Complex food webs coincide with high genetic potential for chemolithoautotrophy in fractured bedrock groundwater.

Groundwater ecosystems face the challenge of energy limitation due to the absence of light-driven primary production. Lack of space and low oxygen availability might further contribute to generally assumed low food web complexity. Chemolithoautotrophy provides additional input of carbon within the subsurface, however, we still do not understand how abundances of chemolithoautotrophs, differences in surface carbon input, and oxygen availability control subsurface food web complexity. Using a molecular approach, we aimed to disentangle the different levels of potential trophic interactions in oligotrophic groundwater along a hillslope setting of alternating mixed carbonate-/siliciclastic bedrock with contrasting hydrochemical conditions and hotspots of chemolithoautotrophy. Across all sites, groundwater harbored diverse protist communities including Ciliophora, Cercozoa, Centroheliozoa, and Amoebozoa but correlations with hydrochemical parameters were less pronounced for eukaryotes compared to bacteria. Ciliophora-affiliated reads dominated the eukaryotic data sets across all sites. DNA-based evidence for the presence of metazoan top predators such as Cyclopoida (Arthropoda) and Stenostomidae (Platyhelminthes) was only found at wells where abundances of functional genes associated with chemolithoautotrophy were 10-100 times higher compared to wells without indications of these top predators. At wells closer to recharge areas with presumably increased inputs of soil-derived substances and biota, fungi accounted for up to 85% of the metazoan-curated eukaryotic sequence data, together with a low potential for chemolithoautotrophy. Although we did not directly observe higher organisms, our results point to the existence of complex food webs with several trophic levels in oligotrophic groundwater. Chemolithoautotrophy appears to provide strong support to more complex trophic interactions, feeding in additional biomass produced by light-independent CO2-fixation.

Attached and Suspended Denitrifier Communities in Pristine Limestone Aquifers Harbor High Fractions of Potential Autotrophs Oxidizing Reduced Iron and Sulfur Compounds.

Oxygen and nitrate availability as well as the presence of suitable organic or inorganic electron donors are strong drivers of denitrification; however, the factors influencing denitrifier abundance and community composition in pristine aquifers are not well understood. We explored the denitrifier community structure of suspended and attached groundwater microorganisms in two superimposed limestone aquifer assemblages with contrasting oxygen regime in the Hainich Critical Zone Exploratory (Germany). Attached communities were retrieved from freshly crushed parent rock material which had been exposed for colonization in two groundwater wells (12.7 and 48 m depth). Quantitative PCR and amplicon pyrosequencing of nirK and nirS genes encoding copper-containing or cytochrome cd1 heme-type nitrite reductase, respectively, and of bacterial 16S ribosomal RNA genes showed a numerical predominance of nirS-type denitrifiers in both attached and suspended groundwater communities and a dominance of nirS-type denitrifiers closely related to the autotrophic thiosulfate- and hydrogen-oxidizing Sulfuritalea hydrogenivorans and the iron- and sulfide-oxidizing Sideroxydans lithotrophicus ES-1. Potential rates of nitrate reduction in association with exposed crushed rock material were higher with an inorganic electron donor (thiosulfate) compared to an organic electron donor (fumarate/acetate) in the upper aquifer assemblage but similar in the lower, oxic aquifer. Our results have clearly demonstrated that groundwater from pristine limestone aquifers harbors diverse denitrifier communities which appear to selectively attach to rock surfaces and harbor a high potential for nitrate reduction. Our findings suggest that the availability of suitable inorganic versus organic electron donors rather than oxygen availability shapes denitrifier communities and their potential activity in these limestone aquifers.

Dominance of 'Gallionella capsiferriformans' and heavy metal association with Gallionella-like stalks in metal-rich pH 6 mine water discharge.

Heavy metal-contaminated, pH 6 mine water discharge created new streams and iron-rich terraces at a creek bank in a former uranium-mining area near Ronneburg, Germany. The transition from microoxic groundwater with ~5 mm Fe(II) to oxic surface water may provide a suitable habitat for microaerobic iron-oxidizing bacteria (FeOB). In this study, we investigated the potential contribution of these FeOB to iron oxidation and metal retention in this high-metal environment. We (i) identified and quantified FeOB in water and sediment at the outflow, terraces, and creek, (ii) studied the composition of biogenic iron oxides (Gallionella-like twisted stalks) with scanning and transmission electron microscopy (SEM, TEM) as well as confocal laser scanning microscopy (CLSM), and (iii) examined the metal distribution in sediments. Using quantitative PCR, a very high abundance of FeOB was demonstrated at all sites over a 6-month study period. Gallionella spp. clearly dominated the communities, accounting for up to 88% of Bacteria, with a minor contribution of other FeOB such as Sideroxydans spp. and 'Ferrovum myxofaciens'. Classical 16S rRNA gene cloning showed that 96% of the Gallionella-related sequences had ≥ 97% identity to the putatively metal-tolerant 'Gallionella capsiferriformans ES-2', in addition to known stalk formers such as Gallionella ferruginea and Gallionellaceae strain R-1. Twisted stalks from glass slides incubated in water and sediment were composed of the Fe(III) oxyhydroxide ferrihydrite, as well as polysaccharides. SEM and scanning TEM-energy-dispersive X-ray spectroscopy revealed that stalk material contained Cu and Sn, demonstrating the association of heavy metals with biogenic iron oxides and the potential for metal retention by these stalks. Sequential extraction of sediments suggested that Cu (52-61% of total sediment Cu) and other heavy metals were primarily bound to the iron oxide fractions. These results show the importance of 'G. capsiferriformans' and biogenic iron oxides in slightly acidic but highly metal-contaminated freshwater environments.

Development of microalgae communities in the Phytotelmata of allochthonous populations of Sarracenia purpurea (Sarraceniaceae).

The phytotelmata of the North American pitcher plant Sarracenia purpurea are colonised by a great variety of aquatic organisms and, thus, provide an ideal model to study trophic interactions in small freshwater ecosystems. Although algae are discussed as a potential food source for predators, little is known about the structure of algae coenoses in pitchers of S. purpurea. This study aims to elucidate temporal shifts in the algae community structure in pitchers of an allochthonous population of S. purpurea in Saxony, Germany. A total of 78 algae taxa was found in the pitchers. Mean algae abundances in new and old pitchers were similar (2.6 x 10(5) and 2.3 x 10(5) algae ml(-1), respectively). Taxa from the orders Chlamydomonadales, Chlorococcales, and Ochromonadales were the primary colonisers. With increasing age of the pitchers the filamentous green algae from the order Klebsormidiales became more abundant. In contrast, pennate diatoms dominated the algae coenoses in the fen. Algae community structure in vase-shaped 50 ml Greiner tubes was similar to those of natural pitchers. Differences in the temporal patterns of algae coenoses in individual pitchers suggested a colonisation of the pitchers by algae via trapped insects, air and rain water rather than via the surrounding fen. Biomass of algae approximated 0.3 mg C ml(-1), which corresponds to 82.8 % of the living biomass (bacteria, heterotrophic nanoflagellates, algae, protozoans and rotifers). Rotifers were abundant in new pitchers; nematodes and mites were seldom found in all pitchers. A similar qualitative and quantitative composition of the aquatic biocoenoses was observed in pitchers of another allochthonous S. purpurea population growing in Blekinge, Sweden. Biomass of algae represented nearly one quarter of the total organic matter content in the pitchers. Thus, nitrogen and phosphorus compounds present in the algae biomass might be used by the carnivorous S. purpurea plant as additional food source in allochthonous populations in Europe lacking top predators.

Microbial processes associated with roots of bulbous rush coated with iron plaques.

Bulbous rush (Juncus bulbosus) is a pioneer species in acidic, iron-rich, coal mining lakes in the eastern part of Germany. Juncus roots are coated with iron plaques, and it has been suggested that microbial processes under the iron plaques might be supportive for Juncus plant growth. The objectives of this work were to enumerate the microbes involved in the turnover of iron and organic root exudates in the rhizoplane, to investigate the effect of oxygen and pH on the utilization of these exudates by the rhizobacteria, and to study the ability of the root-colonizing microbiota to reduce sulfate. Enumeration studies done at pH 3 demonstrated that 10(6) Fe(III) reducers and 10(7) Fe(II) oxidizers g (fresh wt root)(-1) were associated with Juncus roots. When roots were incubated in goethite-containing medium without and with supplemental glucose, Fe(II) was formed at rates approximating 1.1 mmol g (fresh wt root) (-1) d(-1) and 3.6 mmol g (fresh wt root)(-1) d(-1) under anoxic conditions, respectively. These results suggest that a rapid microbially mediated cycling of iron occurs in the rhizosphere of Juncus roots under changing redox conditions. Most-probable-number estimates of aerobes and anaerobes capable of consuming root exudates at pH 3 were similar in the rhizosphere sediment and in Juncus roots, but numbers of aerobes were significantly higher than those of anaerobes. At pH 3, supplemental organic exudates were primarily subject to aerobic oxidation to CO2 and not subject to fermentation. However, at pH 4.5, root exudates were also rapidly utilized under anoxic conditions. Root-associated sulfate reduction was not observed at pH 3 to 4.5 but was observed at pH 4.9. The pH increased during all root-incubation studies both under oxic and anoxic conditions. Thus, as result of the microbial turnover of organic root exudates, pH and CO2 levels might be elevated at the root surface and favor Juncus plants to colonize acidic habitats.

Physiological ecology of Clostridium glycolicum RD-1, an aerotolerant acetogen isolated from sea grass roots.

An anaerobic, H(2)-utilizing bacterium, strain RD-1, was isolated from the highest growth-positive dilution series of a root homogenate prepared from the sea grass Halodule wrightii. Cells of RD-1 were gram-positive, spore-forming, motile rods that were linked by connecting filaments. Acetate was produced in stoichiometries indicative of an acetyl coenzyme A (acetyl-CoA) pathway-dependent metabolism when RD-1 utilized H(2)-CO(2), formate, lactate, or pyruvate. Growth on sugars or ethylene glycol yielded acetate and ethanol as end products. RD-1 grew at the expense of glucose in the presence of low initial concentrations (up to 6% [vol/vol]) of O(2) in the headspace of static, horizontally incubated culture tubes; the concentration of O(2) decreased during growth in such cultures. Peroxidase, NADH oxidase, and superoxide dismutase activities were detected in the cytoplasmic fraction of cells grown in the presence of O(2). In comparison to cultures incubated under strictly anoxic conditions, acetate production decreased, higher amounts of ethanol were produced, and lactate and H(2) became significant end products when RD-1 was grown on glucose in the presence of O(2). Similarly, when RD-1 was grown on fructose in the presence of elevated salt concentrations, lower amounts of acetate and higher amounts of ethanol and H(2) were produced. When the concentration of O(2) in the headspace exceeded 1% (vol/vol), supplemental H(2) was not utilized. The 16S rRNA gene of RD-1 had a 99.7% sequence similarity to that of Clostridium glycolicum DSM 1288(T), an organism characterized as a fermentative anaerobe. Comparative experiments with C. glycolicum DSM 1288(T) demonstrated that it had negligible H(2)- and formate-utilizing capacities. However, carbon monoxide dehydrogenase was detected in both RD-1 and C. glycolicum DSM 1288(T). A 91.4% DNA-DNA hybridization between the genomic DNA of RD-1 and that of C. glycolicum DSM 1288(T) confirmed that RD-1 was a strain of C. glycolicum. These results indicate that (i) RD-1 metabolizes certain substrates via the acetyl-CoA pathway, (ii) RD-1 can tolerate and consume limited amounts of O(2), (iii) oxic conditions favor the production of ethanol, lactate, and H(2) by RD-1, and (iv) the ability of RD-1 to cope with limited amounts of O(2) might contribute to its survival in a habitat subject to daily gradients of photosynthesis-derived O(2).

Effect of Supplemental Electron Donors on the Microbial Reduction of Fe(III), Sulfate, and CO(2) in Coal Mining-Impacted Freshwater Lake Sediments.

In acidic mining-impacted lake sediments, the microbial reduction of Fe(III) is the dominant electron-accepting process, whereas the reduction of sulfate seems to be restricted to a narrow sediment zone of elevated pH and lower amounts of total and reactive iron. To evaluate the microbial heterogeneity and the commensal interactions of the microbial community, the flow of supplemental carbon and reductant was evaluated in four different zones of the sediment in anoxic microcosms at the in situ temperature of 12 degrees C. Substrate consumption, product formation, and the potential to reduce Fe(III) and sulfate were similar with both upper and lower sediment zones. In the upper acidic iron-rich sediment zone, the rate of Fe(II) formation 204 nmol ml(-1) d(-1) was enhanced to 833 nmol ml(-1) d(-1) and 462 nmol ml(-1) d(-1) by supplemental glucose and H(2), respectively. Supplemental lactate and acetate were not consumed under acidic conditions and decreased the rate of Fe(II) formation to 130 nmol ml(-1) d(-1) and 52 nmol ml(-1) d(-1), respectively. When the pH of the upper sediment increased above pH 5, acetate-dependent reduction of sulfate was initiated even though the pool of Fe(III) was not depleted. In deeper sediment zones with elevated pH, the rapid consumption of acetate was always coincident to a decrease in the concentration of sulfate and soluble Fe(II), indicating the formation of Fe(II) sulfides. Although the reduction of Fe(III) was still an ongoing process in deeper sediment zones, the formation of Fe(II) was only slightly enhanced by the consumption of glucose or cellobiose, but not by H(2) or acetate. H(2)-utilizing acetogens seemed to be involved in the consumption of H(2). These collective results indicated (i) that the reduction of Fe(III) predominated over the reduction of sulfate as long as the sediment remained acidic and carbon-limited, and (ii) that the sulfate-reducing microbiota in this heterogeneous sediment were better adapted to the geochemical gradients present than were other neutrophilic dissimilatory Fe(III) reducers.

Clostridium scatologenes strain SL1 isolated as an acetogenic bacterium from acidic sediments.

A strictly anaerobic, H2-utilizing bacterium, strain SL1, was isolated from the sediment of an acidic coal mine pond. Cells of strain SL1 were sporulating, motile, long rods with a multilayer cell wall. Growth was observed at 5-35 degrees C and pH 3.9-7.0. Acetate was the sole end product of H2 utilization and was produced in stoichiometries indicative of an acetyl-CoA-pathway-dependent metabolism. Growth and substrate utilization also occurred with CO/CO2, vanillate, syringate, ferulate, ethanol, propanol, 1-butanol, glycerine, cellobiose, glucose, fructose, mannose, xylose, formate, lactate, pyruvate and gluconate. With most substrates, acetate was the main or sole product formed. Growth in the presence of H2/CO2 or CO/CO2 was difficult to maintain in laboratory cultures. Methoxyl, carboxyl and acrylate groups of various aromatic compounds were O-demethylated, decarboxylated and reduced, respectively. Small amounts of butyrate were produced during the fermentation of sugars. The acrylate group of ferulate was reduced. Nitrate, sulfate, thiosulfate, dimethylsulfoxide and Fe(III) were not utilized as electron acceptors. Analysis of the 16S rRNA gene sequence of strain SL1 demonstrated that it is closely related to Clostridium scatologenes (99.6% sequence similarity), an organism characterized as a fermentative anaerobe but not previously shown to be capable of acetogenic growth. Comparative experiments with C. scatologenes DSM 757T demonstrated that it utilized H2/CO2 (negligible growth), CO/CO2 (negligible growth), formate, ethanol and aromatic compounds according to stoichiometries indicative of the acetyl-CoA pathway. CO dehydrogenase, formate dehydrogenase and hydrogenase activities were present in both strain SL1 and C. scatologenes DSM 757T. These results indicate that (i) sediments of acidic coal mine ponds harbour acetogens and (ii) C. scatologenes is an acetogen that tends to lose its capacity to grow acetogenically under H2/CO2 or CO/CO2 after prolonged laboratory cultivation.

Acetogenic and sulfate-reducing bacteria inhabiting the rhizoplane and deep cortex cells of the sea grass Halodule wrightii.

Recent declines in sea grass distribution underscore the importance of understanding microbial community structure-function relationships in sea grass rhizospheres that might affect the viability of these plants. Phospholipid fatty acid analyses showed that sulfate-reducing bacteria and clostridia were enriched in sediments colonized by the sea grasses Halodule wrightii and Thalassia testudinum compared to an adjacent unvegetated sediment. Most-probable-number analyses found that in contrast to butyrate-producing clostridia, acetogens and acetate-utilizing sulfate reducers were enriched by an order of magnitude in rhizosphere sediments. Although sea grass roots are oxygenated in the daytime, colorimetric root incubation studies demonstrated that acetogenic O-demethylation and sulfidogenic iron precipitation activities were tightly associated with washed, sediment-free H. wrightii roots. This suggests that the associated anaerobes are able to tolerate exposure to oxygen. To localize and quantify the anaerobic microbial colonization, root thin sections were hybridized with newly developed (33)P-labeled probes that targeted (i) low-G+C-content gram-positive bacteria, (ii) cluster I species of clostridia, (iii) species of Acetobacterium, and (iv) species of Desulfovibrio. Microautoradiography revealed intercellular colonization of the roots by Acetobacterium and Desulfovibrio species. Acetogenic bacteria occurred mostly in the rhizoplane and outermost cortex cell layers, and high numbers of sulfate reducers were detected on all epidermal cells and inward, colonizing some 60% of the deepest cortex cells. Approximately 30% of epidermal cells were colonized by bacteria that hybridized with an archaeal probe, strongly suggesting the presence of methanogens. Obligate anaerobes within the roots might contribute to the vitality of sea grasses and other aquatic plants and to the biogeochemistry of the surrounding sediment.

Microbial reduction of Fe(III) in acidic sediments: isolation of Acidiphilium cryptum JF-5 capable of coupling the reduction of Fe(III) to the oxidation of glucose.

To evaluate the microbial populations involved in the reduction of Fe(III) in an acidic, iron-rich sediment, the anaerobic flow of supplemental carbon and reductant was evaluated in sediment microcosms at the in situ temperature of 12 degrees C. Supplemental glucose and cellobiose stimulated the formation of Fe(II); 42 and 21% of the reducing equivalents that were theoretically obtained from glucose and cellobiose, respectively, were recovered in Fe(II). Likewise, supplemental H(2) was consumed by acidic sediments and yielded additional amounts of Fe(II) in a ratio of approximately 1:2. In contrast, supplemental lactate did not stimulate the formation of Fe(II). Supplemental acetate was not consumed and inhibited the formation of Fe(II). Most-probable-number estimates demonstrated that glucose-utilizing acidophilic Fe(III)-reducing bacteria approximated to 1% of the total direct counts of 4', 6-diamidino-2-phenylindole-stained bacteria. From the highest growth-positive dilution of the most-probable-number series at pH 2. 3 supplemented with glucose, an isolate, JF-5, that could dissimilate Fe(III) was obtained. JF-5 was an acidophilic, gram-negative, facultative anaerobe that completely oxidized the following substrates via the dissimilation of Fe(III): glucose, fructose, xylose, ethanol, glycerol, malate, glutamate, fumarate, citrate, succinate, and H(2). Growth and the reduction of Fe(III) did not occur in the presence of acetate. Cells of JF-5 grown under Fe(III)-reducing conditions formed blebs, i.e., protrusions that were still in contact with the cytoplasmic membrane. Analysis of the 16S rRNA gene sequence of JF-5 demonstrated that it was closely related to an Australian isolate of Acidiphilium cryptum (99.6% sequence similarity), an organism not previously shown to couple the complete oxidation of sugars to the reduction of Fe(III). These collective results indicate that the in situ reduction of Fe(III) in acidic sediments can be mediated by heterotrophic Acidiphilium species that are capable of coupling the reduction of Fe(III) to the complete oxidation of a large variety of substrates including glucose and H(2).

Acetogenic bacteria: what are the in situ consequences of their diverse metabolic versatilities?

The four decades of the now classic studies by Harland G. Wood and Lars G. Ljungdahl lead to the resolution of the autotrophic acetyl-CoA 'Wood/Ljungdahl' pathway of acetogenesis. This pathway is the hallmark of acetogens, but is also used by other bacteria, including methanogens and sulfate-reducing bacteria, for both catabolic and anabolic purposes. Thus, the pathway is wide spread in nature and plays an important role in the global turnover of carbon. Because most historical studies with acetogens focused on the biochemistry of the acetyl-CoA pathway, the metabolic diversity and ecology of acetogens remained largely unexplored for many years. Although acetogens were initially conceived to be a somewhat obscure bacteriological group with limited metabolic capabilities, it is now clear that acctogens are arguably the most metabolically diverse group of obligate anaerobes characterized to date. Their anaerobic metabolic arsenal includes the capacity to oxidize diverse substrates, including aromatic, C1, C2, and halogenated compounds, and engage a large number of alternative energy-conserving, terminal electron-accepting processes, including classic fermentations and the dissimilation of inorganic nitrogen. In this regard, one might consider acetogens on a collective basis as the pseudomonads of obligate anaerobes. By virtue of their diverse metabolic talents, acetogens can be found in essentially all habitats. This review evaluates the metabolic versatilities of acetogens relative to both the engagement (regulation) of the acetyl-CoA pathway and the ecological roles likely played by this bacteriogical group.

Anaerobic capacities of leaf litter.

Leaf litter displayed a capacity to spontaneously form organic acids, alcohols, phenolic compounds, H(inf2), and CO(inf2) when incubated anaerobically at 20(deg)C either as buffered suspensions or in a moistened condition in microcosms. Acetate was the predominant organic product formed regardless of the degree of litter decomposition. Initial rates of acetate formation in litter suspensions and microcosms approximated 2.6 and 0.53 (mu)mol of acetate per g (dry weight) of litter per h, respectively. Supplemental H(inf2) was directed towards the apparent acetogenic synthesis of acetate. Acetoclastic methanogenesis was induced by partially decomposed litter after extended lag phases; freshly fallen litter did not display this capacity.

Effects of environmental parameters on the formation and turnover of acetate by forest soils.

The capacity to form acetate from endogenous matter was a common property of diverse forest soils when incubated under anaerobic conditions. At 15 to 20(deg)C, acetate synthesis occurred without appreciable delay when forest soils were incubated as buffered suspensions or in microcosms at various percentages of their maximum water holding capacity. Rates for acetate formation with soil suspensions ranged from 35 to 220 (mu)g of acetate per g (dry weight) of soil per 24 h, and maximal acetate concentrations obtained in soil suspensions were two- to threefold greater than those obtained with soil microcosms at the average water holding capacity of the soil. Cellobiose degradation in soil suspensions yielded H(inf2) as a transient product. Under anaerobic conditions, supplemental H(inf2) and CO(inf2) were directed towards the acetogenic synthesis of acetate, and enrichments yielded a syringate-H(inf2)-consuming acetogenic consortium. At in situ temperatures, acetate was a relatively stable anaerobic end product; however, extended incubation periods induced acetoclastic methanogenesis and sulfate reduction. Higher mesophilic and thermophilic temperatures greatly enhanced the capacity of soils to form methane. Although methanogenic and sulfate-reducing activities under in situ-relevant conditions were negligible, these findings nonetheless demonstrated the occurrence of methanogens and sulfate-reducing bacteria in these aerated terrestrial soils. In contrast to the protracted stability of acetate under anaerobic conditions at 15 to 20(deg)C with unsupplemented soils, acetate formed by forest soils was rapidly consumed in the presence of oxygen and nitrate, and substrate-product stoichiometries indicated that acetate turnover was coupled to oxygen-dependent respiration and denitrification. The collective results suggest that acetate formed under anaerobic conditions might constitute a trophic link between anaerobic and aerobic processes in forest soils.

Acetate synthesis in soil from a bavarian beech forest.

Soil obtained from a beech forest formed significant amounts of acetate when incubated in a bicarbonate-buffered, mineral salt solution under anaerobic conditions at both 5 and 20 degrees C (21 and 38 g of acetate per kg [dry weight] of soil, respectively). At 20 degrees C, following an 18-day lag period, rates of 0.07 mmol of acetate synthesized per g (dry weight) of soil per day were observed. Acetate was not subject to immediate turnover; methane and hydrogen were not formed during the time intervals (5 degrees C, 335 days; 20 degrees C, 95 days) evaluated. The synthesis of acetate from endogenous materials was coincident with acetogenic potentials, i.e., the capacity to catalyze the H(2)-dependent synthesis of acetate. Hydrogen consumption was not directed towards the synthesis of methane. Collectively, these results suggest that acetogenesis may be an underlying microbial activity of this forest soil.