Model system for studies of microbial dynamics at exuding surfaces such as the rhizosphere

An autoclavable all-glass system for studying microbial dynamics at permeable surfaces is described. Standard hydrophobic or hydrophilic membranes (46-mm diameter) of various pore sizes were supported on a glass frit through which nutrient solutions were pumped by a peristaltic pump. The pump provided a precisely controlled flow at speeds of 0.5 to 500 ml of defined or natural cell exudates per h, which passed through the membrane into a receiving vessel. The construction allowed a choice of membranes, which could be modified. The system was tested with a bacterium, isolated from rape plant roots (Brassica napus L.), that was inoculated on a hydrophilic membrane filter and allowed to develop into a biofilm. A defined medium with a composition resembling that of natural rape root exudate was pumped through the membrane at 0.5 ml/h. Scanning electron microscopic examinations indicated that the inoculum formed microcolonies embedded in exopolymers evenly distributed over the membrane surface. The lipid composition and content of poly-beta-hydroxybutyrate in free-living and adhered cells were determined by gas chromatography. The bacterial consumption of amino acids in the exudate was also studied.     

毛竹根际可培养微生物种群多样性分析

[目的]为了了解天然毛竹林根际可培养微生物种群的多样性信息,[方法]采用稀释平板法,对浙江天目山和重庆缙云山天然毛竹林根际细菌和放线菌进行了分离,并对其16S rDNA序列进行了分析.[结果]分别从天目山和缙云山天然毛竹林根际分离得到51株和31株菌落形态差异的细菌和放线菌.16S rDNA序列分析表明,天目山和缙云山毛竹根际细菌主要包括厚壁菌门(Firmicutes,分别为40%和58%)、放线菌门(Actinobacteria,分别为36.7%和10.52%)、变形菌门-亚群(Alphaproteobacteria,分别为10%和5.26%)和变形菌门 --亚群(Gammaproteobacteria,分别为10%和26.32%),其中芽孢杆菌属(Bacillus sp.)为共同的优势菌属(分别为34.38%和42.11%).分离的菌株中,B188、B171和B152等6株与GenBank中已报道16S rRNA基因序列的相似性从90%到96%不等,可能代表着新属或种.[结论]这表明,天然毛竹林根际具有较为丰富的可培养微生物种群多样性,并存在一些潜在的新的微生物菌种资源.     

Solvent-exposed tryptophans probe the dynamics at protein surfaces.

The dynamics of single tryptophan (W) side chain of protease subtilisin Carlsberg (SC) and myelin basic protein (MBP) were used for probing the surface of these proteins. The W side chains are exposed to the solvent, as shown by the extent of quenching of their fluorescence by KI. Time-resolved fluorescence anisotropy measurements showed that the rotational motion of W is completely unhindered in the case of SC and partially hindered in the case of MBP. The rotational correlation time (phi) associated with the fast local motion of W did not scale linearly with the bulk solvent viscosity (eta) in glycerol-water mixtures. In contrast, phi values of either W side chains in the denatured proteins or the free W scaled almost linearly with eta, as expected by the Stokes-Einstein relationship. These results were interpreted as indicating specific partitioning of water at the surface of the proteins in glycerol-water mixtures.     

Temporal dynamics and genetic diversity of chemotactic‐competent microbial populations in the rhizosphere

The contribution of chemotaxis to the competitive colonization of the rhizosphere for the vast majority of the soil community is unknown. We have developed and applied a molecular diagnostic tool, based on a gene encoding the central regulator of bacterial chemotaxis (cheA), to characterize and temporally track specific populations of native microbes with chemotaxis potential that are present in soil exposed to two rhizospheres: wheat and cowpea. The data show that the chemotactic‐competent communities present in the rhizospheres of the two plants are distinct and less diverse than the bulk soil, indicating the development of unique microbial communities. Consistent with the supposition that selection and recruitment of specific soil microbes takes place in the rhizosphere, the dynamics of specific cheA phylotypes provides support for the hypothesis that chemotaxis provides a competitive advantage to some soil microbes. This is the first study to examine and profile the genetic diversity of chemotaxis genes in natural populations. As such, it illustrates our limited understanding of microbial chemotaxis for the majority of soil microbes. It also highlights the value of a culture‐independent approach for examining chemotaxis populations in order to build empirical lines of evidence for its role in structuring of microbial assemblages.     

Nematode-enhanced microbial colonization of the wheat rhizosphere

The mechanisms by which seed-applied bacteria colonize the rhizosphere in the absence of percolating water are poorly understood. Without mass flow, transport of bacteria by growing roots or soil animals, particularly nematodes may be important. We used a sand-based microcosm system to investigate the ability of three species of nematodes (Caenorhabditis elegans, Acrobeloides thornei and a Cruznema sp.) to promote rhizosphere colonization by four strains of beneficial rhizobacteria. In nearly all cases, rhizosphere colonization was substantially increased by the presence of nematodes, irrespective of bacterial or nematode species. Our results suggest that nematodes are important vectors for bacteria rhizosphere colonization in the absence of percolating water.     

小麦-蚕豆间作条件下氮肥施用量对根际微生物区系的影响

通过田间小区试验,研究了小麦/蚕豆间作条件下4种施氮水平(0、90、180和270 kg·hm^-2)对根际微生物区系和多样性的影响.结果表明：在整个生育期,微生物数量有一定的波动,但均以开花期数量最高.与单作相比,间作显著增加了小麦和蚕豆根际的细菌、真菌、放线菌数量和微生物总量,而显著降低了开花期和成熟期蚕豆根际的微生物多样性.在不施氮（N₀）和低氮（N₉₀）水平下,间作与单作在微生物数量上的差异较大.间作对土壤微生物数量的促进效应在分蘖期和开花期最大,成熟期显著降低.小麦根际的微生物数量随施氮量的增加先增加后降低,以常规施氮处理（N₁₈₀）的微生物数量最多,氮肥用量对单作小麦的影响大于间作小麦. 施氮量对蚕豆根际细菌、真菌、放线菌数量和微生物多样性无显著影响,但降低了间作蚕豆根际微生物总量.适量施用氮肥能有效调节根际微生物区系,间作系统地上部植物多样性与地下部微生物区系间存在紧密联系.     

Population dynamics ofFusarium spp. in pea rhizosphere as affected by soil amendments

In nonamended soil, vegetative growth and sporulation ofFusarium spp. were higher in the rhizosphere than in the soil. Sporulation was favoured by young plants and decreased with increasing plant age. Amendments with low C/N oil-cakes enhanced vegetative growth and sporulation in root-free soil. The extent of stimulation varied with the nature of organic matter used and the stage of its deoomposition. Sporulation was suppressed by castor cake and sawdust with urea amendments. Rhizosphere cf pea altered the effect of different amendments.     

Quantitative studies of bacterial chemotaxis and microbial population dynamics

Although there is a long history of conjecture regarding the role and significance of bacterial chemotaxis in microbial ecology, only recently has a significant body of work appeared attempting to address this issue. The purpose of this paper is to provide a concise overview of this work, which combined mathematical modeling of bacterial population migration and experimental measurement of the model parameters with modeling of competitive microbial population dynamics in a nonmixed environment. Predictions from the population dynamics models, based on experimental estimates of the various motility and growth parameter values, are related to the small number of experimental observations available to date dealing with the effects of bacterial motility on competition in a nonmixed environment. Current results indicate that cell motility and chemotaxis properties can be as important to population dynamics as cell growth kinetic properties, so that greater attention to this aspect of microbial behavior is warranted in future studies of microbial ecology.     

Agarose as a suitable substrate for use in the study of Al dynamics in the rhizosphere

Because of experimental difficulties, few authors have studied the dynamics of aluminium in the rhizosphere. The aim of this paper is to present a suitable method for studying rhizosphere Al dynamics. It is based on the use of agarose as a substrate for plant growth. Agar and agarose gels are often used in rhizosphere studies, but most are poorly characterized and occasionally give rise to experimental artefacts, especially with low mobility elements like Al. The results reported here show that agarose is a relatively pure substrate, nearly devoid of phosphorus and other Al-complexing substances. Aqueous extracts of agarose also exhibit Al phytotoxicity equivalent to that of a nutrient solution. Since this substrate has the properties of a variable charge exchange complex, it can be considered as a physico-chemical model for organic matter. Finally, its Al adsorption capacity is high enough for the Al reserve in the substrate not to exert a limiting effect on plants and low enough to allow accurate measurement of Al depletion in the rhizophere.     

Low amounts of herbivory by root-knot nematodes affect microbial community dynamics and carbon allocation in the rhizosphere

Increased carbon translocation to the rhizosphere via 'leakage' induced by low amounts of plant parasitic nematodes can foster microorganisms. The effects of the root-knot nematode Meloidogyne incognita on microbial biomass (C(mic)) and community structure (phospholipid fatty acids) in the rhizosphere of barley were studied. Inoculation densities of 2000, 4000, and 8000 nematodes were well below the threshold level for plant damage. A (13)CO(2) pulse-labelling was performed to assess the distribution of assimilated (13)C in the rhizosphere. Infection with M. incognita increased the carbon concentration in shoots, and enhanced root biomass slightly. The presence of nematodes did not affect microbial biomass, but significantly changed the allocation of the recent photosynthate. Less plant carbon was sequestered by microorganisms with increasing nematode abundance. Microbial community structure was distinctly altered in the early stages of the plant-nematode interactions. Both, bacteria and fungi, showed a positive response with 2000, and a negative one with 4000 and 8000 M. incognita added. The results suggest that low-level root herbivory still imposes a considerable carbon demand, and that proliferation of microorganisms due to increased rhizodeposition may be short-termed. The carbon flow to rhizosphere microbial communities is likely dependent on the specific nematode-plant association and the developmental stage of the nematode in the host.     

Wipe-rinse technique for quantitating microbial contamination on large surfaces.

The Orca Basin is a hypersaline depression in the northern Gulf of Mexico with anoxic conditions observed in the lower 200 m of the water column. Measurements of adenosine 5′-triphosphate, heterotrophic potential, and uridine uptake made above and across the interface into the anoxic zone revealed the presence of an active microbial population approximately 100 m above the interface. Biomass and activity decreased at and just below the interface but increased near the bottom, consistent with similar observations made in the Cariaco Trench. The maximum adenosine 5′-triphosphate concentration above the interface of 5.9 ng/liter (2,173 m) is about eight times greater than the value found in oxygenated waters of corresponding depth in the absence of an anoxic zone. The maximum adenosine 5′-triphosphate concentration in the anoxic zone is approximately 15 times greater than that found in oxygenated water of similar depth, suggesting anoxia will support the development of a larger bacterial population. Our findings suggest that autotrophic bacteria may be the dominant physiological group in the region just above the interface.     

Formation of palladium(0) nanoparticles at microbial surfaces

The increasing demand and limited natural resources for industrially important platinum‐group metal (PGM) catalysts render the recovery from secondary sources such as industrial waste economically interesting. In the process of palladium (Pd) recovery, microorganisms have revealed a strong potential. Hitherto, bacteria with the property of dissimilatory metal reduction have been in focus, although the biochemical reactions linking enzymatic Pd(II) reduction and Pd(0) deposition have not yet been identified. In this study we investigated Pd(II) reduction with formate as the electron donor in the presence of Gram‐negative bacteria with no documented capacity for reducing metals for energy production: Cupriavidus necator, Pseudomonas putida, and Paracoccus denitrificans. Only large and close‐packed Pd(0) aggregates were formed in cell‐free buffer solutions. Pd(II) reduction in the presence of bacteria resulted in smaller, well‐suspended Pd(0) particles that were associated with the cells (called “bioPd(0)” in the following). Nanosize Pd(0) particles (3–30 nm) were only observed in the presence of bacteria, and particles in this size range were located in the periplasmic space. Pd(0) nanoparticles were still deposited on autoclaved cells of C. necator that had no hydrogenase activity, suggesting a hydrogenase‐independent formation mechanism. The catalytic properties of Pd(0) and bioPd(0) were determined by the amount of hydrogen released in a reaction with hypophosphite. Generally, bioPd(0) demonstrated a lower level of activity than the Pd(0) control, possibly due to the inaccessibility of the Pd(0) fraction embedded in the cell envelope. Our results demonstrate the suitability of bacterial cells for the recovery of Pd(0), and formation and immobilization of Pd(0) nanoparticles inside the cell envelope. However, procedures to make periplasmic Pd(0) catalytically accessible need to be developed for future nanobiotechnological applications. Biotechnol. Bioeng. 2010;107: 206–215. © 2010 Wiley Periodicals, Inc.     

Pesticide dissipation and microbial community changes in a biopurification system: influence of the rhizosphere

The dissipation of atrazine, chlorpyrifos and iprodione in a biopurification system and changes in the microbial and some biological parameters influenced by the rhizosphere of Lolium perenne were studied in a column system packed with an organic biomixture. Three column depths were analyzed for residual pesticides, peroxidase, fluorescein diacetate activity and microbial communities. Fungal colonization was analyzed by confocal laser scanning microscopy to assess the extent of its proliferation in wheat straw. The L. perenne rhizosphere enhanced pesticide dissipation and negligible pesticide residues were detected at 20–30 cm column depth. Atrazine, chlorpyrifos and iprodione removal was 82, 89 and 74% respectively in the first 10 cm depth for columns with vegetal cover. The presence of L. perenne in contaminated columns stimulated peroxidase activity in all three column depth sections. Fluorescein diacetate activity decreased over time in all column sections with the highest values in biomixtures with vegetal cover. Microbial communities, analyzed by PCR-DGGE, were not affected by the pesticide mixture application, presenting high values of similarity (>65%) with and without vegetal cover. Microbial abundance of Actinobacteria varied according to treatment and no clear link was observed. However, bacterial abundance increased over time and was similar with and without vegetal cover. On the other hand, fungal abundance decreased in all sections of columns after 40 days, but an increase was observed in response to pesticide application. Fungal colonization and straw degradation during pesticide dissipation were verified by monitoring the lignin autofluorescence loss.     

Soil microbial diversity as affected by the rhizosphere of the hyperaccumulator Thlaspi caerulescens under natural conditions

It is hypothesized that metal hyperaccumulator plants have specific rhizosphere conditions, potentially modifying the bioavailability of soil metals. This article aims to further the knowledge about the rhizosphere of the hyperaccumulator Thlaspi caerulescens, focusing on its microflora isolated from metalliferous soils collected in situ where the plants grow naturally. We characterized the cultivable microbial communities isolated from the rhizosphere of one population of this Ni hyperaccumulator species grown on a serpentine soil. The rhizosphere soil harbored a wide variety of microorganisms, predominantly bacteria, confirming the stimulatory effect of the T. caerulescens rhizosphere on microbial growth and proliferation. We tested the hypothesis that the rhizosphere of T. caerulescens influences (1) the metabolic diversity of the bacterial community and (2) the bacterial resistance to metals. The principal component analysis of the Biolog plate's data confirmed a structural effect of the rhizosphere of T. caerulescens on bacterial communities. The percentage of Ni-resistant bacteria was higher in the rhizosphere than in the bulk soil, suggesting a direct effect of the rhizosphere on Ni tolerance, reflecting a greater bacterial tolerance to Ni in the rhizosphere.     

Molecular dynamics of microbial lipases as determined from their intrinsic tryptophan fluorescence.

We have studied the intrinsic tryptophan fluorescence of the lipases from Chromobacterium viscosum (CVL), Pseudomonas species (PSL), and Rhizopus oryzae (ROL) in aqueous buffer, zwitterionic detergent micelles, and isopropanol-water mixtures. It was the purpose of this study to obtain information about biophysical properties of the respective enzymes under conditions that modulate enzyme activities and stereoselectivities to a significant extent. According to their decay-associated emission spectra, CVL tryptophans are located in the hydrophobic interior of the protein. In contrast, the PSL and ROL tryptophans are probably confined to the core and the surface of the lipase. From the tryptophan lifetime distributions it can be concluded that the conformation of CVL is not much affected by detergent or organic solvent (isopropanol). Accordingly, CVL is enzymatically active in these systems and most active in the presence of isopropanol. In contrast, ROL and PSL show high conformational mobility, depending on the solvent, because their lifetime distributions are very different in the presence and absence of detergent or isopropanol. Time-resolved anisotropy studies provided evidence that the lipases exhibit very high internal molecular flexibility. This peculiar feature of lipases is perhaps the key to the great differences in activity and stereoselectivity observed in different reaction media. Furthermore, information about self-association of the lipases in different solvents could be obtained. PSL, but not CVL and ROL, forms aggregates in water. Lipase aggregation can be reversed by the addition of detergent or isopropanol, which competes for the hydrophobic surface domains of this protein. This dissociation could efficiently contribute to the increase in lipase activity in the presence of a detergent or isopropanol.     

Optically transparent porous medium for nondestructive studies of microbial biofilm architecture and transport dynamics

We describe a novel and noninvasive, microscopy-based method for visualizing the structure and dynamics of microbial biofilms, individual fluorescent microbial cells, and inorganic colloids within a model porous medium. Biofilms growing in flow cells packed with granules of an amorphous fluoropolymer could be visualized as a consequence of refractive index matching between the solid fluoropolymer grains and the aqueous immersion medium. In conjunction with the capabilities of confocal microscopy for nondestructive optical sectioning, the use of amorphous fluoropolymers as a solid matrix permits observation of organisms and dynamic processes to a depth of 2 to 3 mm, whereas sediment biofilms growing in sand-filled flow cells can only be visualized in the region adjacent to the flow cell wall. This method differs fundamentally from other refractive index-matching applications in that optical transparency was achieved by matching a solid phase to water (and not vice versa), thereby permitting real-time microscopic studies of particulate-containing, low-refractive-index media such as biological and chromatographic systems.     

Biogenic volatile organic compounds as potential carbon sources for microbial communities in soil from the rhizosphere of Populus tremula

Catabolism of a (14)C-labelled volatile monoterpene compound (geraniol) to (14)CO(2) was investigated in soils taken from the rhizosphere at distances up to 200 cm from the trunks of three small Populus tremula trees growing at different sites in Slovenia. Emissions of limonene of up to 18 microg m(-2) h(-1) were detected from the soil surface at each site. Evolution of (14)C-labelled CO(2) was measured as a product of catabolism of (14)C-labelled geraniol introduced into the soil samples. Indigenous soil microorganisms degraded the geraniol rapidly. There was a significant difference in relative lag times and rates of catabolism along the gradient from the tree trunks, with relatively longer lag times and lower rates occurring in soil samples from the farthest point from the tree.