Effect of Pyocyanin on a Crude-Oil-Degrading Microbial Community

Pseudomonas aeruginosa is an n-alkane degrader that is frequently isolated from petroleum-contaminated sites and produces factors that enhance its competitiveness and survival in many environments. In this study, one such factor, pyocyanin, has been detected in an oil-degrading culture containing P. aeruginosa and is a redox-active compound capable of inhibiting microbial growth. To examine the effects of pyocyanin further, an oil-degrading culture was grown with and without 9.5 μM pyocyanin and microbial community structure and oil degradation were monitored for 50 days. Denaturing gradient gel electrophoresis (DGGE) analysis of cultures revealed a decrease in the microbial community diversity in the pyocyanin-amended cultures compared to that of the unamended cultures. Two members of the microbial community in pure culture exhibited intermediate and high sensitivities to pyocyanin corresponding to intermediate and low levels of activity for the antioxidant enzymes catalase and superoxide dismutase, respectively. Another member of the community that remained constant in the DGGE gels over the 50-day culture incubation period exhibited no sensitivity to pyocyanin, corresponding to a high level of catalase and superoxide dismutase when examined in pure culture. Pyocyanin also affected the overall degradation of the crude oil. At 50 days, the culture without pyocyanin had decreased polycyclic aromatic hydrocarbons compared to the pyocyanin-amended culture, with a specific reduction in the degradation of dibenzothiophenes, naphthalenes, and C₂₉ and C₃₀ hopanes. This study demonstrated that pyocyanin influenced the diversity of the microbial community and suggests the importance of understanding how interspecies interactions influence the degradation capability of a microbial community.     

Long-Term Explosive Contamination in Soil: Effects on Soil Microbial Community and Bioremediation

Explosive contamination in soil is a great concern for environmental health. Following 50 years of munitions manufacturing and loading, soils from two different sites contained ≥ 6,435 mg 2,4,6-trinitrotoluene (TNT), 2,933 mg hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and 2,135 mg octahydrol-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) kg^{? 1} soil. Extractable nitrate-N was as high as 315 and ammonium-N reached 150 mg N kg^{? 1} soil. Water leachates in the highly contaminated soils showed near saturation levels of TNT and RDX, suggesting great risk to water quality. The long-term contamination resulted in undetectable fungal populations and as low as 180 bacterial colony forming units (CFU) g^–1 soil. In the most severely contaminated soil, dehydrogenase activity was undetectable and microbial biomass carbon was very low (< 3.4 mg C _mic kg^–1 soil). The diminished biological activity was a consequence of long-term contamination because short-term (14 d) contamination of TNT at up to 5000 mg TNT kg^–1 soil did not cause a decline in the culturable bacterial population. Natural attenuation may not be a feasible remediation strategy in soils with long-term contamination by high concentrations of explosives.     

Assessing the Microbial Community and Functional Genes in a Vertical Soil Profile with Long-Term Arsenic Contamination

Arsenic (As) contamination in soil and groundwater has become a serious problem to public health. To examine how microbial communities and functional genes respond to long-term arsenic contamination in vertical soil profile, soil samples were collected from the surface to the depth of 4 m (with an interval of 1 m) after 16-year arsenic downward infiltration. Integrating BioLog and functional gene microarray (GeoChip 3.0) technologies, we showed that microbial metabolic potential and diversity substantially decreased, and community structure was markedly distinct along the depth. Variations in microbial community functional genes, including genes responsible for As resistance, carbon and nitrogen cycling, phosphorus utilization and cytochrome c oxidases were detected. In particular, changes in community structures and activities were correlated with the biogeochemical features along the vertical soil profile when using the rbcL and nifH genes as biomarkers, evident for a gradual transition from aerobic to anaerobic lifestyles. The C/N showed marginally significant correlations with arsenic resistance (p = 0.069) and carbon cycling genes (p = 0.073), and significant correlation with nitrogen fixation genes (p = 0.024). The combination of C/N, NO₃ ⁻ and P showed the highest correlation (r = 0.779, p = 0.062) with the microbial community structure. Contradict to our hypotheses, a long-term arsenic downward infiltration was not the primary factor, while the spatial isolation and nutrient availability were the key forces in shaping the community structure. This study provides new insights about the heterogeneity of microbial community metabolic potential and future biodiversity preservation for arsenic bioremediation management.     

Effect of Nutrient Periodicity on Microbial Community Dynamics

When microbes are subjected to temporal changes in nutrient availability, growth rate and substrate affinity can contribute to competitive fitness and thereby affect microbial community structure. This hypothesis was tested using planktonic bacterial communities exposed to nutrient additions at 1-, 3-, 7-, or 14-day intervals. Growth rates after nutrient addition were inversely proportional to the pulse interval and declined from 0.5 h⁻¹ to 0.15 h⁻¹ as the pulse interval increased from 1 to 14 days. The dynamics of community structure were monitored by 16S rRNA gene PCR-denaturing gradient gel electrophoresis. At pulse intervals of more than 1 day, the community composition continued to change over 130 days. Although replicate systems exposed to the same pulse interval were physiologically similar, their community compositions could exhibit as much dissimilarity (Dice similarity coefficients of <0.5) as did systems operated at different intervals. Bacteria were cultivated from the systems to determine if the physiological characteristics of individual members were consistent with the measured performance of the systems. The isolates fell into three bacterial divisions, Bacteroidetes, Proteobacteria, and Actinobacteria. In agreement with community results, bacteria isolated from systems pulsed every day with nutrients had higher growth rates and ectoaminopeptidase specific activities than isolates from systems pulsed every 14 days. However, the latter isolates did not survive starvation longer than those provided with nutrients every day. The present study demonstrates the dynamic nature of microbial communities exposed to even simple and regular environmental discontinuities when a substantial pool of species that can catabolize the limiting substrate is present.     

Microbial Interactions within a Cheese Microbial Community

The interactions that occur during the ripening of smear cheeses are not well understood. Yeast-yeast interactions and yeast-bacterium interactions were investigated within a microbial community composed of three yeasts and six bacteria found in cheese. The growth dynamics of this community was precisely described during the ripening of a model cheese, and the Lotka-Volterra model was used to evaluate species interactions. Subsequently, the effects on ecosystem functioning of yeast omissions in the microbial community were evaluated. It was found both in the Lotka-Volterra model and in the omission study that negative interactions occurred between yeasts. Yarrowia lipolytica inhibited mycelial expansion of Geotrichum candidum, whereas Y. lipolytica and G. candidum inhibited Debaryomyces hansenii cell viability during the stationary phase. However, the mechanisms involved in these interactions remain unclear. It was also shown that yeast-bacterium interactions played a significant role in the establishment of this multispecies ecosystem on the cheese surface. Yeasts were key species in bacterial development, but their influences on the bacteria differed. It appeared that the growth of Arthrobacter arilaitensis or Hafnia alvei relied less on a specific yeast function because these species dominated the bacterial flora, regardless of which yeasts were present in the ecosystem. For other bacteria, such as Leucobacter sp. or Brevibacterium aurantiacum, growth relied on a specific yeast, i.e., G. candidum. Furthermore, B. aurantiacum, Corynebacterium casei, and Staphylococcus xylosus showed reduced colonization capacities in comparison with the other bacteria in this model cheese. Bacterium-bacterium interactions could not be clearly identified.     

Effect of Microbial Species Richness on Community Stability and Community Function in a Model Plant-Based Wastewater Processing System

Microorganisms will be an integral part of biologically based waste processing systems used for water purification or nutrient recycling on long-term space missions planned by the National Aeronautics and Space Administration. In this study, the function and stability of microbial inocula of different diversities were evaluated after inoculation into plant-based waste processing systems. The microbial inocula were from a constructed community of plant rhizosphere-associated bacteria and a complexity gradient of communities derived from industrial wastewater treatment plant-activated sludge. Community stability and community function were defined as the ability of the community to resist invasion by a competitor (Pseudomonas fluorescens 5RL) and the ability to degrade surfactant, respectively. Carbon source utilization was evaluated by measuring surfactant degradation and through Biolog and BD oxygen biosensor community level physiological profiling. Community profiles were obtained from a 16S–23S rDNA intergenic spacer region array. A wastewater treatment plant-derived community with the greatest species richness was the least susceptible to invasion and was able to degrade surfactant to a greater extent than the other complexity gradient communities. All communities resisted invasion by a competitor to a greater extent than the plant rhizosphere isolate constructed community. However, the constructed community degraded surfactant to a greater extent than any of the other communities and utilized the same number of carbon sources as many of the other communities. These results demonstrate that community function (carbon source utilization) and community stability (resistance to invasion) are a function of the structural composition of the community irrespective of species richness or functional richness.     

A Long-Term Cultivation of an Anaerobic Methane-Oxidizing Microbial Community from Deep-Sea Methane-Seep Sediment Using a Continuous-Flow Bioreactor

Anaerobic oxidation of methane (AOM) in marine sediments is an important global methane sink, but the physiological characteristics of AOM-associated microorganisms remain poorly understood. Here we report the cultivation of an AOM microbial community from deep-sea methane-seep sediment using a continuous-flow bioreactor with polyurethane sponges, called the down-flow hanging sponge (DHS) bioreactor. We anaerobically incubated deep-sea methane-seep sediment collected from the Nankai Trough, Japan, for 2,013 days in the bioreactor at 10°C. Following incubation, an active AOM activity was confirmed by a tracer experiment using ¹³C-labeled methane. Phylogenetic analyses demonstrated that phylogenetically diverse Archaea and Bacteria grew in the bioreactor. After 2,013 days of incubation, the predominant archaeal components were anaerobic methanotroph (ANME)-2a, Deep-Sea Archaeal Group, and Marine Benthic Group-D, and Gammaproteobacteria was the dominant bacterial lineage. Fluorescence in situ hybridization analysis showed that ANME-1 and -2a, and most ANME-2c cells occurred without close physical interaction with potential bacterial partners. Our data demonstrate that the DHS bioreactor system is a useful system for cultivating fastidious methane-seep-associated sedimentary microorganisms.     

Effect of Metal-Rich Sludge Amendments on the Soil Microbial Community

The effects of heavy-metal-containing sewage sludge on the soil microbial community were studied in two agricultural soils of different textures, which had been contaminated separately with three predominantly single metals (Cu, Zn, and Ni) at two different levels more than 20 years ago. We compared three community-based microbiological measurements, namely, phospholipid fatty acid (PLFA) analysis to reveal changes in species composition, the Biolog system to indicate metabolic fingerprints of microbial communities, and the thymidine incorporation technique to measure bacterial community tolerance. In the Luddington soil, bacterial community tolerance increased in all metal treatments compared to an unpolluted-sludge-treated control soil. Community tolerance to specific metals increased the most when the same metal was added to the soil; for example, tolerance to Cu increased most in Cu-polluted treatments. A dose-response effect was also evident. There were also indications of cotolerance to metals whose concentration had not been elevated by the sludge treatment. The PLFA pattern changed in all metal treatments, but the interpretation was complicated by the soil moisture content, which also affected the results. The Biolog measurements indicated similar effects of metals and moisture to the PLFA measurements, but due to high variation between replicates, no significant differences compared to the uncontaminated control were found. In the Lee Valley soil, significant increases in community tolerance were found for the high levels of Cu and Zn, while the PLFA pattern was significantly altered for the soils with high levels of Cu, Ni, and Zn. No effects on the Biolog measurements were found in this soil.     

菌肥的作用研究及其资源开发

论述了菌肥对作物及土壤的作用 ,分别归纳了菌肥中自生性肥料、共生性肥料、抗生性肥料各自的作用 ,以及菌肥资源的开发现状及前景     

Rice to Vegetables: Short- Versus Long-Term Impact of Land-Use Change on the Indigenous Soil Microbial Community

Land-use change is known to have a significant effect on the indigenous soil microbial community, but it is unknown if there are any general trends regarding how this effect varies over time. Here, we describe a comparative analysis of microbial communities from three adjacent agricultural fields: one-century-old paddy field (OP) and two vegetable fields (new vegetable field (NV) and old vegetable field (OV)) that were established on traditional paddy fields 10 and 100 years ago, respectively. Soil chemical and physical analysis showed that both vegetable fields were more nutrient rich than the paddy field in terms of organic C, total N, total P, and available K. The vegetable fields possessed relatively higher abundance of culturable bacteria, fungi, and specific groups of bacteria (Actinomyces, nitrifying bacteria, and cellulose-decomposing bacteria) but lower levels of microbial biomass C and N. Notably, the decrease of biomass was further confirmed by analysis of seven additional soils in chronosequence sampled from the same area. Next we examined the metabolic diversity of the microbial community using the EcoPlate^TM system from Biolog Inc. (Hayward, CA, USA). The utilization patterns of 31 unique C substrates (i.e., community-level physiological profile) showed that microorganisms in vegetable soil and paddy soil prefer to use different C substrates (polymeric compounds for NV and OV soils, phenolic acids for OP soil). Principal component analysis and the average well color development data showed that the NV is metabolically more distinct from the OV and OP. The effect was likely attributable to the elevated soil pH in NV soil. Furthermore, we assessed the diversity of soil bacterial populations using the cultivation-independent technology of amplified ribosomal DNA restriction analysis (ARDRA). Results showed that levels of bacterial diversity in OP and NV soils were similar (Shannon’s diversity index H = 4.83 and 4.79, respectively), whereas bacteria in OV soil have the lowest score of diversity (H = 3.48). The low level of bacterial diversity in OV soil was supported by sequencing of ten randomly selected 16S rDNA clones from each of the three rDNA libraries. Phylogenetic analysis showed that all the ten OV clones belonged to Proteobacteria with eight in the gamma-subdivision and two in the alpha-subdivision. In contrast, the ten clones from NV and OP soils were classified into four and eight bacterial classes or unclassified groups, respectively. Taken together, our data suggest that land-use change from rice to vegetables resulted in a decrease of bacterial diversity and soil biomass despite an increase in the abundance of culturable microorganisms and, moreover, the decrease of bacterial diversity occurred during long-term rather than short-term vegetable cultivation.     

Shifting Species Interaction in Soil Microbial Community and Its Influence on Ecosystem Functions Modulating

The supportive and negative evidence for the stress gradient hypothesis (SGH) led to an ongoing debate among ecologists and called for new empirical and theoretical work. In this study, we took various biological soil crust (BSCs) samples along a spatial gradient with four environmental stress levels to examine the fitness of SGH in microbial interactions and evaluate its influence on biodiversity–function relationships in BSCs. A new assessment method of species interactions within hard-cultured invisible soil community was employed, directly based on denaturing gradient gel electrophoresis fingerprint images. The results showed that biotic interactions in soil phototroph community dramatically shifted from facilitation to dominant competition with the improvement of microhabitats. It offered new evidence, which presented a different perspective on the hypothesis that the relative importance of facilitation and competition varies inversely along the gradient of abiotic stress. The path analysis indicated that influence of biotic interactions (r?=?0.19, p?<?0.05) on ecosystem functions is lower than other community properties (r?=?0.62, p?<?0.001), including soil moisture, crust coverage, and biodiversity. Furthermore, the correlation between species interactions and community properties was non-significant with low negative influence (r?=??0.27, p?>?0.05). We demonstrate that the inversion of biotic interaction as a response to the gradient of abiotic stresses existed not only in the visible plant community but also in the soil microbial community.     

The Effect of Biochar and Its Interaction with the Earthworm Pontoscolex corethrurus on Soil Microbial Community Structure in Tropical Soils

Biochar effects on soil microbial abundance and community structure are keys for understanding the biogeochemical cycling of nutrients and organic matter turnover, but are poorly understood, in particular in tropical areas. We conducted a greenhouse experiment in which we added biochars produced from four different feedstocks [sewage sludge (B1), deinking sewage sludge (B2), Miscanthus (B3) and pine wood (B4)] at a rate of 3% (w/w) to two tropical soils (an Acrisol and a Ferralsol) planted with proso millet (Panicum milliaceum L.). The interactive effect of the addition of earthworms was also addressed. For this purpose we utilized soil samples from pots with or without the earthworm Pontoscolex corethrurus, which is a ubiquitous earthworm in tropical soils. Phospholipid fatty acid (PLFA) measurements showed that biochar type, soil type and the presence of earthworms significantly affected soil microbial community size and structure. In general, biochar addition affected fungal but not bacterial populations. Overall, biochars rich in ash (B1 and B2) resulted in a marked increase in the fungi to bacteria ratio, while this ratio was unaltered after addition of biochars with a high fixed carbon content (B3 and B4). Our study remarked the contrasting effect that both, biochar prepared from different materials and macrofauna, can have on soil microbial community. Such changes might end up with ecosystem-level effects.     

Characterizing the Microbial Colonization of a Dolostone Quarry: Implications for Stone Biodeterioration and Response to Biocide Treatments

This study examines the microbial colonization of three fronts of an abandoned dolostone quarry (Redueña, Madrid, Spain) exposed to atmospheric conditions for different time periods since Roman times to the present. Through scanning electron microscopy in backscattered electron mode (SEM-BSE), endolithic colonization was predominantly detected in the most recently exposed front, while in the longer exposed quarry fronts, epilithic forms of growth were most often observed. These observations were confirmed by denaturing gradient gel electrophoresis (DGGE) analysis. Based on the distribution pattern of microbial colonization in the different quarry fronts, we then established a sequence of colonization events that took place over this long time frame. Bioalteration processes related to this sequential colonization were also identified. Characterizing these sequential processes can be useful for interpreting biodeterioration processes in historic dolostone monuments, especially those affecting constructions in the area of the Redueña stone quarry. In a second experimental stage, different biocide treatments were tested on this quarry rock to find the best way to avoid the microbial colonization effects identified. Through combined SEM-BSE/DGGE analysis, the efficacy of several biocides against the microorganisms inhabiting the dolostones was assessed after 4 and 16 months treatment. In general, all treatments were effective at reducing around 80% of the lichen cover, although effects on endolithic lithobiontic communities were dependant on how well the rock surface had been mechanically cleaned prior to treatment and gradually disappeared over time.     

MAR-FISH技术及其在环境微生物群落与功能研究中的应用

对复杂环境中微生物群落结构和功能的研究是微生物生态学的重要任务。尽管现代分子生物学技术已经成功地用于解析环境中微生物的群落结构, 但是这些方法并不能提供微生物的原位生理学信息。而一种新的方法, 微观放射自显影和荧光原位杂交集成技术(MAR-FISH)则能够同时在单细胞水平上, 检测复杂环境中微生物的系统发育信息及其生理特性。本文总结了MAR-FISH方法的原理, 实验步骤及其在环境微生物群落与功能研究中的应用。     

Evaluation of Methyl Bromide Alternatives Efficacy against Soil-Borne Pathogens,Nematodes and Soil Microbial Community

Methyl bromide (MB) and other alternatives were evaluated for suppression of Fusarium spp., Phytophthora spp., and Meloidogyne spp. and their influence on soil microbial communities. Both Fusarium spp. and Phytophthora spp. were significantly reduced by the MB (30.74 mg kg^-1), methyl iodide (MI: 45.58 mg kg^-1), metham sodium (MS: 53.92 mg kg^-1) treatments. MS exhibited comparable effectiveness to MB in controlling Meloidogyne spp. and total nematodes, followed by MI at the tested rate. By contrast, sulfuryl fluoride (SF: 33.04 mg kg^-1) and chloroform (CF: 23.68 mg kg^-1) showed low efficacy in controlling Fusarium spp., Phytophthora spp., and Meloidogyne spp. MB, MI and MS significantly lowered the abundance of different microbial populations and microbial biomass in soil, whereas SF and CF had limited influence on them compared with the control. Diversity indices in Biolog studies decreased in response to fumigation, but no significant difference was found among treatments in PLFA studies. Principal component and cluster analyses of Biolog and PLFA data sets revealed that MB and MI treatments greatly influenced the soil microbial community functional and structural diversity compared with SF treatment. These results suggest that fumigants with high effectiveness in suppressing soil-borne disease could significantly influence soil microbial community.     

Microbial Responses to Long-Term N Deposition in a Semiarid Grassland

Nitrogen (N) enrichment of the biosphere is an expanding problem to which arid ecosystems may be particularly sensitive. In semiarid grasslands, scarce precipitation uncouples plant and microbial activities, and creates within the soil a spatial mosaic of rhizosphere and cyanobacterial crust communities. We investigated the impact of elevated N deposition on these soil microbial communities at a grama-dominated study site located incentral New Mexico (USA). The study plots were established in 1995 and receive 10 kg ha⁻¹ year⁻¹ of supplemental N in the form of NH₄NO₃. Soil samples were collected in July 2004, following 2 years of severe drought, and again in March 2005 following a winter of record high precipitation. Soils were assayed for potential activities of 20 extracellular enzymes and N₂O production. The rhizosphere and crust-associated soils had peptidase and peroxidase potentials that were extreme in relation to those of temperate soils. N addition enhanced glycosidase and phosphatase activities and depressed peptidase. In contrast to temperate forest soils, oxidative enzyme activity did not respond to N treatment. Across sampling dates, extracellular enzyme activity responses correlated with inorganic N concentrations. N₂O generation did not vary significantly with soil cover or N treatment. Microbial responses to N deposition in this semiarid grassland were distinct from those of forest ecosystems and appear to be modulated by inorganic N accumulation, which is linked to precipitation patterns.     

Microbial Diversity and Community Structure on Corroding Concretes

The objective of this study was to analyze bacterial diversity in two different concrete samples to understand the dominant types of bacteria that may contribute to concrete corrosion. Two concrete samples, HN-1 from the sunny side and HN-2 from dark and damp side, were collected from Zijin Mountain in Nanjing and genomic DNA was extracted. The partial bacterial 16S rRNA gene fragment was PCR amplified and two clone libraries were constructed. Amplified ribosomal DNA restriction analysis (ARDRA) was performed by digestion of the 16S rRNA gene and each unique restriction fragment polymorphism pattern was designated as an operational taxonomic unit (OTU). Phylogenetic trees of bacterial 16S rDNA nucleotide sequences were constructed. Sample HN-1 and HN-2 contained 21 OTUs and 26 OTUs, respectively. Proteobacteria and Planctomycetes were the predominant bacteria in both samples, and they are distributed among Herbaspirillum, Archangium, Phyllobacteriaceae and Planctomycetaceae. Cyanobacteria and Rubrobacter sp. are dominant in HN-1; while Acidobacteriaceae, Adhaeribacter sp. and Nitrospira sp. are predominant in HN-2. This distribution pattern was consistent with local environmental conditions of these two samples. The inferred physiological characteristics of these bacteria, based on relatedness of the DNA clone sequences to cultivated species, revealed different mechanisms of concrete corrosion depending on the local environmental conditions.     

Effect of a Sinorhizobium meliloti Strain with a Modified putA Gene on the Rhizosphere Microbial Community of Alfalfa

The success of a rhizobial inoculant in the soil depends to a large extent on its capacity to compete against indigenous strains. M403, a Sinorhizobium meliloti strain with enhanced competitiveness for nodule occupancy, was recently constructed by introducing a plasmid containing an extra copy of a modified putA (proline dehydrogenase) gene. This strain and M401, a control strain carrying the same plasmid without the modified gene, were used as soil inoculants for alfalfa in a contained field release experiment at León, Spain. In this study, we determined the effects of these two strains on the indigenous microbial community. 16S rRNA genes were obtained from the rhizosphere of alfalfa inoculated with strain M403 or strain M401 or from noninoculated plants by amplification of DNA from soil with bacterial group-specific primers. These genes were analyzed and compared by restriction fragment length polymorphism and temperature gradient gel electrophoresis. The results allowed us to differentiate between alterations in the microbial community apparently caused by inoculation and by the rhizosphere effect and seasonal fluctuations induced by the alfalfa plants and by the environment. Only moderate inoculation-dependent effects could be detected, while the alfalfa plants appeared to have a much stronger influence on the microbial community.