Ammonia-oxidizing communities in agricultural soil incubated with organic waste residues

The impact of organic compounds present in different kinds of organic fertilizers, i.e., anaerobically digested household waste, composted organic household waste, swine manure, and cow manure, on microbial communities in arable soil was investigated using microcosms. Soil was amended with dried residues or organic extracts of the residues and incubated for 12 weeks at 25°C. The microbial community composition was investigated by phospholipid fatty acid (PLFA) analysis, and the community of ammonia-oxidizing bacteria (AOB) was assessed by denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments, followed by sequencing. All dried residues increased the AOB activity, determined as potential ammonia oxidation, whereas the organic extracts from the thermophilically digested waste and the swine manure caused a decreased potential activity. However, no differences in the DGGE banding patterns were detected, and the same AOB sequences were present in all samples treated with the residue extracts. Moreover, the PLFA composition showed that none of the residue additions affected the overall microbial community structure in the soil. We conclude that the AOB community composition was not affected by the organic compounds in the fertilizers, although the activity in some cases was.     

Characterization of developing microbial communities in Mount St. Helens pyroclastic substrate

The 1980 eruption of Mount St. Helens created a unique opportunity to study microbial communities in a developing soil ecosystem containing little total carbon (C) or total nitrogen (N). We collected surface samples (0-5 cm) from areas near Mount St. Helens National Volcanic Monument 17 years after the eruption. The samples were from bare soil with no plant development, soil under living prairie lupine (Lupinus lepidus) and dead prairie lupine in the pyroclastic plain near Spirit Lake, Washington. We also collected soil from a nearby forested area. Phospholipid fatty acids (PLFAs) from pyroclastic materials were analyzed to determine changes in soil microbial composition. Total bacterial DNA was also extracted from the soils and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes and DNA sequence analysis of cloned 16S rRNA gene libraries were used to determine the influence of plants on microbial development. Both principal components analysis (PCA) of PLFA fingerprints and non-metric multidimensional scaling (NMS) of DGGE fingerprints distinguished the four soils. Lupine plants influenced the PLFA and DGGE fingerprints depending on the distance of the samples from the plant. DGGE and PLFA profiles from the forest soil were significantly different (P=0.001, based on Monte Carlo permutation test) from those of the bare soil and soil with live lupine. Bacterial clone libraries were constructed, and 800 clones were analyzed by amplified ribosomal DNA restriction analysis (ARDRA) and grouped into operational taxonomic units (OTUs). A total of 51, 77, 58, and 42 different OTUs were obtained from forest soil, soil with live and dead lupine, and bare soil, respectively. Phylogenetic analysis revealed that 62% of the 228 OTUs were classified as Proteobacteria, Actinobacteria, Acidobacterium, Verrucomicrobia, Bacteroides, Cyanobacteria, Planctomycetes, and candidate divisions TM7 and OP10. Members of Proteobacteria represented 29% of the OTUs. Thirty-eight percent of the OTUs could not be classified into known bacterial divisions. This study emphasized the role of prairie lupine in the establishment of pioneering microbial communities and the subsequent roles the biotic components played in improving the quality of pyroclastic soil.     

黄瓜连作土壤微生物区系变化研究

研究了黄瓜根部土壤主要微生物类群随连作茬次的反应,并着重用变性梯度凝胶电泳(DGGE)监测黄瓜根际未培养优势菌群的动态变化。结果表明:随着连作茬次增加,土壤可培养微生物数量减少,其中细菌数量降低更为明显,对连作表现出较高的敏感性,放线菌对黄瓜连作反应稍滞后,至第三茬时开始呈现降低趋势。黄瓜连作致使少数真菌种群富集,同时多种真菌类群数量减少,种群变化呈现单一化趋势,多样性水平降低。DGGE分析结果表明黄瓜连作引起根际Bacillus sp.、Pseudom onas sp.和另一未培养细菌种群数量减少,而同时引起Sphingom onas sp.类群数量增加。实验结果暗示,黄瓜连作破坏根部微生物种群生态平衡,使其多样性水平降低。     

不同年限毛竹林土壤固氮菌群落结构和丰度的演变

应用变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)和荧光定量PCR(real time fluorescent quantitative PCR,q PCR)方法研究了不同年限毛竹林土壤固氮菌群落结构和丰度的变化。结果表明:土壤p H、有机质、有效磷、速效钾和铵态氮含量在马尾松林改造成毛竹林5 a后明显提高,而后逐渐降低,并趋于稳定;土壤固氮菌多样性和nif H基因丰度也呈现相似的趋势。条带测序分析表明,毛竹林土壤固氮菌均为不可培养的固氮菌,与慢生根瘤菌(Bradyrhizobium sp.)具有较高的相似度。冗余分析结果表明,不同栽培年限毛竹林地土壤固氮菌群落组成发生了明显变化,长期栽培毛竹林引起的土壤养分变化对土壤固氮菌多样性具有重要影响。     

菇菜套作对土壤微生物群落的影响

套作是防治连作障碍的有效方法之一,但是蔬菜和可食用菌之间的套作机理研究鲜见报道,尤其是其土壤微生物学机制。本研究建立菇菜套作体系,利用实时荧光定量PCR和PCR-DGGE技术研究土壤细菌和真菌群落的变化。结果表明,菇菜套作显著提高了番茄生物量,且其番茄果实产量最高,硝酸盐含量最低。与对照相比,菇菜套作下土壤细菌和真菌基因拷贝数量均无显著变化;DGGE指纹图谱表明,不同处理下的细菌群落无明显差异,但是菇菜套作下真菌群落结构发生了分异,主要表现为尖孢镰刀菌(Fusarium oxysporum)和稻黑孢菌(Nigrospora oryzae)代表型条带的强度的下降。     

Community structure of microaerophilic iron-oxidizing bacteria in Japanese paddy field soils

ABSTRACT

Redox cycle of iron (Fe) is the central process in the biogeochemistry of paddy field soil. Although Fe(II)-oxidizing process is mediated by both abiotic and biotic reactions, microorganisms involved in the process have not been well studied in paddy field soil. The present study investigated the community structure of microaerophilic Fe(II)-oxidizing bacteria (FeOB) in the family Gallionellaceae in the plow layer of paddy fields located in the central (Anjo) and northeastern (Omagari) Japan since the members in the family are the typical FeOB in circumneutral freshwater environments and possibly have the significant role for Fe(II) oxidation in paddy field soils. A primer set targeting 16S rRNA gene of Gallionella-related FeOB was newly designed for the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative PCR (qPCR) analyses. DGGE analysis showed significant differences in the band patterns between the field sites. Besides, minor differences were observed in the patterns between the soil depths (0–1 cm and below 1 cm) in the Anjo field, while the patterns were relatively stable in the Omagari field during the annual rice cultivation practices. In total 54 bands were sequenced and clustered into 20 operational taxonomic units (OTUs) on the basis of the 97% similarity. Eighteen out of twenty OTUs (50 of 54 bands) were affiliated within the FeOB cluster of Gallionellaceae, some of which were clustered with known microaerophilic FeOB, Ferrigenium kumadai, Ferriphaselus amnicola, ‘Sideroxydans lithotrophicus’ and ‘S. paludicola’. The number of the 16S rRNA gene copies was 10⁵–10⁷ and 10⁶–10⁸ copies g^?1 dried soil in the two paddy fields and negatively correlated to the contents of acetate-extractable Fe(II) in the soils during the rice cultivation period. These results suggested inhabitance of considerable number of diverse Gallionella-related FeOB and their potential involvement in the Fe(II)-oxidizing process of soil, especially during the rice cultivation period in the paddy field soils.     

Can the extent of degradation of soil fungal mycelium during soil incubation be used to estimate ectomycorrhizal biomass in soil?

The extent of degradation of the fungal biomass in forest soil during laboratory incubation was investigated as a measure of ectomycorrhizal (EM) biomass. The method simulates the disappearance of fungal mycelium after root trenching, where the EM fungi, deprived of its energy source (the tree), will start to die off. Incubating a forest humus soil at 25 °C resulted in a decrease in the relative proportion (mol%) of the phospholipid fatty acid 18:2ω6,9 (a fungal marker molecule) within 3-6 months, indicating that fungal biomass was disappearing. Incubation at 5 °C resulted in essentially no change in the amount of 18:2ω6,9. The measurement of ergosterol, another fungal marker molecule, gave similar results. Incubation of different forest soils (pine, spruce and spruce/oak), and assuming that the disappearance of fungal biomass during this period of time was entirely due to EM fungi, resulted in an estimation of EM biomass of between 47 and 84% of the total fungal biomass in these soils. The humus layer had more EM biomass than deeper mineral layers.     

Phospholipid fatty acid composition of a 2,4,6-trinitrotolune contaminated soil and an uncontaminated soil as affected by a humification remediation process

Analysis of phospholipid fatty acids (PLFAs) was performed to investigate effects of 2,4,6-trinitrotoluene (TNT) contamination and soil remediation on microbial biomass and community structure. A TNT-contaminated and an uncontaminated soil from a former ammunition plant were analysed before and after a humification/remediation process. TNT contamination reduced microbial biomass but indicated only minor differences in PLFA composition between the contaminated and uncontaminated soils. The humification process increased microbial biomass and altered soil PLFA patterns to a larger degree than did TNT contamination.     

石油污染土壤鞘氨醇单胞菌遗传多样性16S rDNA-PCR-DGGE分析

鞘氨醇单胞菌是降解多环芳烃(PAHs)的重要功能微生物。然而,采用属特异引物直接从PAHs污染土壤中检测鞘氨醇单胞菌进而研究鞘氨醇单胞菌在污染土壤中的多样性、种群结构及其与PAHs污染的关系鲜有报道。本研究自行设计鞘氨醇单胞菌属特异性引物,通过16S rDNA-PCR-DGGE技术对沈抚灌区石油污染土壤鞘氨醇单胞菌遗传多样性进行了研究。结果表明,自行设计的鞘氨醇单胞菌属引物732f-982r能够特异、灵敏的从环境样品中检出鞘氨醇单胞菌。当沈抚灌区原位污染土壤的PAHs浓度小于5 mg kg-1时,鞘氨醇单胞菌多样性随着PAHs浓度的增加而增加;而初始PAHs浓度高的实验室模拟实验土壤,其鞘氨醇单胞菌多样性较低。鞘氨醇单胞菌种群结构受污染土壤总石油烃(TPH)含量的影响,TPH含量接近的土壤,鞘氨醇单胞菌的群落结构较为相似。     

Structure and function of the soil microbial community in a long-term fertilizer experiment

The effect of organic and inorganic fertiliser amendments is often studied shortly after addition of a single dose to the soil but less is known about the long-term effects of amendments. We conducted a study to determine the effects of long-term addition of organic and inorganic fertiliser amendments at low rates on soil chemical and biological properties. Surface soil samples were taken from an experimental field site near Cologne, Germany in summer 2000. At this site, five different treatments were established in 1969: mineral fertiliser (NPK), crop residues removed (mineral only); mineral fertiliser with crop residues; manure 5.2 t ha⁻¹ yr⁻¹; sewage sludge 7.6 t ha⁻¹ yr⁻¹ or straw 4.0 t ha⁻¹ yr⁻¹ with 10 kg N as CaCN₂ t straw⁻¹. The organic amendments increased the C_org content of the soil but had no significant effect on the dissolved organic C (DOC) content. The C/N ratio was highest in the straw treatment and lowest in the mineral only treatment. Of the enzymes studied, only protease activity was affected by the different amendments. It was highest after sewage amendment and lowest in the mineral only treatment. The ratios of Gram+ to Gram− bacteria and of bacteria to fungi, as determined by signature phospholipid fatty acids, were higher in the organic treatments than in the inorganic treatments. The community structure of bacteria and eukaryotic microorganisms was assessed by denaturing gradient gel electrophoresis (DGGE) and redundancy discriminate analyses of the DGGE banding patterns. While the bacterial community structure was affected by the treatments this was not the case for the eukaryotes. Bacterial and eukaryotic community structures were significantly affected by C_org content and C/N ratio.     

Anammox反应器的启动及其菌群演变的研究

为了研究工艺条件对反应器内微生物多样性的影响,该论文采用城市污水处理厂活性污泥接种,通过培育硝化污泥,进行了启动厌氧氨氧化(Anammox)反应器的试验,并对启动过程中细菌的多样性变化作了跟踪研究。研究结果表明,以好氧活性污泥作为接种物,可成功地培育硝化生物膜;通过反应器运行条件的控制,硝化生物膜可从进行好氧氨氧化反应过渡到进行厌氧氨氧化反应。在此过程中,异养型细菌的数量大幅度下降,硝化细菌、反硝化细菌和厌氧氨氧化细菌的数量增大,推测它们都与厌氧氨氧化作用有关。运用PCR-DGGE技术证明,随着反应器运行时间的延长,菌群发生明显变化并呈现简化趋势。     

Does the depletion of pentachlorophenol in root–soil interface follow a simple linear dependence on the distance to root surfaces?

To understand root–soil–microbe interactions in rhizo-depletion of xenobiotics, we conducted a glasshouse study using specially designed laminar rhizoboxes which allow intact layers of near- (1–5 mm) and far- (>5 mm) rhizosphere soil to be harvested separately from root surfaces without the removal of the root material itself. Plant (Lolium perenne L.) seedlings were grown for 90 days in a soil treated with PCP at 20 and 50 mg kg⁻¹. Changes in PCP depletion, soil microbial biomass and community structure (as indicated by phospholipid fatty acids (PLFAs) profiles) with increasing distance from the root surfaces were then assessed after harvesting. Surprisingly, depletion of PCP in the planted rhizoboxes exhibited a nonlinear dependence on the distance to root surfaces, with the most rapid loss in the 2 or 3 mm near-rhizosphere layers, contrasting to the well-known linear gradient of root exudates and mineral nutrients etc. (generally, the extent gradually decreased with increasing distance from the root surface). Soil microbial biomass carbon, however, decreased linearly as expected with increasing distance from the roots. The microbial community structures as indicated by PLFA profiles showed distance-dependent selective enrichment of competent species that may be responsible for efficient PCP depletion. The results suggest that root exudates induced modifications of microbial communities in the PCP contaminated rhizosphere and spatially modified the dominant species within these communities, resulting in the nonlinear PCP depletion pattern.     

Distribution of light and heavy fractions of soil organic carbon as related to land use and tillage practice

Mass distributions of different soil organic carbon (SOC) fractions are influenced by land use and management. Concentrations of C and N in light- and heavy fractions of bulk soils and aggregates in 0–20 cm were determined to evaluate the role of aggregation in SOC sequestration under conventional tillage (CT), no-till (NT), and forest treatments. Light- and heavy fractions of SOC were separated using 1.85 g mL⁻¹ sodium polytungstate solution. Soils under forest and NT preserved, respectively, 167% and 94% more light fraction than those under CT. The mass of light fraction decreased with an increase in soil depth, but significantly increased with an increase in aggregate size. C concentrations of light fraction in all aggregate classes were significantly higher under NT and forest than under CT. C concentrations in heavy fraction averaged 20, 10, and 8 g kg⁻¹ under forest, NT, and CT, respectively. Of the total SOC pool, heavy fraction C accounted for 76% in CT soils and 63% in forest and NT soils. These data suggest that there is a greater protection of SOC by aggregates in the light fraction of minimally disturbed soils than that of disturbed soil, and the SOC loss following conversion from forest to agriculture is attributed to reduction in C concentrations in both heavy and light fractions. In contrast, the SOC gain upon conversion from CT to NT is primarily attributed to an increase in C concentration in the light fraction.     

长期秸秆覆盖对免耕稻-麦产量、土壤氮组分及微生物群落的影响

为探究长期秸秆覆盖对免耕区作物产量、土壤氮素组分及微生物群落特征的影响,以稻–麦定位免耕试验为研究对象,选取了其中免耕且秸秆移除和免耕且秸秆覆盖2个处理,于试验开展第12年（2018年）小麦收获后,统计分析近五年产量数据,并采集各处理0～5、5～10、10～20、20～30 cm的土壤样品,测定土壤全氮及活性氮组分,利用磷脂脂肪酸（PLFA）方法表征土壤微生物群落。结果表明：（1）秸秆覆盖显著提高了小麦产量（增幅为6.49%）,对水稻产量影响不显著。（2）秸秆覆盖对土壤氮组分的影响略有差异：它显著提高了土壤0～5 cm全氮、硝态氮和铵态氮以及0～10 cm颗粒有机氮、0～5 cm和10～20 cm可溶性有机氮含量,对微生物生物量氮无显著影响;它提高了0～5 cm和10～20 cm可溶性有机氮占全氮的比例,对其他组分占全氮比例无显著影响。（3）秸秆覆盖显著提高了土壤微生物总PLFA和细菌PLFA丰度,对真菌PLFA和放线菌PLFA无影响,降低了土壤真菌/细菌比;微生物生物量氮、土壤全氮、颗粒有机碳/颗粒有机氮比是显著影响土壤微生物群落组成的关键土壤环境因子。（4）无论秸秆覆盖与否,土壤全...     

利用PCR-DGGE技术研究长期自然恢复、种植作物和裸地条件下黑土土壤细菌群落结构变化

Soil microbial biomass and community structures are commonly used as indicators for soil quality and fertility. A investigation was performed to study the effects of long-term natural restoration, cropping, and bare fallow managements on the soil microbial biomass and bacterial community structures in depths of 0--10, 20--30, and 40--50 cm in a black soil (Mollisol). Microbial biomass was estimated from chloroform fumigation-extraction, and bacterial community structures were determined by analysis of 16S rDNA using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Experimental results showed that microbial biomass significantly declined with soil depth in the managements of restoration and cropping, but not in the bare fallow. DGGE profiles indicated that the band number in top 0--10 cm soils was less than that in depth of 20--30 or 40--50 cm. These suggested that the microbial population was high but the bacterial community structure was simple in the topsoil. Cluster and principle component analysis based on DGGE banding patterns showed that the bacterial community structure was affected by soil depth more primarily than by managements, and the succession of bacterial community as increase of soil depth has a similar tendency in the three managements. Fourteen predominating DGGE bands were excised and sequenced, in which 6 bands were identified as the taxa of Verrucomicrobia, 2 bands as Actinobacteria, 2 bands as α-Proteobacteria, and the other 4 bands as δ-Proteobacteria, Acidobacteria, Nitrospira, and unclassified bacteria. In addition, the sequences of 11 DGGE bands were closely related to uncultured bacteria. Thus, the bacterial community structure in black soil was stable, and the predominating bacterial groups were uncultured.     

Application of DGGE analysis to the study of bacterial community structure in plant roots and in nonrhizosphere soil

To analyze the structure of bacterial communities in spinach roots and in the nonrhizosphere soil, we used PeR-amplified 16S rRNA gene fragments separated by denaturing gradient gel electrophoresis (DGGE). DGGE revealed a large number of band patterns, which were ascribed to various bacterial species composing each of the bacterial communities. The pattern from the roots was less complex than that from the soil. It is considered that DGGE analysis is suitable for studies of bacterial community structure in soil-plant ecosystems.     

Forest floor microbial communities in relation to stand composition and timber harvesting in northern Alberta

With the growing interest in silvicultural techniques that more closely emulate natural disturbance regimes, there is a need to better understand how partial harvesting affects the soil microbial community in stands with varying ecological characteristics, e.g., tree species composition. Four and a half and 5.5 years post-harvest, we used phospholipid fatty acid (PLFA) and substrate-induced respiration (SIR) analyses to compare the microbial biomass and microbial community structure of forest floors from stands dominated by white spruce (Picea glauca; SPRUCE) or by trembling aspen (Populus tremuloides; ASPEN) and from mixed-species (MIXED) stands in northern Alberta, Canada, that had been clearcut, partial-cut with 20% retention, partial-cut with 50% retention or left uncut (controls). PLFA and SIR analyses revealed that ASPEN forest floors supported a larger microbial biomass with a very different community structure than MIXED or SPRUCE forest floors. The microbial community structure of these soils appeared to be strongly affected by the presence of white spruce and the composition of the understory vegetation. There were no effects of timber harvesting detected within or across stand types on any of the variables measured, with the exception of the PLFA 16:1ω5, which was relatively more abundant in the clearcuts and 50% retention treatments than in the uncut controls, perhaps in response to an increased forest floor pH and grass cover in the disturbed areas. The resilience to timber harvesting of the forest floors from these stands may be the result of efforts to minimize soil disturbance during harvesting and to allow vegetation to regenerate naturally. From the perspective of the forest floor microbial community, partial harvesting does not appear to have any benefit over clearcut harvesting at these boreal forest sites.     

Microbial composition and diversity of an upland red soil under long-term fertilization treatments as revealed by culture-dependent and culture-independent approaches

Background, aim, and scope  Fertilization is an important agricultural practice for increasing crop yields. In order to maintain the soil sustainability, it is important to monitor the effects of fertilizer applications on the shifts of soil microorganisms, which control the cycling of many nutrients in the soil. Here, culture-dependent and culture-independent approaches were used to analyze the soil bacterial and fungal quantities and community structure under seven fertilization treatments, including Control, Manure, Return (harvested peanut straw was returned to the plot), and chemical fertilizers of NPK, NP, NK, and PK. The objective of this study was to examine the effects on soil microbial composition and diversity of long-term organic and chemical fertilizer regimes in a Chinese upland red soil. Materials and methods  Soil samples were collected from a long-term experiment station at Yingtan (28°15′N, 116°55′E), Jiangxi Province of China. The soil samples (0–20 cm) from four individual plots per treatment were collected. The total numbers of culturable bacteria and fungi were determined as colony forming units (CFUs) and selected colonies were identified on agar plates by dilution plate methods. Moreover, soil DNAs were extracted and bacterial 16S rRNA genes and fungal 18S rRNA genes were polymerase chain reaction amplified, and then analyzed by denaturing gradient gel electrophoresis (DGGE), cloning, and sequencing. Results  The organic fertilizers, especially manure, induced the least culturable bacterial CFUs, but the highest bacterial diversity ascertained by DGGE banding patterns. Chemical fertilizers, on the other hand, had less effect on the bacterial composition and diversity, with the NK treatment having the lowest CFUs. For the fungal community, the manure treatment had the largest CFUs but much fewer DGGE bands, also with the NK treatment having the lowest CFUs. The conventional identification of representative bacterial and fungal genera showed that long-term fertilization treatments resulted in differences in soil microbial composition and diversity. In particular, 42.4% of the identified bacterial isolates were classified into members of Arthrobacter. For fungi, Aspergillus, Penicillium, and Mucor were the most prevalent three genera, which accounted for 46.6% of the total identified fungi. The long-term fertilization treatments resulted in different bacterial and fungal compositions ascertained by the culture-dependent and also the culture-independent approaches. Discussion  It was evident that more representative fungal genera appeared in organic treatments than other treatments, indicating that culturable fungi were more sensitive to organic than to chemical fertilizers. A very notable finding was that fungal CFUs appeared maximal in organic manure treatments. This was quite different from the bacterial CFUs in the manure, indicating that bacteria and fungi responded differently to the fertilization. Similar to bacteria, the minimum fungal CFUs were also observed in the NK treatment. This result provided evidence that phosphorus could be a key factor for microorganisms in the soil. Thus, despite the fact that culture-dependent techniques are not ideal for studies of the composition of natural microbial communities when used alone, they provide one of the more useful means of understanding the growth habit, development, and potential function of microorganisms from soil habitats. A combination of culture-dependent and culture-independent approaches is likely to reveal more complete information regarding the composition of soil microbial communities. Conclusions  Long-term fertilization had great effects on the soil bacterial and fungal communities. Organic fertilizer applications induced the least culturable bacterial CFUs but the highest bacterial diversity, while chemical fertilizer applications had less impact on soil bacterial community. The largest fungal CFUs were obtained, but much lower diversity was detected in the manure treatment. The lowest bacterial and also fungal CFUs were observed in the NK treatment. The long-term fertilization treatments resulted in different bacterial and fungal compositions ascertained by the culture-dependent and also the culture-independent approaches. Phosphorus fertilizer could be considered as a key factor to control the microbial CFUs and diversity in this Chinese upland red soil. Recommendations and perspectives  Soil fungi seem to be a more sensitive indicator of soil fertility than soil bacteria. Since the major limitation of molecular methods in soil microbial studies is the lack of discrimination between the living and dead, or active and dormant microorganisms, both culture-dependent and culture-independent methods should be used to appropriately characterize soil microbial diversity.