Biochemical markers for measurement of predation effects on the biomass,community structure,nutritional status,and metabolic activity of microbial biofilms

Chemical measures for the biomass, community structure, nutritional status, and metabolic activities of microbes in biofilms attached to detrital or sediment surfaces based on analysis of components of cells and extracellular polymers represent a quantitative and sensitive method for the analysis of predation. These methods require neither the quantitative removal of the organisms from the surfaces nor the efficient culture of each group of microbes for analysis of predation effects on the biofilm. The biomass of microbes can be determined by measuring the content of cellular components found universally in relatively constant amounts. If these components have a high natural turnover or are rapidly lost from viable cells, they can be utilized to measure the viable cell mass. The membrane phospholipids have a naturally high turnover, are found in all cellular membranes, are rapidly hydrolyzed on cell death, and are found in reasonably constant amounts in bacterial cells as they occur in nature. Estimates of the viable biomass by phospholipid content correspond to estimates from the content of muramic acid, ATP, several enzyme activities, direct cell counts, and in some cases viable counts of subsurface sediments. The analysis of the ester-linked fatty acids of the phospholipids (PLFA) using capillary gas chromatography/mass spectrometry (GC/MS) provides sufficient information for the detection of specific subsets of the microbiota based on patterns of PLFA. With this technique shifts in community structure can be quantitatively assayed. Some of the microbiota form specific components such as poly beta-hydroxyalkanoate (PHA) under conditions of unbalanced growth. Others form polysaccharide glycocalyx when subjected to mechanical or chemical stress. The combination of analysis of phospholipids, PLFA, PHA, and glycocalyx provides a definition of the biomass, community structure, and metabolic status of complex microbial communities. These methods involve chromatographic separation and analysis so rates of incorporation or turnover into specific components can be utilized as measures of metabolic activities. With these methods it has proved possible to show that amphipod grazing can induce shifts in biofilm community structure, nutritional status, and metabolic activities. With this technology it proved possible to show resource partitioning amongst sympatric detrital feeding amphipods, prey specificity of feeding of benthic microvores, effects of sedimentary microtopology on predation, and shifts in the microbiota by exclusion of top epibenthic predators.     

Polymeric Beta-Hydroxyalkanoates from Environmental Samples and Bacillus megaterium

The procaryotic endogenous storage polymer known as poly-beta-hydroxybutyrate is actually a mixed polymer of short-chain beta-hydroxy fatty acids. A method for the quantitative recovery of this mixed polymer, called poly-beta-hydroxyalkanoate (PHA), with analysis by capillary gas-liquid chromatography, showed the presence of at least 11 short-chain beta-hydroxy acids in polymers extracted from marine sediments. Polymers extracted from Bacillus megaterium monocultures were also a complex mixture of beta-hydroxy acids with chain lengths between four and eight carbons. Lyophilized sediments were extracted in a modified Soxhlet extractor, and the polymer was purified with ethanol and diethyl ether washes. The purified polymer was treated with ethanol-chloroform-hydrochloric acid (8.5:2.5:1) for 4 h at 100°C, a treatment which resulted in the formation of the ethyl esters of the constituent beta-hydroxy acids. Subsequent assay of the products by gas-liquid chromatography indicated excellent reproducibility and sensitivity (detection limit, 100 fmol). Disturbing sediments mechanically or adding natural chelators increased all major PHA components relative to the bacterial biomass. Gardening of sedimentary microbes by Clymenella sp., an annelid worm, induced decreases in PHA, with changes in the relative proportion of component beta-hydroxy acids. The concentration of PHA relative to the bacterial biomass can reflect the recent metabolic status of the microbiota.     

Enumeration and Characterization of Iron(III)-Reducing Microbial Communities from Acidic Subsurface Sediments Contaminated with Uranium(VI)

Iron(III)-reducing bacteria have been demonstrated to rapidly catalyze the reduction and immobilization of uranium(VI) from contaminated subsurface sediments. Thus, these organisms may aid in the development of bioremediation strategies for uranium contamination, which is prevalent in acidic subsurface sediments at U.S. government facilities. Iron(III)-reducing enrichment cultures were initiated from pristine and contaminated (high in uranium, nitrate; low pH) subsurface sediments at pH 7 and pH 4 to 5. Enumeration of Fe(III)-reducing bacteria yielded cell counts of up to 240 cells ml⁻¹ for the contaminated and background sediments at both pHs with a range of different carbon sources (glycerol, acetate, lactate, and glucose). In enrichments where nitrate contamination was removed from the sediment by washing, MPN counts of Fe(III)-reducing bacteria increased substantially. Sediments of lower pH typically yielded lower counts of Fe(III)-reducing bacteria in lactate- and acetate-amended enrichments, but higher counts were observed when glucose was used as an electron donor in acidic enrichments. Phylogenetic analysis of 16S rRNA gene sequences extracted from the highest positive MPN dilutions revealed that the predominant members of Fe(III)-reducing consortia from background sediments were closely related to members of the Geobacteraceae family, whereas a recently characterized Fe(III) reducer (Anaeromyxobacter sp.) and organisms not previously shown to reduce Fe(III) (Paenibacillus and Brevibacillus spp.) predominated in the Fe(III)-reducing consortia of contaminated sediments. Analysis of enrichment cultures by terminal restriction fragment length polymorphism (T-RFLP) strongly supported the cloning and sequencing results. Dominant members of the Fe(III)-reducing consortia were observed to be stable over several enrichment culture transfers by T-RFLP in conjunction with measurements of Fe(III) reduction activity and carbon substrate utilization. Enrichment cultures from contaminated sites were also shown to rapidly reduce millimolar amounts of U(VI) in comparison to killed controls. With DNA extracted directly from subsurface sediments, quantitative analysis of 16S rRNA gene sequences with MPN-PCR indicated that Geobacteraceae sequences were more abundant in pristine compared to contaminated environments,whereas Anaeromyxobacter sequences were more abundant in contaminated sediments. Thus, results from a combination of cultivation-based and cultivation-independent approaches indicate that the abundance/community composition of Fe(III)-reducing consortia in subsurface sediments is dependent upon geochemical parameters (pH, nitrate concentration) and that microorganisms capable of producing spores (gram positive) or spore-like bodies (Anaeromyxobacter) were representative of acidic subsurface environments.     

Microbial formation of esters

Small aliphatic esters are important natural flavor and fragrance compounds and have numerous uses as solvents and as chemical intermediates. Besides the chemical or lipase-catalyzed formation of esters from alcohols and organic acids, small volatile esters are made by several biochemical routes in microbes. This short review will cover the biosynthesis of esters from acyl-CoA and alcohol condensation, from oxidation of hemiacetals formed from aldehydes and alcohols, and from the insertion of oxygen adjacent to the carbonyl group in a straight chain or cyclic ketone by Baeyer–Villiger monooxygenases. The physiological role of the ester-forming reactions can allow degradation of ketones for use as a carbon source and may play a role in detoxification of aldehydes or recycling cofactors. The enzymes catalyzing each of these processes have been isolated and characterized, and a number of genes encoding the proteins from various microbes have been cloned and functionally expressed. The use of these ester-forming organisms or recombinant organisms expressing the appropriate genes as biocatalysts in biotechnology to make specific esters and chiral lactones has been studied in recent years.     

Aging in Brachionus plicatilis: The evolution of swimming as a function of age at two different calcium concentrations

Species composition and density of cladoceran populations changed in Lakes Zürich, St. Mortiz and Baldegg as human populations increased in these watersheds. Lake Zürich sediments became annually laminated in the 1890's as a result of increased organic input as the size of the cities surrounding the lake grew. At the same time, the Bosmina species changed from a oligotrophic form (longispina) to a eutrophic form (longirostris). An increase in Daphnia spp. populations also occurred at this time in the lake's history. Bosmina longispina reappeared in the lake in 1965 as the lake's trophic status changed from eutrophic to mesotrophic due to effective sewage treatment facilities. Annual laminations appear in the Lake St. Moritz sediments about 1910. Shortly thereafter, a shift from B. longispina to B. longirostris occurred. This change in trophic status is associated with increased tourism in the area. Lake Baldegg sediments also show annual laminations beginning in 1885 and a similar shift in the Bosmina species. Other cladoceran remains were too scarce to be useful in interpreting the histories of these lakes.     

Archaeal Diversity and Spatial Distribution in the Surface Sediment of the South China Sea

Archaea represent a significant portion of biomass in the marine sediments and may play an important role in global carbon cycle. However, the identity and composition of deep sea sediment Archaea are unclear. Here, we used the archaeal 16S rRNA gene primers to determine the diversity and community structure of Archaea from shallow water (<100 m) and deep water (>1500 m) sediments in the South China Sea. Phylogenetically the archaeal community is separated between the shallow- and deep sea sediments, with the former being dominated by the Thaumarchaeota and the latter by the Marine Benthic Group B, E and the South African GoldMine Euryarchaeotal Group as well as Thaumarchaeota. Sand content showed significant correlation with Thaumarchaeota, suggesting that the porous media may create an oxic environment that allowed these aerobic organisms to thrive in the surface sediments. The carbon isotope composition of total organic carbon was significantly correlated to the distribution of archaeal groups, suggesting that Archaea overall may be constrained by the availability or sources of organic carbon in the sediments of the South China Sea.     

Exploration of deep intraterrestrial microbial life: current perspectives

Intraterrestrial life has been found at depths of several thousand metres in deep sub-sea floor sediments and in the basement crust beneath the sediments. It has also been found at up to 2800-m depth in continental sedimentary rocks, 5300-m depth in igneous rock aquifers and in fluid inclusions in ancient salt deposits from salt mines. The biomass of these intraterrestrial organisms may be equal to the total weight of all marine and terrestrial plants. The intraterrestrial microbes generally seem to be active at very low but significant rates and several investigations indicate chemolithoautotrophs to form a chemosynthetic base. Hydrogen, methane and carbon dioxide gases are continuously generated in the interior of our planet and probably constitute sustainable sources of carbon and energy for deep intraterrestrial biosphere ecosystems. Several prospective research areas are foreseen to focus on the importance of microbial communities for metabolic processes such as anaerobic utilisation of hydrocarbons and anaerobic methane oxidation.     

Biogeochemical evidence that thermophilic archaea mediate the anaerobic oxidation of methane

Distributions and isotopic analyses of lipids from sediment cores at a hydrothermally active site in the Guaymas Basin with a steep sedimentary temperature gradient revealed the presence of archaea that oxidize methane anaerobically. The presence of strongly (13)C-depleted lipids at greater depths in the sediments suggests that microbes involved in anaerobic oxidation of methane are present and presumably active at environmental temperatures of >30 degrees C, indicating that this process can occur not only at cold seeps but also at hydrothermal sites. The distribution of the membrane tetraether lipids of the methanotrophic archaea shows that these organisms have adapted their membrane composition to these high environmental temperatures.     

Highly Divergent Genes for Methanopterin-Linked C1 Transfer Reactions in Lake Washington, Assessed via Metagenomic Analysis and mRNA Detection

The origins and the evolutionary history of tetrahydromethanopterin-linked C₁ transfer reactions that are part of two environmentally important biotransformations, methylotrophy and methanogenesis, are still not well understood. In previous studies, we have expanded the known phylogenetic diversity of these reactions by identifying genes highly diverging from the ones associated with cultivated Proteobacteria, Planctomycetes, or Archaea (M. G. Kalyuzhnaya, M. E. Lidstrom, and L. Chistoserdova, Microb. Ecol. 48:463-472, 2004; M. G. Kalyuzhnaya, O. Nercessian, M. E. Lidstrom, and L. Chistoserdova, Environ. Microbiol. 7:1269-1274, 2005). Here we used a metagenomic approach to demonstrate that these divergent genes are present with high abundance in the microbial community inhabiting Lake Washington sediment. We also gained preliminary insights into the genomic composition of the organisms possessing these genes by sequencing genomic fragments from three uncultured microbes possessing the genes of interest. Phylogenetic analyses suggested that, although distantly related to each other, these organisms deeply diverge from known Bacteria and Archaea, with more relation to the former, suggesting their affiliation with a new bacterial phylum. We also demonstrate, via specific mRNA detection, that these divergent genes are expressed in the environment, pointing toward their potential role in local carbon cycling.     

Biogeochemical Evidence that Thermophilic Archaea Mediate the Anaerobic Oxidation of Methane

Distributions and isotopic analyses of lipids from sediment cores at a hydrothermally active site in the Guaymas Basin with a steep sedimentary temperature gradient revealed the presence of archaea that oxidize methane anaerobically. The presence of strongly ¹³C-depleted lipids at greater depths in the sediments suggests that microbes involved in anaerobic oxidation of methane are present and presumably active at environmental temperatures of >30°C, indicating that this process can occur not only at cold seeps but also at hydrothermal sites. The distribution of the membrane tetraether lipids of the methanotrophic archaea shows that these organisms have adapted their membrane composition to these high environmental temperatures.     

Metagenomic analysis of microbial communities of the sediments of the Kara Sea shelf and the Yenisei Bay

Microbial diversity in the sediments of the Kara Sea shelf and the southern Yenisei Bay, differing in pore water mineralization, was studied using massive parallel pyrosequencing according to the 454 (Roche) technology. Members of the same phyla (Cyanobacteria, Verrucomicrobia, Actinobacteria, Proteobacteria, and Bacteroidetes) predominated in bacterial communities of the sediments, while their ratio and taxonomic composition varied within the phyla and depended on pore water mineralization. Increasing salinity gradient was found to coincide with increased share of the γ-Proteobacteria and decreased abundance of α- and β-Proteobacteria, as well as of the phyla Verrucomicrobia, Chloroflexi, Chlorobi, and Acidobacteria. Archaeal diversity was lower, with Thaumarchaeota predominant in the sediments with high and low mineralization, while Crenarchaeota predominated in moderately mineralized sediments. Microbial communities of the Kara Sea shelf and Yenisei Bay sediments were found to contain the organisms capable of utilization of a broad spectrum of carbon sources, including gaseous and petroleum hydrocarbons.     

Effects of Dissolved Sulfide, pH, and Temperature on Growth and Survival of Marine Hyperthermophilic Archaea

The ability of metabolically diverse hyperthermophilic archaea to withstand high temperatures, low pHs, high sulfide concentrations, and the absence of carbon and energy sources was investigated. Close relatives of our study organisms, Methanocaldococcus jannaschii, Archaeoglobus profundus, Thermococcus fumicolans, and Pyrococcus sp. strain GB-D, are commonly found in hydrothermal vent chimney walls and hot sediments and possibly deeper in the subsurface, where highly dynamic hydrothermal flow patterns and steep chemical and temperature gradients provide an ever-changing mosaic of microhabitats. These organisms (with the possible exception of Pyrococcus strain GB-D) tolerated greater extremes of low pH, high sulfide concentration, and high temperature when actively growing and metabolizing than when starved of carbon sources and electron donors/acceptors. Therefore these organisms must be actively metabolizing in the hydrothermal vent chimneys, sediments, and subsurface in order to withstand at least 24 h of exposure to extremes of pH, sulfide, and temperature that occur in these environments.     

Seagrass (Zostera marina) Colonization Promotes the Accumulation of Diazotrophic Bacteria and Alters the Relative Abundances of Specific Bacterial Lineages Involved in Benthic Carbon and Sulfur Cycling

Seagrass colonization changes the chemistry and biogeochemical cycles mediated by microbes in coastal sediments. In this study, we molecularly characterized the diazotrophic assemblages and entire bacterial community in surface sediments of a Zostera marina-colonized coastal lagoon in northern China. Higher nitrogenase gene (nifH) copy numbers were detected in the sediments from the vegetated region than in the sediments from the unvegetated region nearby. The nifH phylotypes detected were mostly affiliated with the Geobacteraceae, Desulfobulbus, Desulfocapsa, and Pseudomonas. Redundancy analysis based on terminal restriction fragment length polymorphism analysis showed that the distribution of nifH genotypes was mostly shaped by the ratio of total organic carbon to total organic nitrogen, the concentration of cadmium in the sediments, and the pH of the overlying water. High-throughput sequencing and phylogenetic analyses of bacterial 16S rRNA genes also indicated the presence of Geobacteraceae and Desulfobulbaceae phylotypes in these samples. A comparison of these results with those of previous studies suggests the prevalence and predominance of iron(III)-reducing Geobacteraceae and sulfate-reducing Desulfobulbaceae diazotrophs in coastal sedimentary environments. Although the entire bacterial community structure was not significantly different between these two niches, Desulfococcus (Deltaproteobacteria) and Anaerolineae (Chloroflexi) presented with much higher proportions in the vegetated sediments, and Flavobacteriaceae (Bacteroidetes) occurred more frequently in the bare sediments. These data suggest that the high bioavailability of organic matter (indicated by relatively lower carbon-to-nitrogen ratios) and the less-reducing anaerobic condition in vegetated sediments may favor Desulfococcus and Anaerolineae lineages, which are potentially important populations in benthic carbon and sulfur cycling in the highly productive seagrass ecosystem.     

Effects of Iron and Nitrogen Limitation on Sulfur Isotope Fractionation during Microbial Sulfate Reduction

Sulfate-reducing microbes utilize sulfate as an electron acceptor and produce sulfide that is depleted in heavy isotopes of sulfur relative to sulfate. Thus, the distribution of sulfur isotopes in sediments can trace microbial sulfate reduction (MSR), and it also has the potential to reflect the physiology of sulfate-reducing microbes. This study investigates the relationship between the availability of iron and reduced nitrogen and the magnitude of S-isotope fractionation during MSR by a marine sulfate-reducing bacterium, DMSS-1, a Desulfovibrio species, isolated from salt marsh in Cape Cod, MA. Submicromolar levels of iron increase sulfur isotope fractionation by about 50% relative to iron-replete cultures of DMSS-1. Iron-limited cultures also exhibit decreased cytochrome c-to-total protein ratios and cell-specific sulfate reduction rates (csSRR), implying changes in the electron transport chain that couples carbon and sulfur metabolisms. When DMSS-1 fixes nitrogen in ammonium-deficient medium, it also produces larger fractionation, but it occurs at faster csSRRs than in the ammonium-replete control cultures. The energy and reducing power required for nitrogen fixation may be responsible for the reverse trend between S-isotope fractionation and csSRR in this case. Iron deficiency and nitrogen fixation by sulfate-reducing microbes may lead to the large observed S-isotope effects in some euxinic basins and various anoxic sediments.     

The early evolution of land plants,from fossils to genomics: a commentary on Lang (1937) ‘On the plant-remains from the Downtonian of England and Wales'

During the 1920s, the botanist W. H. Lang set out to collect and investigate some very unpromising fossils of uncertain affinity, which predated the known geological record of life on land. His discoveries led to a landmark publication in 1937, ‘On the plant-remains from the Downtonian of England and Wales’, in which he revealed a diversity of small fossil organisms of great simplicity that shed light on the nature of the earliest known land plants. These and subsequent discoveries have taken on new relevance as botanists seek to understand the plant genome and the early evolution of fundamental organ systems. Also, our developing knowledge of the composition of early land-based ecosystems and the interactions among their various components is contributing to our understanding of how life on land affects key Earth Systems (e.g. carbon cycle). The emerging paradigm is one of early life on land dominated by microbes, small bryophyte-like organisms and lichens. Collectively called cryptogamic covers, these are comparable with those that dominate certain ecosystems today. This commentary was written to celebrate the 350th anniversary of the journal Philosophical Transactions of the Royal Society.     

Exopolymer Diversity and the Role of Levan in Bacillus subtilis Biofilms

Exopolymeric substances (EPS) are important for biofilm formation and their chemical composition may influence biofilm properties. To explore these relationships the chemical composition of EPS from Bacillus subtilis NCIB 3610 biofilms grown in sucrose-rich (SYM) and sucrose-poor (MSgg and Czapek) media was studied. We observed marked differences in composition of EPS polymers isolated from all three biofilms or from spent media below the biofilms. The polysaccharide levan dominated the EPS of SYM grown biofilms, while EPS from biofilms grown in sucrose-poor media contained significant amounts of proteins and DNA in addition to polysaccharides. The EPS polymers differed also in size with very large polymers (Mw>2000 kDa) found only in biofilms, while small polymers (Mw<200 kD) dominated in the EPS isolated from spent media. Biofilms of the eps knockout were significantly thinner than those of the tasA knockout in all media. The biofilm defective phenotypes of tasA and eps mutants were, however, partially compensated in the sucrose-rich SYM medium. Sucrose supplementation of Czapek and MSgg media increased the thickness and stability of biofilms compared to non-supplemented controls. Since sucrose is essential for synthesis of levan and the presence of levan was confirmed in all biofilms grown in media containing sucrose, this study for the first time shows that levan, although not essential for biofilm formation, can be a structural and possibly stabilizing component of B. subtilis floating biofilms. In addition, we propose that this polysaccharide, when incorporated into the biofilm EPS, may also serve as a nutritional reserve.     

Uncovering the Cultivable Microbial Diversity of Costa Rican Beetles and Its Ability to Break Down Plant Cell Wall Components

Coleopterans are the most diverse insect order described to date. These organisms have acquired an array of survival mechanisms through their evolution, including highly efficient digestive systems. Therefore, the coleopteran intestinal microbiota constitutes an important source of novel plant cell wall-degrading enzymes with potential biotechnological applications. We isolated and described the cultivable fungi, actinomycetes and aerobic eubacteria associated with the gut of larvae and adults from six different beetle families colonizing decomposing logs in protected Costa Rican ecosystems. We obtained 611 isolates and performed phylogenetic analyses using the ITS region (fungi) and 16S rDNA (bacteria). The majority of fungal isolates belonged to the order Hypocreales (26% of 169 total), while the majority of actinomycetes belonged to the genus Streptomyces (86% of 241 total). Finally, we isolated 201 bacteria spanning 19 different families belonging into four phyla: Firmicutes, α, β and γ-proteobacteria. Subsequently, we focused on microbes isolated from Passalid beetles to test their ability to degrade plant cell wall polymers. Highest scores in these assays were achieved by a fungal isolate (Anthostomella sp.), two Streptomyces and one Bacillus bacterial isolates. Our study demonstrates that Costa Rican beetles harbor several types of cultivable microbes, some of which may be involved in symbiotic relationships that enable the insect to digest complex polymers such as lignocellulose.     

Production and fate of methylated sulfur compounds from methionine and dimethylsulfoniopropionate in anoxic salt marsh sediments

Anoxic salt marsh sediments were amended with dl-methionine and dimethylsulfoniopropionate (DMSP). Microbial metabolism of methionine yielded methane thiol (MSH) as the major volatile organosulfur product, with the formation of lesser amounts of dimethylsulfide (DMS). Biological transformation of DMSP resulted in the rapid release of DMS and only small amounts of MSH. Experiments with microbial inhibitors indicated that production of MSH from methionine was carried out by procaryotic organisms, probably sulfate-reducing bacteria. Methane-producing bacteria did not metabolize methionine. The involvement of specific groups of organisms in DMSP hydrolysis could not be determined with the inhibitors used, because DMSP was hydrolyzed in all samples except those which were autoclaved. Unamended sediment slurries, prepared from Spartina alterniflora sediments, contained significant (1 to 10 muM) concentrations of DMS. Endogenous methylated sulfur compounds and those produced from added methionine and DMSP were consumed by sediment microbes. Both sulfate-reducing and methane-producing bacteria were involved in DMS and MSH consumption. Methanogenesis was stimulated by the volatile organosulfur compounds released from methionine and DMSP. However, apparent competition for these compounds exists between methanogens and sulfate reducers. At low (1 muM) concentrations of methionine, the terminal S-methyl group was metabolized almost exclusively to CO(2) and only small amounts of CH(4). At higher (>100 muM) concentrations of methionine, the proportion of the methyl-sulfur group converted to CH(4) increased. The results of this study demonstrate that methionine and DMSP are potential precursors of methylated sulfur compounds in anoxic sediments and that the microbial community is capable of metabolizing volatile methylated sulfur compounds.     

Physiological responses of the seagrass Posidonia oceanica to elevated organic matter content in sediments: An experimental assessment

Here we studied the effects of adding organic carbon and nutrients to sediment on the physiology and survival of the seagrass Posidonia oceanica in a field experiment in the Medes Islands (NE Spain). Nine randomly selected plots were established at a depth of 10 m; three were enriched with organic carbon (OM treatment), three with organic carbon and nutrients (OMN treatment), and three were kept as controls (CON). The experiment was performed over 5 months and sampling of plants and sediments was done in March, May and July 2002. Sediment sulfide pools and pore water ammonium concentrations increased significantly in OM and OMN plots, both treatments showing increased reducing conditions in the sediment. Plants in these two treatments showed higher mortality and lower biomass compared to plants from CON plots. The greatest effects on seagrass occurred in the OMN plots, indicating a synergistic effect of organic carbon and nutrient additions. Treatments had significant effects on plant nitrogen (N) and phosphorous (P) metabolism, shown by an increase in free amino acid (FAA) content, a change in FAA composition and a lack of increase in N and P tissue content. Treated plants showed higher g-aminobutyric acid (GABA) and malate concentrations and lower concentrations of non-structural carbohydrates compared to CON, indicating that anaerobic respiration in below-ground tissues occurred. Several of the physiological changes shown by P. oceanica can be interpreted as adaptations to anoxia exposure. However, the increased mortality in treated plots demonstrates that this seagrass does not tolerate highly reduced sediments.     

Effects of Artificial Defoliation of Pines on the Structure and Physiology of the Soil Fungal Community of a Mixed Pine-Spruce Forest

Loss of photosynthetic area can affect soil microbial communities by altering the availability of fixed carbon. We used denaturing gradient gel electrophoresis (DGGE) and Biolog filamentous-fungus plates to determine the effects of artificial defoliation of pines in a mixed pine-spruce forest on the composition of the fungal community in a forest soil. As measured by DGGE, two fungal species were affected significantly by the defoliation of pines (P < 0.001); the frequency of members of the ectomycorrhizal fungus genus Cenococcum decreased significantly, while the frequency of organisms of an unidentified soil fungus increased. The decrease in the amount of Cenococcum organisms may have occurred because of the formation of extensive hyphal networks by species of this genus, which require more of the carbon fixed by their host, or because this fungus is dependent upon quantitative differences in spruce root exudates. The defoliation of pines did not affect the overall composition of the soil fungal community or fungal-species richness (number of species per core). Biolog filamentous-fungus plate assays indicated a significant increase (P < 0.001) in the number of carbon substrates utilized by the soil fungi and the rate at which these substrates were used, which could indicate an increase in fungal-species richness. Thus, either small changes in the soil fungal community give rise to significant increases in physiological capabilities or PCR bias limits the reliability of the DGGE results. These data indicate that combined genetic and physiological assessments of the soil fungal community are needed to accurately assess the effect of disturbance on indigenous microbial systems.