Seasonal controls on grassland microbial biogeography: Are they governed by plants, abiotic properties or both?

Temporal dynamics create unique and often ephemeral conditions that can influence soil microbial biogeography at different spatial scales. This study investigated the relation between decimeter to meter spatial variability of soil microbial community structure, plant diversity, and soil properties at six dates from April through November. We also explored the robustness of these interactions over time. An historically unfertilized, unplowed grassland in southwest Germany was selected to characterize how seasonal variability in the composition of plant communities and substrate quality changed the biogeography of soil microorganisms at the plot scale (10 m x 10 m). Microbial community spatial structure was positively correlated with the local environment, i.e. physical and chemical soil properties, in spring and autumn, while the density and diversity of plants had an additional effect in the summer period. Spatial relationships among plant and microbial communities were detected only in the early summer and autumn periods when aboveground biomass increase was most rapid and its influence on soil microbial communities was greatest due to increased demand by plants for nutrients. Individual properties exhibited varying degrees of spatial structure over the season. Differential responses of Gram positive and Gram negative bacterial communities to seasonal shifts in soil nutrients were detected. We concluded that spatial distribution patterns of soil microorganisms change over a season and that chemical soil properties are more important controlling factors than plant density and diversity. Finer spatial resolution, such as the mm to cm scale, as well as taxonomic resolution of microbial groups, could help determine the importance of plant species density, composition, and growth stage in shaping microbial community composition and spatial patterns. (C) 2014 The Authors. Published by Elsevier Ltd. All rights reserved.

Plant diversity drives soil microbial biomass carbon in grasslands irrespective of global environmental change factors

Soil microbial biomass is a key determinant of carbon dynamics in the soil. Several studies have shown that soil microbial biomass significantly increases with plant species diversity, but it remains unclear whether plant species diversity can also stabilize soil microbial biomass in a changing environment. This question is particularly relevant as many global environmental change (GEC) factors, such as drought and nutrient enrichment, have been shown to reduce soil microbial biomass. Experiments with orthogonal manipulations of plant diversity and GEC factors can provide insights whether plant diversity can attenuate such detrimental effects on soil microbial biomass. Here, we present the analysis of 12 different studies with 14 unique orthogonal plant diversity × GEC manipulations in grasslands, where plant diversity and at least one GEC factor (elevated CO2, nutrient enrichment, drought, earthworm presence, or warming) were manipulated. Our results show that higher plant diversity significantly enhances soil microbial biomass with the strongest effects in long-term field experiments. In contrast, GEC factors had inconsistent effects with only drought having a significant negative effect. Importantly, we report consistent non-significant effects for all 14 interactions between plant diversity and GEC factors, which indicates a limited potential of plant diversity to attenuate the effects of GEC factors on soil microbial biomass. We highlight that plant diversity is a major determinant of soil microbial biomass in experimental grasslands that can influence soil carbon dynamics irrespective of GEC.

Shifts in the microbial community structure in different temperatures during sample storage from IODP Hole 325-M0058A

The objective of this study was to determine shifts in the microbial community structure and potential function based on standard Integrated Ocean Drilling Program (IODP) storage procedures for sediment cores. Standard long-term storage protocols maintain sediment temperature at 4°C for mineralogy, geochemical, and/or geotechnical analysis whereas standard microbiological sampling immediately preserves sediments at -80°C. Storage at 4°C does not take into account populations may remain active over geologic time scales at temperatures similar to storage conditions. Identification of active populations within the stored core would suggest geochemical and geophysical conditions within the core change over time. To test this potential, the metabolically active fraction of the total microbial community was characterized from IODP Expedition 325 Great Barrier Reef sediment cores prior to and following a 3-month storage period. Total RNA was extracted from complementary 2, 20, and 40 m below sea floor sediment samples, reverse transcribed to complementary DNA and then sequenced using 454 FLX sequencing technology, yielding over 14,800 sequences from the six samples. Interestingly, 97.3% of the sequences detected were associated with lineages that changed in detection frequency during the storage period including key biogeochemically relevant lineages associated with nitrogen, iron, and sulfur cycling. These lineages have the potential to permanently alter the physical and chemical characteristics of the sediment promoting misleading conclusions about the in situ biogeochemical environment. In addition, the detection of new lineages after storage increases the potential for a wider range of viable lineages within the subsurface that may be underestimated during standard community characterizations.

Microbial community composition in fresh water at different stations

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes was used to investigate the phylogenetic composition of bacterioplankton communities in several freshwater and marine samples. An average of about 50% of the cells were detected by probes for the domains Bacteria and Archaea. Cells were concentrated from water samples (1 to 100 ml) on white polycarbonate filters (diameter, 47 mm; pore size, 0.2 mm; type GTTP 4700 [Millipore, Eschborn, Germany]) by applying a vacuum of <25 kPa. They were subsequently fixed by covering the filter with 3 ml of a freshly prepared, phosphate-buffered saline (pH 7.2)-4% paraformaldehyde (Sigma, Deisenhofen, Germany) solution for 30 min at room temperature. Airdried filters are ready for hybridization and can be stored at 220°C or room temperature for several months without showing apparent changes. Probes BET42a, GAM42a, and PLA886 were used with competitor oligonucleotides as described previously amongst others in Manz et al., (1992; doi:10.1016/S0723-2020(11)80121-9). The filters were transferred to a vial containing 50 ml of prewarmed (48°C) washing solution (70 mM NaCl, 20 mM Tris-HCl [pH 7.4], 5 mM EDTA, 0.01% sodium dodecyl sulfate) and incubated freely floating without shaking at 48°C for 15 min. The filter sections were dried on Whatman 3M paper (Whatman Ltd., Maidstone, United Kingdom) and covered with 50 ml of DAPI solution (1 mg/ml in distilled water filtered through at 0.2-mm filter) for 5 min at room temperature in the dark. For each sample and probe, more than 500 cells were enumerated; for the DAPI examination, more than 1,500 cells were counted per sample. All probe-specific cell counts are presented as the percentage of cells visualized by DAPI. The mean abundances and standard deviations were calculated from the counts of 10 to 20 randomly chosen fields on each filter section. All counts were corrected by subtracting the counts obtained with the negative control NON338. Mean and standard deviation were calculated from the counts of 10 to 20 randomly chosen fields on each filter section.

Chemical analysis and Microbial community composition of surface water samples from the Southern Lagoon of Venice

The Lagoon of Venice is a large water basin that exchanges water with the Northern Adriatic Sea through three large inlets. We examined two adjacent sites within the Southern Basin and at the Chioggia inlet in autumn 2007 and summer 2008. A pilot study in June 2007 on a surface water sample from Chioggia with a rather high salinity of 36.9 PSU had revealed a conspicuous bloom of CF319a-positive cells likely affiliated with the Cytophaga /Flavobacteria cluster of Bacteroidetes. These flavobacterial abundances were one to two orders of magnitude higher than in other marine surface waters. DAPI-stained cells were identified as bacteria with the general bacterial probe mixture EUB338 I-III. CARD-FISH counts with group-specific probes confirmed the dominance of Bacteroidetes (CF319a), Alphaproteobacteria (ALF968), and Gammaproteobacteria (GAM42a). CARD-FISH showed thatBetaproteobacteria and Planctomycetes were minor components of the bacterioplankton in the Lagoon of Venice.

Microbial community, biomarker concentrations and their isotopic signature in sediment of Gullfaks and Tommeliten methane seeps in the Northern North Sea

Gullfaks is one of the four major Norwegian oil and gas fields, located in the northeastern edge of the North Sea Plateau. Tommeliten lies in the greater Ekofisk area in the central North Sea. During the cruises HE 208 and AL 267 several seep locations of the North Sea were visited. At the Heincke seep at Gullfaks, sediments were sampled in May 2004 (HE 208) using a video-guided multiple corer system (MUC; Octopus, Kiel). The samples were recovered from an area densely covered with bacterial mats where gas ebullition was observed. The coarse sands limited MUC penetration depth to maximal 30 centimeters and the highly permeable sands did not allow for a high-resolution, vertical subsampling because of pore water loss. The gas flare mapping and videographic observation at Tommeliten indicated an area of gas emission with a few small patches of bacterial mats with diameters <50 cm from most of which a single stream of gas bubbles emerged. The patches were spaced apart by 10-100 m. Sampling of sediments covered by bacterial mats was only possible with 3 small push cores (3.8 cm diameter) mounted to ROV Cherokee. These cores were sampled in 3 cm intervals. Lipid biomarker extraction from 10 -17 g wet sediment was carried out as described in detail elsewhere (Elvert et al., 2003; doi:10.1080/01490450303894). Briefly, defined concentrations of cholestane, nonadecanol and nonadecanolic acid with known delta 13C-values were added to the sediments prior to extraction as internal standards for the hydrocarbon, alcohol and fatty acid fraction, respectively. Total lipid extracts were obtained from the sediment by ultrasonification with organic solvents of decreasing polarity. Esterified fatty acids (FAs) were cleaved from the glycerol head group by saponification with methanolic KOH solution. From this mixture, the neutral fraction was extracted with hexane. After subsequent acidification, FAs were extracted with hexane. For analysis, FAs were methylated using BF3 in methanol yielding fatty acid methyl esters (FAMES). The fixation for total cell counts and CARD-FISH were performed on-board directly after sampling. For both methods, sediments were fixed in formaldehyde solution. After two hours, aliquots for CARD-FISH staining were washed with 1* PBS (10mmol/l sodium phosphate solution, 130mmol/l NaCl, adjusted to a pH of 7.2) and finally stored in a 1:1 PBS:ethanol solution at -20°C until further processing. Samples for total cell counts were stored in formalin at 4°C until analysis. For sandy samples, the total cell count/CARD-FISH protocol was optimized to separate sand particles from the cells. Cells were dislodged from sediment grains and brought into solution with the supernatant by sonicating each sample onice for 2 minutes at 50W. This procedure was repeated four times and supernatants were combined. The sediment samples were brought to a final dilution of 1:2000 to 1:4000 and filtered onto 0.2µm GTTP filters (Millipore, Eschbonn, Germany).

Kinetic and phylogenetic characterization of an anaerobic dechlorinating microbial community

The reductive dechlorination (RD) of tetrachloroethene (PCE) to vinyl chloride (VC) and, to a lesser extent, to ethene (ETH) by an anaerobic microbial community has been investigated by studying the processes and kinetics of the main physiological components of the consortium. Molecular hydrogen, produced by methanol-utilizing acetogens, was the electron donor for the PCE RD to VC and ETH without forming any appreciable amount of other chlorinated intermediates and in the near absence of methanogenic activity. The microbial community structure of the consortium was investigated by preparing a 1 6S rDNA clone library and by fluorescence in situ hybridization (FISH). The PCR primers used in the clone library allowed the harvest of 16SrDNA from both bacterial and archaeal members in the community. A total of 616 clones were screened by RFLP analysis of the clone inserts followed by the sequencing of RFLP group representatives and phylogenetic analysis. The clone library contained sequences mostly from hitherto undescribed bacteria. No sequences similar to those of the known RD bacteria like 'Dehalococcoides ethenogenes' or Dehalobacter restrictus were found in the clone library, and none of these bacteria was present in the RD consortium according to FISH. Almost all clones fell into six previously described phyla of the bacterial domain, with the majority (56(.)6%) being deep-branching members of the Spirochaetes phylum. Other clones were in the Firmicutes phylum (18(.)5%), the Chloroflexi phylum (16(.)4%), the Bacteroidetes phylum (6(.)3%), the Synergistes genus (11(.)1%) and a lineage that could not be affiliated with existing phyla (11(.)1%). No archaeal clones were found in the clone library. Owing to the phylogenetic novelty of the microbial community with regard to previously cultured microorganisms, no specific microbial component(s) could be hypothetically affiliated with the RD phenotype. The predominance of Spirochaetes in the microbial consortium, the main group revealed by clone library analysis, was confirmed by FISH using a purposely developed probe.

The influence of substrate kinetics on the microbial community structure in granular anaerobic biomass

The development of a strong, active granular sludge bed is necessary for optimal operation of upflow anaerobic sludge blanket reactors. The microbial and mechanical structure of the granules may have a strong influence on desirable properties such as growth rate, settling velocity and shear strength. Theories have been proposed for granule microbial structure based on the relative kinetics of substrate degradation, but contradict some observations from both modelling and microscopic studies. In this paper, the structures of four granule types were examined from full-scale UASB reactors, treating wastewater from a cannery, a slaughterhouse, and two breweries. Microbial structure was determined using fluorescence in situ hybridisation probing with 16S rRNA-directed oligonucleotide probes, and superficial structure and microbial density (volume occupied by cells and microbial debris) assessed using scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The granules were also modelled using a distributed parameter biofilm model, with a previously published biochemical model structure, biofilm modelling approach, and model parameters. The model results reflected the trophic structures observed, indicating that the structures were possibly determined by kinetics. Of particular interest were results from simulations of the protein grown granules, which were predicted to have slow growth rates, low microbial density, and no trophic layers, the last two of which were reflected by microscopic observations. The primary cause of this structure, as assessed by modelling, was the particulate nature of the wastewater, and the slow rate of particulate hydrolysis, rather than the presence of proteins in the wastewater. Because solids hydrolysis was rate limiting, soluble substrate concentrations were very low (below Monod half saturation concentration), which caused low growth rates. (C) 2003 Elsevier Ltd. All rights reserved.

Use of stable-isotope probing, full-cycle rRNA analysis, and fluorescence in situ hybridization-microautoradiography to study a methanol-fed denitrifying microbial community

A denitrifying microbial consortium was enriched in an anoxically operated, methanol-fed sequencing batch reactor (SBR) fed with a mineral salts medium containing methanol as the sole carbon source and nitrate as the electron acceptor. The SBR was inoculated with sludge from a biological nutrient removal activated sludge plant exhibiting good denitrification. The SBR denitrification rate improved from less than 0.02 mg of NO3-.N mg of mixed-liquor volatile suspended solids (MLVSS)(-1) h(-1) to a steady-state value of 0.06 mg of NO3-.N mg of MLVSS-1 h(-1) over a 7-month operational period. At this time, the enriched microbial community was subjected to stable-isotope probing (SIP) with [C-13] methanol to biomark the DNA of the denitrifiers. The extracted [C-13]DNA and [C-12]DNA from the SIP experiment were separately subjected to full-cycle rRNA analysis. The dominant 16S rRNA gene phylotype (group A clones) in the [C-13]DNA clone library was closely related to those of the obligate methylotrophs Methylobacillus and Methylophilus in the order Methylophilales of the Betaproteobacteria (96 to 97% sequence identities), while the most abundant clone groups in the [C-12]DNA clone library mostly belonged to the family Saprospiraceae in the Bacteroidetes phylum. Oligonucleotide probes for use in fluorescence in situ hybridization (FISH) were designed to specifically target the group A clones and Methylophilales (probes DEN67 and MET1216, respectively) and the Saprospiraceae clones (probe SAP553). Application of these probes to the SBR biomass over the enrichment period demonstrated a strong correlation between the level of SBR denitrification and relative abundance of DEN67-targeted bacteria in the SBR community. By contrast, there was no correlation between the denitrification rate and the relative abundances of the well-known denitrifying genera Hyphomicrobium and Paracoccus or the Saprospiraceae clones visualized by FISH in the SBR biomass. FISH combined with microautoradiography independently confirmed that the DEN67-targeted cells were the dominant bacterial group capable of anoxic [C-14] methanol uptake in the enriched biomass. The well-known denitrification lag period in the methanol-fed SBR was shown to coincide with a lag phase in growth of the DEN67-targeted denitrifying population. We conclude that Methylophilales bacteria are the dominant denitrifiers in our SBR system and likely are important denitrifiers in full-scale methanol-fed denitrifying sludges.

Investigation of an acetate-fed denitrifying microbial community by stable isotope probing, full-cycle rRNA analysis, and fluorescent in situ hybridization-microautoradiography

The acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (SIP). [C-13]acetate was used in SIP to label the DNA of the denitrifiers. The [C-13]DNA fraction that was extracted was subjected to a full-cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the C-13 library were closely related to the bacterial families Comamonadaceae and Rhodocyclaceae in the class Betaproteobacteria. Seven oligonucleotide probes for use in fluorescent in situ hybridization (FISH) were designed to specifically target these clones. Application of these probes to the sludge of a continuously fed denitrifying sequencing batch reactor (CFDSBR) operated for 16 days revealed that there was a significant positive correlation between the CFDSBR denitrification rate and the relative abundance of all probe-targeted bacteria in the CFDSBR community. FISH-microautoradiography demonstrated that the DEN581 and DEN124 probe-targeted cells that dominated the CFDSBR were capable of taking Up [C-14] acetate under anoxic conditions. Initially, DEN444 and DEN1454 probe-targeted bacteria also dominated the CFDSBR biomass, but eventually DEN581 and DEN124 probe-targeted bacteria were the dominant bacterial groups. All probe-targeted bacteria assessed in this study were denitrifiers capable of utilizing acetate as a source of carbon. The rapid increase in the number of organisms positively correlated with the immediate increase in denitrification rates observed by plant operators when acetate is used as an external source of carbon to enhance denitrification. We suggest that the impact of bacteria on activated sludge subjected to intermittent acetate supplementation should be assessed prior to the widespread use of acetate in the waste-water industry to enhance denitrification.