列测定的方法,对我国淡水铜绿微囊藻有毒株(M8641)和另一低毒的种类惠氏微囊藻(M574)rDNA16S-23S基因间隔区进行了序列的测定和分析,结果表明:rDNA16S-23S基因间隔区可以作为一个精细且稳定的指标,用于微囊藻的分类和鉴定。并从分子水平提出了铜绿微囊藻与惠氏微囊藻在种系发生上有较近缘的关系。本文首次对微囊藻属Microcystis rDNA基因间隔区全序列作了报道,为微囊藻属的鉴定及系统学研究提供了分子基础。       

CO_2浓度升高对水体硝化、反硝化作用的影响研究进展

大气CO_2浓度升高潜移默化地影响着水体生态系统的碳循环过程.然而,该过程如何影响与其耦合的氮循环过程仍不明确.水体硝化、反硝化过程作为水体氮循环的重要环节,必然会对大气CO_2浓度升高产生一系列的响应.本文总结了国内外关于大气CO_2浓度升高对水体理化性质、硝化作用、反硝化作用及N形态转化影响方面的研究工作,发现大气CO_2浓度升高会降低水体的p H,增加水中CO_2和HCO_3^-含量,但对富营养化与寡营养化水体中硝化、反硝化作用的影响具有明显差异.大气CO_2浓度升高抑制寡营养化水体的硝化作用和反硝化作用,降低N2_O的释放通量,抑制富营养化水体的硝化作用,但当水体pH在7～9时,可能促进反硝化作用,增加N2_O的释放通量,最终可能导致水体中NH_4^+的积累及NO_3^-浓度的降低,影响水体中微生物的多样性.在此基础上提出目前相关研究存在的瓶颈问题及值得深入探讨的科学问题,为进一步深入理解温室效应背景下全球CO_2浓度升高对水体生态系统N循环的影响提供参考.     

CO2浓度升高对水体硝化、反硝化作用的影响研究进展

大气CO₂浓度升高潜移默化地影响着水体生态系统的碳循环过程.然而,该过程如何影响与其耦合的氮循环过程仍不明确.水体硝化、反硝化过程作为水体氮循环的重要环节,必然会对大气CO₂浓度升高产生一系列的响应.本文总结了国内外关于大气CO₂浓度升高对水体理化性质、硝化作用、反硝化作用及N形态转化影响方面的研究工作,发现大气CO₂浓度升高会降低水体的pH,增加水中CO₂和HCO₃^-含量,但对富营养化与寡营养化水体中硝化、反硝化作用的影响具有明显差异.大气CO₂浓度升高抑制寡营养化水体的硝化作用和反硝化作用,降低N₂O的释放通量,抑制富营养化水体的硝化作用,但当水体pH在7~9时,可能促进反硝化作用,增加N₂O的释放通量,最终可能导致水体中NH₄⁺的积累及NO₃^-浓度的降低,影响水体中微生物的多样性.在此基础上提出目前相关研究存在的瓶颈问题及值得深入探讨的科学问题,为进一步深入理解温室效应背景下全球CO₂浓度升高对水体生态系统N循环的影响提供参考.     

不同氮源下好氧反硝化菌Defluvibacter lusatiensis str.DN7的脱氮特性

研究了不同氮源下好氧反硝化菌Defluvibacter lusatiensis str.DN7的脱氮特性。结果表明:菌株均能以硝酸盐和亚硝酸盐为唯一氮源进行好氧反硝化作用。反应4 h,NO-3-N和NO-2-N的去除率分别达83.35%和85.72%。亚硝酸盐完全还原比硝酸盐提前42 h。硝酸盐还原过程中基本无亚硝酸盐积累,而亚硝酸盐还原过程中则检测到明显的硝酸盐积累,反应4 h,NO-3-N积累量达到21.83 mg/L。培养液中同时存在硝酸盐和亚硝酸盐时,菌株优先选择硝酸盐作电子受体。亚硝酸盐共存对硝酸盐还原无显著影响,但培养液中残留的NO-2-N随亚硝酸盐比例上升而增加,当亚硝酸盐比例从10%升至50%时,NO-2-N残留量由3.38 mg/L增至7.60 mg/L。少量硝酸盐的加入对亚硝酸盐的还原产生抑制作用。当硝酸盐比例为10%时,72 h NO-2-N的去除率仅为74.79%,远低于以亚硝酸盐为唯一氮源情况(去除率100%)。以氨氮为唯一氮源时,菌株同时进行异养硝化和好氧反硝化反应,72 h,NH+4-N去除率达85.66%,且基本无硝酸盐或亚硝酸盐积累。少量氨氮共存(氨氮比例<30%)有利于促进菌株的好氧反硝化作用,反之亦然。     

氮浓度对铜绿微囊藻、大型溞和金鱼藻三者相互作用的影响

为了解氮浓度对生物操纵和草-藻竞争的影响, 选取铜绿微囊藻、大型溞和金鱼藻分别作为浮游植物、浮游动物和沉水植物的代表, 在温度25℃, 光强2600 lx, 光暗比14h﹕10h, 磷浓度1.5 mg/L时, 研究5种氮浓度(0.5、2、4、8和16 mg/L, 用KNO₃溶液配制)下, 溞-藻, 草-藻和溞-草-藻共培养时各自的增长率和培养液中氮磷削减率的变化。结果表明: 在单独培养铜绿微囊藻时, 氮浓度控制在1.97 mg/L以下, 可有效降低培养液中藻的增长率。在溞-藻共培养时, 大型溞有效控藻的氮浓度范围为0.5—4 mg/L; 在草-藻共培养时, 有效控藻的氮浓度范围为0.5—2 mg/L, 对应氮浓度下(0.5和2 mg/L), 实验末期铜绿微囊藻细胞密度分别是溞-藻共培养的23.89%和21.51%, 控藻效果更好; 在溞-草-藻三者共培养时, 有效控藻的氮浓度范围为0.5—16 mg/L, 且氮浓度为0.5—4 mg/L时, 大型溞和金鱼藻的增长率均显著大于铜绿微囊藻, 铜绿微囊藻的增长率均为负值, 控藻效果最好。大型沉水植物的加入, 可以有效提高生物操纵的控藻效果, 减少水中氮磷含量, 长期有效地改善水质。     

低强度超声波抑制铜绿微囊藻生长的研究

铜绿微囊藻(Microcystis aeruginosa)是常见的水华蓝藻,会对湖库的生态环境造成严重危害.室内研究了低强度超声波在不同藻生长时相、藻细胞浓度、水体pH、水温和二次超声条件下对铜绿微囊藻的抑制效果.结果表明:藻细胞浓度和pH对超声抑藻效果无明显影响,藻生长时相、水温和超声次数对超声抑藻效果有明显影响.15℃、20℃和25℃时的超声抑藻效果好于30℃和35℃,不经超声作用40℃高温即已不利于藻细胞生长;低强度超声对延滞期、稳定期和衰退期铜绿微囊藻抑制效果好于指数生长期铜绿微囊藻;二次超声可有效延长残余藻细胞的恢复时间,稳定抑藻效果.     

不同氮、磷浓度对铜绿微囊藻生长、光合及产毒的影响

对一株从野外分离得到的铜绿微囊藻产毒株进行分批培养,在不同的氮磷条件下研究其生长、光合荧光及毒素含量的变化。结果表明:正磷酸盐浓度不变时,铵氮浓度的改变对铜绿微囊藻的生长有明显影响。叶绿素a(Chl.a)含量在铵氮浓度为1.83-18.3mg/L时明显较大;微囊藻毒素(包括MC-LR和MC-RR)的含量在铵氮浓度为1.83mg/L时达到最大;当铵氮浓度为0-1.83mg/L时,随着铵氮浓度升高,可变荧光FV和MC的产量均增大,同时MC异构体的种类增多;铵氮浓度过大对M.aeruginosa的生长、生理和产毒均有抑制作用。在另一组实验中,即铵氮浓度不变而正磷酸盐浓度增大时,Chl.a含量呈总体下降的趋势,并且与FV/Fm呈显著正相关关系(P<0.01,r=0.97),MC(MC-LR和MC-RR)的含量在正磷酸盐浓度小于0.56mg/L时明显升高,MC-LR与FV/Fm呈显著正相关关系(P<0.01,r=0.967)。       

一株耐氧反硝化细菌的筛选及脱氮特性研究

从鱼塘中分离到1株高效的有氧反硝化菌,经初步鉴定,为芽孢杆菌。在溶解氧(OD)达到2mg/L时,除氮率达97%,OD达到4～5mg/L,仍有一定的反硝化作用,除氮率为85%以上。与典型的好氧反硝化菌Pseudomonasstutzeri[1]相比,有更强的耐溶解氧的优势。同时初步探讨了水体中不同溶解氧、碳源、pH、温度对该芽孢杆菌W2菌株反硝化作用的影响,水体中存在一定量有机碳源有利于反硝化,当以葡萄糖为碳源,pH为7.0～7.5,温度为32℃时,W2菌株具有最佳的降解人工废水反硝化能力。实验结果表明,在好氧条件下,菌体浓度为1000个/mL时,对自然水体中高达1mg/L亚硝酸浓度也能发挥高效的反硝化作用。     

不同外源条件对4种白腐真菌溶藻效果的影响

【目的】评价白腐真菌Irpex lacteus XX-5、Trichaptum abietinum 1302BG、Ceriporia lacerata P2、Bjerkandera adusta XX-2处理铜绿微囊藻废水的应用潜力。【方法】采用分批次实验研究pH、温度、铜绿微囊藻浓度、金属离子、氮源、磷源对白腐真菌I. lacteus XX-5、T. abietinum 1302BG、C. lacerata P2、B. adusta XX-2溶解铜绿微囊藻的影响。【结果】在不同外源条件下,4种白腐真菌对铜绿微囊藻的抑制效果明显,均达60%以上。菌株C. lacerata P2和B. adusta XX-2受外源条件的影响很小,菌株C. lacerata P2的抑制率达70%以上,菌株B. adusta XX-2的抑制率达60%以上;菌株T. abietinum 1302BG、I. lacteus XX-5在不同外源条件下抑制率均会发生相应的变化,但抑藻率均可达60%以上。【结论】研究所使用的4种白腐真菌对抑制铜绿微囊藻具有较好的应用潜力,尤其是菌株C. lacerata P2和B. adusta XX-2。     

三株铜绿微囊藻对外源无机碳利用的研究

本文研究了三株铜绿微囊藻在不同条件下光合放氧对外源溶解无机碳的响应。当温度从20℃上升到30℃时,三株铜绿微囊藻的光合放氧速率都显著增加。随着反应介质pH值从7．0上升到9．0,铜绿微囊藻的光合放氧逐渐增强,其最大光合放氧速率显著增大。胞外碳酸酐酶抑制剂乙酰唑胺（acetazolamide,AZ）对铜绿微囊藻的光合放氧无显著影响,而碳酸酐酶抑制剂乙氧苯丙噻唑磺胺（ethoxyzolamide,EZ）则抑制其光合放氧。通过酶动力学方程拟合,得出这三株铜绿微囊藻的表观无机碳亲和力常数K0.5（DIC）均低于50Lmol／L,显示出铜绿微囊藻对外源无机碳的亲和力较高。在20-30℃范围内,温度的变化对微囊藻无机碳亲和力无显著影响,表明环境温度不影响其对外源无机碳利用的能力。当反应介质pH值升高,铜绿微囊藻FACHB905和PCC7806的K0.5（DIC）增大,而铜绿微囊藻FACHB469的K0.5（DIC）值却减小。在加入AZ和EZ后铜绿微囊藻的K0.5（DIC）值均无显著变化。本文还比较了铜绿微囊藻与莱氏衣藻在介质pH值变化和加入碳酸酐酶抑制剂条件下光合放氧的不同响应。结果表明,莱氏衣藻的光合放氧随反应介质pH值的升高而减弱,而AZ和EZ均抑制莱氏衣藻的光合放氧。当反应介质pH值升高以及加入AZ或EZ后,莱氏衣藻的K0.5（DIC）值均减小。     

一株海水异养硝化-好氧反硝化菌系统发育及脱氮特性

【目的】确定一株分离自海水的异养硝化-好氧反硝化菌的系统发育地位并探索其脱氮特性和机理,以期为解释异养硝化-好氧反硝化机理以及改进海水养殖及废水的生物脱氮工艺提供理论依据。【方法】通过形态观察、生理生化实验和16S rRNA基因序列分析,鉴定该菌株;通过测定菌株在不同无机氮源降解测试液中的生长和脱氮效率,分析其异养硝化和好氧反硝化性能。【结果】经鉴定该菌株属于盐单胞菌属(Halomonas);最适生长条件为盐度3%、pH 8.5、温度28℃、碳氮比10:1,在盐度为15%的培养液中仍能生长;可以同时去除氨氮、亚硝酸氮和硝酸氮,24 h时对NH4+-N、NO2--N、和NO3--N的去除率可分别达到98.29%、99.07%、96.48%,3种形态无机氮同时存在时,会优先利用NH4+-N,且总无机氮去除率较单一存在时更高,说明该菌株可实现同步硝化反硝化。【结论】该分离自海水的异养硝化-好氧反硝化菌属于盐单胞菌属(Halomonas),在高盐环境中仍能生长,同时具有高效的异养硝化和好氧反硝化能力,能够独立完成脱氮的全部过程。     

The effect of oxygen on denitrification in Paracoccus denitrificans and Pseudomonas aeruginosa

Denitrification by Paracoccus denitrificans and Pseudomonas aeruginosa was studied using quadrupole membrane-inlet mass spectrometry to measure simultaneously and continuously dissolved gases. Evidence was provided for aerobic denitrification by both species: in the presence of O2, N2O production increased in Pa. denitrificans, while that of N2 decreased; with Ps. aeruginosa, the concentrations of both N2 and N2O increased on introducing O2 into the gas phase. Disappearance of NO-3 was monitored in anaerobically and aerobically grown cells which were maintained either anaerobically or aerobically: the rate and extent of NO-3 utilization by both species depended on growth and maintenance conditions. The initial rate of disappearance was most rapid under completely anaerobic conditions, and lowest rates occurred when cells were grown anaerobically and maintained aerobically. In nitrogen balance experiments both species converted over 87% of the added NO-3 to N2 and N2O under both anaerobic and aerobic maintenance conditions.     

Fixation of dinitrogen derived from denitrification of nitrate in a photosynthetic bacterium, Rhodopseudomonas sphaeroides forma sp. denitrificans.

Studies with 15N demonstrated that the phototrophic bacterium Rhodopseudomonas sphaeroides forma sp. denitrificans strain IL106 cannot assimilate NO-3 but rather denitrifies it to N2. This strain also fixed N2 into cell protein, although nitrogenase activity was partially inhibited in the presence of NO-3. Strain IL106 did not assimilate NO-3, but growing cultures and washed cell suspensions incorporated the tracer from 15NO-3 via denitrification to 15N2 and then via nitrogenase into cell nitrogen. This incorporation was inhibited in cells supplied with NH4+ or in the absence of light, thus confirming the participation of nitrogenase in the assimilation of nitrogen from nitrate. This represents a novel type of N2 recycling in a photodiazotrophic denitrifying bacterium.     

Denitrification of high concentrations of nitrites and nitrates in synthetic medium with different sources of organic carbon. III. Methanol

The denitrification of nitrites and nitrates (1000 mg N/l) in medium containing methanol as a source of organic carbon was studied. Continuous cultures of mixed population of autochtonic microflora from bottom sludge of nitrogenous wastewater reservoir were set up in a chemostat-type column and packed bed reactor. The efficiency of denitrification of nitrates in packed bed reactor was 506.7 mg N/l/h whereas denitrification of nitrites was from 8.7 to 16.0 mg N/l/h depending on the granulation of the filing material. In the latter case 83% nitrogen was removed from the medium. One of the factors causing low efficiency of denitrification of nitrites is excessive alkalization of the medium in the bed. The use of a three-step bed with adjusted pH resulted in complete denitrification of nitrites with efficiency 60 mg N/l/h. The bacteria inside the bed were dominated by Paracoccus denitrificans and by Pseudomonas aeruginosa when nitrates were present. The sensitivity of P. denitrificans to high concentrations of nitrites seems to be the second factor contributing to low efficiency of denitrification with methanol as organic substrate.     

不同氮效率水稻全生育期内对增硝营养的响应及其生理机制

通过添加硝化抑制剂（二氰胺,DCD）来控制硝化作用的水培试验方法,研究了氮高效水稻品种南光和氮低效水稻品种ELIO的籽粒产量对增硝营养（NH4＋∶NO3-比例为100∶0和75∶25）的响应,同时从产量构成、不同生育时期水稻生长、氮素吸收和同化4个方面研究了造成其产量差异的生理机制。结果表明：增NO3-营养可以显著促进氮高效水稻品种南光的生长,从而使其籽粒产量水平提高21%,而对氮低效水稻品种ELIO的籽粒产量没有显著影响。进一步分析表明：在增NO3-营养条件下,南光的穗粒数增加了25%,结实率增加了16%,而氮低效水稻品种ELIO的结实率和穗粒数在两种营养条件下没有显著变化;增NO3-营养可以促进南光对氮素的吸收,使其在苗期、分蘖盛期、齐穗期和成熟期对氮素的吸收量平均增加了36%,进而促进了其生长,干物质积累量在四个生育时期平均增加了30%;南光叶片硝酸还原酶和根系谷氨酰胺合成酶的活力在增硝营养条件下分别增加了100%和95%,说明增硝营养促进了南光对NH4＋和NO3-的同化利用。与氮低效水稻品种（ELIO）相比,氮高效水稻品种（南光）对增硝营养表现出较强的生理响应。     

重要环境因子对小球藻去除污水中氮磷的影响

对小球藻去除污水中氮磷的性能进行了研究,考察了初始氮磷浓度、氮磷比、光照条件和pH等因素对其去除效率的影响。结果表明,在初始氮磷浓度分别在35 mg/L和7 mg/L以下时去除率接近100%;氮磷比为5∶1和10∶1,小球藻第4 d基本完全去除了污水中的氨态氮,而氮磷比对小球藻的除磷能力没有显著影响;在初始氨态氮或总磷浓度相同的条件下,光照条件(L/D为24 h∶0 h和12 h∶12 h)对氮磷去除效果的无明显差异性（P＞0.05）,而随着氮磷浓度的增加,连续光照条件逐渐展现出去除氮磷的优势;小球藻在pH 7～8范围内对氨态氮的去除率最佳,pH 5～7范围内,对总磷的去除率最佳。     

Effect of reduced concentrations of carbon, nitrogen, and phosphorus in a medium on growth of three dissociants of Pseudomonas aeruginosa]

The effect of lowered concentrations of carbon, nitrogen, and phosphorus sources in the medium on the specific growth rate mu of the R, S, and M dissociants of the hydrocarbon-oxidizing strain Pseudomonas aeruginosa K-2, culture pH, and the population composition was studied. Within the first 16 hours of cultivation in all of the four media tested, the R, S, and M dissociants had virtually identical mu. The maximal values of mu were reached by the 20th h of growth in the basal medium (R and S dissociants) and in the carbon-deficient medium containing 0.4% glucose (M dissociant). The R and M dissociants showed the most rapid decrease in mu in the nitrogen-deficient medium containing 0.55% NaNO3. By the end of cultivation in the basal medium, the pH of the R, S, and M cultures decreased to 6.3, 5.3, and 3.3, respectively. In the case of the carbon-deficient medium, the drop in the culture pH was lower. After a 2.5-day incubation of the S dissociant in the phosphorus-deficient medium containing 0.028% NaH2PO4.2H2O and of the M dissociant in the basal medium supplemented with chalk powder, these dissociants were completely displaced from the media.     

Effect of various sources of organic carbon and high nitrite and nitrate concentrations on the selection of denitrifying bacteria. I. Stationary cultures

The effect of methanol, ethanol, acetic acid and glucose together with NaNO2 or KNO3 (1,000 mg N/l) on the intensity of denitrification and selection of denitrifying bacteria from the bottom sludge of nitrogenous wastewater reservoir was examined. Denitrification was found to be the most efficient in medium with ethanol or acetic acid. The presence of glucose facilitated the selection of Alcaligenes faecalis whereas the other carbon sources enabled the selection of bacteria of the genus Pseudomonas: methanol -- P. fluorescens, ethanol -- P. mendocina. In medium with acetic acid species selection depends on the form of nitrogen: NaNO2 -- P. fluorescens, KNO3 -- P. aeruginosa.     

Isotope labeling studies on the mechanism of N-N bond formation in denitrification

The mechanism of the denitrification and nitrosation reactions catalyzed by the heme cd-containing nitrite reductase from Pseudomonas stutzeri JM 300 has been studied with whole cell suspensions using H2(18)O, 15NO, and 15NO-2. The extent of H2(18)O exchange with the enzyme-bound nitrosyl intermediate, as determined by the 18O content of product N2O, decreased with increasing nitrite concentration, which is consistent with production of N2O by sequential reaction of two nitrite ions with the enzyme. Reaction of NO with whole cells in H2(18)O gave amounts of 18O in the N2O product consistent with equilibration of nitric oxide with a small pool of free nitrite. Using 15NO and NH2OH, competition between denitrification and nitrosation reactions was demonstrated, as is required if the enzyme-nitrosyl complex is an intermediate in both nitrosation and denitrification reactions. The first evidence for exchange of 18O between H2(18)O and a nitrosation intermediate occurring after the enzyme-nitrosyl complex, presumably an enzyme-bound nitrosamine, has been obtained. The collective results are most consistent with denitrification N2O originating via attack of NO-2 on a coordinated nitrosyl, as proposed earlier (Averill, B. A., and Tiedje, J. M. (1982) FEBS Lett. 138, 8-11).     

Standard electromotive force of the H2---AgCl;Ag cell in 30, 40, and 50 mass% dimethyl sulfoxide/water from −20 to 25 °C: pK2 and pH values for a standard “Bicine” buffer solution at subzero temperatures

The establishment of the pH (designated pH*) of a standard buffer solution suitable as a pH reference in 30, 40, and 50 mass% dimethyl sulfoxide (DMSO)/H₂O mixtures at temperatures in the range −20 to 0 °C is reported. The buffer material selected was the ampholyte Bicine (N,N-bis(2-hydroxyethyl)glycine), and the reference standard consists of equal molal quantities of Bicine and its sodium salt. The assignment of pH* values rests on measurements of the emf of cells without liquid junction, Pt;H₂(g, 1 atm) ¦Bicine, Na Bicinate, NaCl ¦AgCl;Ag, and the pH* was derived from a determination of K₂, the equilibrium constant for the dissociation process (Bicine) ^± (Bicinate)⁻ + H⁺. The standard emf in the DMSO/H₂O solvents at subzero temperatures was determined from emf measurements of the cell with solutions of HCl replacing the buffer-chloride mixture.