蓝藻群体颗粒驱动元素地球化学循环研究进展

在天然淡水和半咸水水体中,水华蓝藻常以群体颗粒的形态存在。在蓝藻群体颗粒中聚集着大量异养细菌,和蓝藻共同构成了具有独特生态功能的基本单元。与蓝藻单体细胞相比,蓝藻群体颗粒呈现出许多独有的特性,如内部丰富的有机质、急剧的氧化还原梯度、密切的种间互作关系等等。这些特质使得蓝藻群体颗粒在水体中成为元素地球化学循环的反应热点。同时,在蓝藻群体颗粒中也存在着远比单细胞藻类-浮游细菌之间更为密切的种间互作。本综述围绕蓝藻群体颗粒的这些特点,结合当前的研究进展,重点阐述蓝藻群体颗粒中的生物、生理、化学过程,讨论其驱动宏观生态现象的微观机制。未来蓝藻群体颗粒组学研究和多组学微生态数据库的构建或成为探索蓝藻群体颗粒中生命过程及揭示蓝藻水华暴发机制的突破口之一。     

A cyanobacterial recombination study, involving an efficient N2-fixing non-heterocystous partner

Many filamentous cyanobacteria fix atmospheric nitrogen under natural conditions in specialized anaerobic compartments, heterocysts, interspersed between vegetative cells, which provide protection to the O2-sensitive nitrogenase. A few unicellular cyanobacterial strains are also known to fix nitrogen aerobically at a slower rate. Filamentous cyanobacteria lacking heterocysts are not known so far to fix nitrogen. We describe the isolation and purification of a non-heterocystous filamentous cyanobacterium from the fronds of the water-fern Azolla, fixing nitrogen at 18.7+/-0.2 n moles ethylene microg Chl. a(-1) h(-1) when grown in nitrogen-free medium at a low level of oxygen between two layers of agar. This strain of Anabaena azollae has been designated as het- nif+ (non-heterocystous and nitrogen-fixing), and is found to be easily and effectively preserved in nitrogen-free medium in standard synthetic cyanobacterial nutrient medium (pH 8.5) at a continuous light intensity of 2800 lx at 25+/-1 degrees C. This het- nif+ strain is an effective donor of the nif+ marker to a het+ nif- strain of another cyanobacterium, Nostoc muscorum, when both are grown together in a recombination study.     

The nitrogen physiology of the marine N2-fixing cyanobacteria Trichodesmium spp

Trichodesmium spp. have proved to be enigmatic organisms, and their ecology and physiology are unusual among diazotrophs. Recent research shows that they can simultaneously fix N2 and take up combined nitrogen. The co-occurrence of these two processes is thought to be incompatible, but they could be obligatory in Trichodesmium spp. if only a small fraction of cells within a colony or along a filament are capable of N2 fixation. Combined nitrogen is released from cells during periods of active growth and N2 fixation, and concomitantly taken up by Trichodesmium spp. or cells living in association with colonies. Although the nitrogenase of Trichodesmium spp. is affected by high concentrations of combined nitrogen, it might be relatively less sensitive to low concentrations of combined nitrogen typical of the oligotrophic ocean and culture conditions. Nitrogenase activity and synthesis exhibits an endogenous rhythm in Trichodesmium spp. cultures, which is affected by the addition of nitrogen.     

Ecological factors and adaptive processes in N2-fixing bacterial populations of the plant environment

Summary Physiology and genetics of non-symbiotic N₂-fixing bacteria have made much progress in recent years, especially in the case of a few reference strains. Nevertheless, understanding the ecology of diazotrophs cannot be achieved by studying only laboratory microorganisms. It is necessary to study naturally-occurring populations, to characterize their densities, size, composition, variability and variations in order to understand how a plant can select a rhizosphere population from a soil population. Very few comparisons of phenotypic diversity and dominant phenotypes in these two habitats have been made up to now. More studies of this type would allow a better knowledge of the selective pressures which actually drive the shift of population and they would permit investigation of the underlying mechanisms. These can vary from mere metabolic adaptation to selection of pre-adapted genotypes. A third mechanism is possible in which pre-adapted genes are maintained in soil populations at very low frequencies and energy costs, and whose transfer is triggered by the selective factor to which they constitute an adaptation.     

Natural 15N abundance of presumed N2-fixing and non-N2-fixing plants from selected ecosystems

Summary The ¹⁵N/¹⁴N ratios of plant and soil samples from Northern California ecosystems were determined by mass spectrometry. The ¹⁵N abundance of 176 plant foliar samples averaged 0.0008 atom % ¹⁵N excess relative to atmospheric N₂ and ranged from-0.0028 to 0.0064 atom % ¹⁵N excess relative to atmospheric N₂. Foliage from reported N₂-fixing species had significantly lower mean ¹⁵N abundance (relative to atmospheric N₂ and total soil N) and significantly higher N concentration (% N dry wt.) than did presumed non-N₂-fixing plants growing on the same sites. The mean difference between N₂-fixing species and other plants was 0.0007 atom % ¹⁵N. N₂-fixing species had lower ¹⁵N abundance than the other plants on most sites examined despite large differences between sites in vegetation, soil, and climate. The mean ¹⁵N abundance of N₂-fixing plants varied little between sites and was close to that of atmospheric N₂. The ¹⁵N abundance of presumed non-N₂-fixing species was highest at coastal sites and may reflect an input of marine spray N having relatively high ¹⁵N abundance. The ¹⁵N abundance of N₂-fixing species was not related to growth form but was for other plants. Annual herbaceous plants had highest ¹⁵N abundance followed in decreasing order by perennial herbs, shrubs, and trees. Several terrestrial ferns (Pteridaceae) had ¹⁵N abundances comparable to N₂-fixing legumes suggesting N₂-fixation by these ferns. On sites where the ¹⁵N abundance of soil N differs from that of the atmosphere, N₂-fixing plants can be identified by the natural ¹⁵N abundance of their foliage. This approach can be useful in detecting and perhaps measuring N₂-fixation on sites where direct recovery of nodules is not possible.     

Estimation of N2 fixation based on differences in the natural abundance of 15N among freshwater N2-fixing and non-N2-fixing algae

The hypothesis that small mammal burrows can increase the amount of water infiltrating into the soil profile was tested. The amount of water added to the soil profile from spring recharge in areas adjacent to ground squirrel (Spermophilus townsendii and S. elegans) burrows was compared to nearby areas without burrows. Recharge amounts in burrow areas were significantly higher than nonburrow areas. An average of 21% more of the winter precipitation infiltrated into the soil near burrows. The amount of recharge was also found to be positively related to burrow density. Burrows also affected the distribution of the recharge by adding significantly more water to the deeper portions (>50 cm) of the soil profile.     

New, man-made N2-fixing systems

The major inputs of fixed N into the global nitrogen cycle are assessed and compared as indicators of both the need for and the likely basis of new, complementary, man-made N2-fixing processes. The development, since 1964, of the purely chemical, highly reactive systems for the reduction of N2, including those driven electro- and photochemically, is traced, along with the parallel efforts to synthesize metal-N2 complexes (the first step in any likely fixation process) and subsequently protonate them to produce hydrazine or ammonia. These experimental approaches are convergent. Successful cycling or catalysing of some of these N2-binding systems has been achieved. The advantages and limitations of the more successful systems are noted. Approaches to this problem via direct modelling of the nitrogenase active site are outlined, as is the one successful use of such complexes in achieving N2 reduction. This wealth of effort on the reductive approaches contrasts vividly with the almost complete absence of research on N2 oxidation. Currently, only a re-evaluation of the arc discharge process is continuing. Finally, the author's studies of the extruded molybdenum-containing prosthetic group of nitrogenase, the enzymic N2-reducing site, are described in relation to future N2-fixing systems.     

Mechanistic studies of O2-resistant N2-fixation in N2-fixing Escherichia coli

Escherichia coli carrying the entire nif gene cluster from Klebsiella pneumoniae on a multicopy plasmid becomes more O2-resistant in a N-free medium as a result of the integration of the nif gene cluster into the chromosome and the loss of the plasmid (H.Iwahashi and J.Someya, Biochem. Biophys. Res. Comm. 1990, 168: 288–294). Our purpose is to characterize the physiological reason why the strain became O2-resistant by measuring the levels of nif proteins in cells under microaerobic conditions. The O2-resistant strain had a higher amount of NifH and a lower amount of NifL under microaerobic conditions (compared to that under anaerobic conditions), while the parent strain showed the opposite characteristics. Thus, the biochemical mechanism of the O2-resistant strain is attributed to the strain's ability to synthesize and maintain a high amount of NifH and a low amount of NifL under microaerobic conditions. © Rapid Science Ltd. 1998     

The purification of hydrogenase II (uptake hydrogenase) from the anaerobic N2-fixing bacterium Clostridium pasteurianum

The H₂ uptake activity (units/mg protein) of Clostridium pasteurianum cells with methylene blue as the electron acceptor increases with cell density independent of the growth conditions. The H₂ evolution activity (units/mg protein) of the same cells with reduced methyl viologen as the electron donor remains fairly constant under all growth conditions tested. Cells grown under N₂-fixing conditions have the highest H₂ uptake activity and were used for the purification of hydrogenase II (uptake hydrogenase). Attempts to separate hydrogenase II from hydrogenase I (bidirectional hydrogenase) by a previously published method were unreliable. We report here a new large-scale purification procedure which employs a rapid membrane filtration system to fractionate cell-free extracts. Hydrogenases I and II were easily filtered into the low-molecular-weight fraction (M_r less than 100 000), and from this, hydrogenase II was further purified to a homogeneous state. Hydrogenase II is a monomeric iron-sulfur protein of molecular weight 53 000 containing eight iron atoms and eight acid-labile sulfur atoms per molecule. Hydrogenase II catalyzes both H₂ oxidation and H₂ evolution at rates of 3000 and 5.9 μmol H₂ consumed or evolved/min per mg protein, respectively. The purification procedure for hydrogenase II using the filtration system described greatly facilitates the large-scale purification of hydrogenase I and other enzymes from cell-free extracts of C. pasteurianum.     

The contribution of nitrogen transformation processes to total N2O emissions from soils used for intensive vegetable cultivation

The rapid expansion of intensively farmed vegetable fields has substantially contributed to the total N₂O emissions from croplands in China. However, to date, the mechanisms underlying this phenomenon have not been completely understood. To quantify the contributions of autotrophic nitrification, heterotrophic nitrification, and denitrification to N₂O production from the intensive vegetable fields and to identify the affecting factors, a ¹⁵N tracing experiment was conducted using five soil samples collected from adjacent fields used for rice-wheat rotation system (WF), or for consecutive vegetable cultivation (VF) for 0.5 (VF1), 6 (VF2), 8 (VF3), and 10 (VF4) years. Soil was incubated under 50% water holding capacity (WHC) at 25°C for 96 h after being labeled with ¹⁵NH₄NO₃ or NH ₄ ¹⁵ NO₃. The average N₂O emission rate was 24.2 ng N?kg^?1 h^?1 in WF soil, but it ranged from 69.6 to 507 ng N?kg^?1 h^?1 in VF soils. Autotrophic nitrification, heterotrophic nitrification and denitrification accounted for 0.3–31.4%, 25.4–54.4% and 22.5–57.7% of the N₂O emissions, respectively. When vegetable soils were moderately acidified (pH, 6.2 to ?≥?5.7), the increased N₂O emissions resulted from the increase of both the gross autotrophic and heterotrophic nitrification rates and the N₂O product ratio of autotrophic nitrification. However, once severe acidification occurred (as in VF4, pH?≤?4.3) and salt stress increased, both autotrophic and heterotrophic nitrification rates were inhibited to levels similar to those of WF soil. The enhanced N₂O product ratios of heterotrophic nitrification (4.84‰), autotrophic nitrification (0.93‰) and denitrification processes were the most important factors explaining high N₂O emission in VF4 soil. Data from this study showed that various soil conditions (e.g., soil salinity and concentration of NO ₃ ^- or NH ₄ ⁺ ) could also significantly affect the sources and rates of N₂O emission.     

Oxygen resistant strain of N2-fixing Escherichia coli

Oxygen resistant N2-fixing Escherichia coli, which can grow at a high oxygen concentration in a nitrogen-free medium, were produced by several times of cultivation under a condition of 0.03% oxygen. Six isolated resistant strains could grow at an oxygen concentration ten times that of the parent strain. The nif-genes of these six strains were integrated into a chromosome. At low oxygen, they showed one third the nitrogenase activity to the parent strain, but more so at a high oxygen concentration. It is thus evident that the amount of nitrogenase protein in a cell is a factor determining the oxygen resistance of nitrogenase.     

Inoculation of forage grasses with N2-fixing Enterobacteriaceae

Summary Previous investigations indicated some forage grass roots in Texas are heavily colonized with N₂-fixing bacteria. The most numerous N₂-fixing bacteria were in the genera Klebsiella and Enterobacter. In the present investigation inoculation experiments were conducted using 18 isolates of these bacteria to determine if a N₂-fixing association could be established between the bacteria and the grassesCynodon dactylon andPanicum coloratum. Plants were grown in soil for approximately 5 months in a greenhouse and were measured periodically for dry matter, nitrogen accumulation, and acetylene reduction activity. Results of the investigation indicated that 25% of the plant-soil systems were active in acetylene reduction and the activity was high enough to indicate agronomically significant quantities of N₂ were being fixed (>8kg N ha⁻¹). However, plant systems extrapolated to fix>8 kg N ha⁻¹ contained less nitrogen and accumulated less dry matter than plants less active in acetylene reduction. Inocula could not be re-isolated from healthy grass roots indicating that the N₂-fixing activity may have not have been closely assiciated with plant roots. Future research is needed to determine factors limiting colonization of grass roots.     

Nitrogenase activity and N2 fixation are stimulated by elevated CO2 in a tropical N2-fixing tree

 Seeds of Gliricidia sepium, a fast-growing woody legume native to seasonal tropical forests of Central America, were inoculated with N₂-fixing Rhizobium bacteria and grown in environmentally controlled glasshouses for 67–71 days under ambient CO₂ (35 Pa) and elevated CO₂ (70 Pa) conditions. Seedlings were watered with an N-free, but otherwise complete, nutrient solution such that bacterial N₂ fixation was the only source of N available to the plant. The primary objective of our study was to quantify the effect of CO₂ enrichment on the kinetics of photosynthate transport to nodules and determine its subsequent effect on N₂ fixation. Photosynthetic rates and carbon storage in leaves were higher in elevated CO₂ plants indicating that more carbon was available for transport to nodules. A ¹⁴CO₂ pulse-chase experiment demonstrated that photosynthetically fixed carbon was supplied by leaves to nodules at a faster rate when plants were grown in elevated CO₂. Greater rates of carbon supply to nodules did not affect nodule mass per plant, but did increase specific nitrogenase activity (SNA) and total nitrogenase activity (TNA) resulting in greater N₂ fixation. In fact, a 23% increase in the rate of carbon supplied to nodules coincided with a 23% increase in SNA for plants grown in elevated CO₂, suggesting a direct correlation between carbon supply and nitrogenase activity. The improvement in plant N status produced much larger plants when grown in elevated CO₂. These results suggest that Gliricidia, and possibly other N₂-fixing trees, may show an early and positive growth response to elevated CO₂, even in severely N-deficient soils, due to increased nitrogenase activity. Received: 27 February 1996 / Accepted: 19 June 1996     

土壤氮素转化的关键微生物过程及机制

微生物是驱动土壤元素生物地球化学循环的引擎.氮循环是土壤生态系统元素循环的核心之一,其四个主要过程,即生物固氮作用、氨化作用、硝化作用、反硝化作用,均由微生物所驱动.近10年来,随着免培养的分子生态学技术和高通量测序技术等的发展,在硝化微生物多样性及其作用机理、厌氧氨氧化过程和机理等研究方面取得了突破性进展.本文重点阐述了我国有关土壤硝化微生物方面的研究进展,在此基础上,简要介绍了反硝化微生物和厌氧氨氧化及硝酸盐异化还原成铵作用的研究进展,并对今后的研究工作提出了展望.今后土壤氮素转化微生物生态学的研究,应瞄准国际微生生态学发展的前沿,加强新技术新方法的应用,结合我国农业可持续发展、资源环境保护和全球变化研究的重大需求,重点开展以下几方面的工作:(1)开展大尺度上土壤硝化作用及氨氧化微生物分布的时空演变特征及驱动因子的研究;(2)加强氮素转化关键微生物过程与机理的研究,并与相关过程的通量(如氨挥发、N2O释放)和反应速率(如矿化速率、硝化速率)关联起来;(3)在特定生态系统中系统研究各个氮转化过程的耦合关系,构建相关氮素转化和氮素平衡模型,为定向调控土壤氮素转化过程,提高氮素利用效率并减少其负面效应提供科学依据.     

Rice response to inoculation with N2-fixing and P-solubilizing microorganisms

Summary Inoculation effect ofA. chroococcum, P. striata andA. awamorii on yield and nutrients uptake in rice was studied under green house conditions. The organisms appreciably increased the yield and uptake of nutrients with or without chemical fertilizers. Phosphorussolubilizing microorganisms and a mixture of the three showed better response than the rest of the treatments among single and mixed culture inoculations respectively. Chemical fertilizers further improved the yield and nutrients uptake. The yield response remained unaffected by replacing superphosphate with rock phosphate and microbial inoculations.     

The uptake and metabolism of methylamine by N2-fixing cyanobacteria

The cyanobacteria Anabaena variabilis and Nostoc CAN showed a biphasic pattern of ¹⁴CH₃NH ₃ ⁺ uptake at external pH values of 7.0 and 9.0. The initial phase of uptake, which was independent of metabolism of ¹⁴CH₃NH ₃ ⁺ , was attributed to uptake via a CH₃NH ₃ ⁺ (NH ₄ ⁺ ) transport system at pH 7.0 and probably to passive diffusion of uncharged CH₃NH₂ and trapping by protonation at pH 9.0. The second slower phase of uptake was attributed to metabolism of CH₃NH ₃ ⁺ via glutamine synthetase to form -methylglutamine which accumulates. Anabaena cylindrica showed an initial rapid uptake at pH 7.0 and pH 9.0 but metabolism of ¹⁴CH₃NH ₃ ⁺ was undetectable at pH 7.0 and was barely detectable at pH 9.0. Pretreatment of A. variabilis with l-methionine-d,l-sulphoximine to inactivate glutamine synthetase, inhibited the second phase of ¹⁴CH₃NH ₃ ⁺ uptake at both pH 7.0 and pH 9.0 and the accumulation of -methylglutamine but had no effect on the first phase of uptake. Following transfer of A. variabilis to darkness the initial phase of ¹⁴CH₃NH ₃ ⁺ uptake at pH 7.0 and 9.0 was unaffected but the subsequent metabolism via glutamine synthetase was inhibited.Abbreviations MSX l-methionine-d,l-sulphoximine - GS glutamine synthetase     

Untraditional N2-fixing bacteria as biofertilizers for wheat and barley

The screening of 27 isolates grown on nitrogen-free medium for nitrogen-fixing ability resulted in the isolation of five organisms belonging toBacillaceae, Enterobacteriaceae andPseudomonadaceae. Estimates of N₂-fixation efficiencies of these isolates indicated that they may be responsible for low rates of N₂-fixation in soil. The possible association of these isolates as well as ofAzotobacter andAzospirillum with wheat and barley was investigated in a greenhouse experiment. The highest values of nitrogenase activity on plant root were recorded in treatments inoculated with composite inocula of the isolated N₂-fixers, particularly whenAzotobacter and/orAzospirillum were added in combination. Inoculation with single inoculum of each of the N₂-fixing isolates had no significant influence on plant growth, except withPseudomonas andBacillus for wheat and barley, respectively. Highly significant increases in growth of both plants were recorded in all cases of multistrain inoculation.     

H2-dependent mixotrophic growth of N2-fixing Azotobacter vinelandii.

Azotobacter vinelandii can grow with a variety of organic carbon sources and fix N2 without the need for added H2. However, due to an active H2-oxidizing system, H2-dependent mixotrophic growth in an N-free medium was demonstrated when mannose was provided as the carbon source. There was no appreciable growth with either H2 or mannose alone. Both the growth rate and the cell yield were dependent on the concentrations of both substrates, H2 and mannose. Cultures growing mixotrophically with H2 and mannose consumed approximately 4.8 mmol of O2 and produced 4.6 mmol of CO2 per mmol of mannose consumed. In the absence of H2, less CO2 was produced, less O2 was consumed, and cell growth was negligible. The rate of acetylene reduction in mixotrophic cultures was comparable to the rate in cultures grown in N-free sucrose medium. The rate of [14C]mannose uptake of cultures with H2 was greater than with argon, whereas [14C]sucrose uptake was unaffected by the addition of H2; therefore, the role of H2 in mixotrophic metabolism may be to provide energy for mannose uptake. A. vinelandii is not an autotroph, as attempts to grow the organism chemoautotrophically with H2 or to detect ribulose bisphosphate carboxylase activity were unsuccessful.     

Aerobic and anaerobic metabolism in Entamoeba histolytica

Respiration by Entamoeba histolylica is confirmed. A doubling of the rate of oxygen uptake was observed upon the addition of d-glucose to cells in which the glycogen reserve had been partially depleted. In cells metabolizing endogenous substrates the rate of oxygen uptake was not influenced by sodium cyanide or sodium succinate. It was slightly depressed when d-mannose was the added sugar. The end products, CO₂, ethanol, and acetate accounted for essentially all of the glucose carbon utilized in both aerobic and anaerobic experiments. The radioactivity from uniformly labelled ¹⁴C-glucose was found in these products. Three times as much ethanol as acetate was produced in the anaerobic experiments and in the aerobic experiments this ratio was approximately reversed.