Microbially induced carbonate precipitation via methanogenesis pathway by a microbial consortium enriched from activated anaerobic sludge.

AIMS: Various applications of microbially induced carbonate precipitation (MICP) has been proposed. However, most studies use cultured pure strains to obtain MICP, ignoring advantages of microbial consortia. The aims of this study were to: (i) test the feasibility of a microbial consortium to produce MICP; (ii) identify functional micro-organisms and their relationship; (iii) explain the MICP mechanism; (iv) propose a way of applying the MICP technique to soil media. METHODS AND RESULTS: Anaerobic sludge was used as the source of the microbial consortium. A laboratory anaerobic sequencing batch reactor and beaker were used to perform precipitation experiment. The microbial consortium produced MICP with an efficiency of 96·6%. XRD and SEM analysis showed that the precipitation composed of different-size calcite crystals. According to high-throughput 16S rRNA gene sequencing, the functional micro-organisms included acetogenic bacteria, acetate-oxidizing bacteria and archaea Methanosaeta and Methanobacterium beijingense. The methanogenesis acetate degradation provides dissolved inorganic carbon and increases pH for MICP. A series of reactions catalysed by many enzymes and cofactors of methanogens and acetate-oxidizers are involved in the acetate degradation. CONCLUSION: This work demonstrates the feasibility of using the microbial consortium to achieve MICP from an experimental and theoretical perspective. SIGNIFICANCE AND IMPACT OF THE STUDY: A method of applying the microbial-consortium MICP to soil media is proposed. It has the advantages of low cost, low environmental impact, treatment uniformity and less limitations from natural soils. This method could be used to improve mechanical properties, plug pores and fix harmful elements of soil media, etc.

Short-Term Stable Isotope Probing of Proteins Reveals Taxa Incorporating Inorganic Carbon in a Hot Spring Microbial Mat.

The upper green layer of the chlorophototrophic microbial mats associated with the alkaline siliceous hot springs of Yellowstone National Park consists of oxygenic cyanobacteria (Synechococcus spp.), anoxygenic Roseiflexus spp., and several other anoxygenic chlorophototrophs. Synechococcus spp. are believed to be the main fixers of inorganic carbon (Ci), but some evidence suggests that Roseiflexus spp. also contribute to inorganic carbon fixation during low-light, anoxic morning periods. Contributions of other phototrophic taxa have not been investigated. In order to follow the pathway of Ci incorporation into different taxa, mat samples were incubated with [13C]bicarbonate for 3 h during the early-morning, low-light anoxic period. Extracted proteins were treated with trypsin and analyzed by mass spectrometry, leading to peptide identifications and peptide isotopic profile signatures containing evidence of 13C label incorporation. A total of 25,483 peptides, corresponding to 7,221 proteins, were identified from spectral features and associated with mat taxa by comparison to metagenomic assembly sequences. A total of 1,417 peptides, derived from 720 proteins, were detectably labeled with 13C. Most 13C-labeled peptides were derived from proteins of Synechococcus spp. and Roseiflexus spp. Chaperones and proteins of carbohydrate metabolism were most abundantly labeled. Proteins involved in photosynthesis, Ci fixation, and N2 fixation were also labeled in Synechococcus spp. Importantly, most proteins of the 3-hydroxypropionate bi-cycle for Ci fixation in Roseiflexus spp. were labeled, establishing that members of this taxocene contribute to Ci fixation. Other taxa showed much lower [13C]bicarbonate incorporation.IMPORTANCE Yellowstone hot spring mats have been studied as natural models for understanding microbial community ecology and as modern analogs of stromatolites, the earliest community fossils on Earth. Stable-isotope probing of proteins (Pro-SIP) permitted short-term interrogation of the taxa that are involved in the important process of light-driven Ci fixation in this highly active community and will be useful in linking other metabolic processes to mat taxa. Here, evidence is presented that Roseiflexus spp., which use the 3-hydroxypropionate bi-cycle, are active in Ci fixation. Because this pathway imparts a lower degree of selection of isotopically heavy Ci than does the Calvin-Benson-Bassham cycle, the results suggest a mechanism to explain why the natural abundance of 13C in mat biomass is greater than expected if only the latter pathway were involved. Understanding how mat community members influence the 13C/12C ratios of mat biomass will help geochemists interpret the 13C/12C ratios of organic carbon in the fossil record.

Rubisco carboxylation kinetics and inorganic carbon utilization in polar versus cold-temperate seaweeds.

Despite the high productivity and ecological importance of seaweeds in polar coastal regions, little is known about their carbon utilization mechanisms, especially the kinetics of the CO2-fixing enzyme Rubisco. We analyzed Rubisco carboxylation kinetics at 4 °C and 25 °C in 12 diverse polar seaweed species (including cold-temperate populations of the same species) and the relationship with their ability to use bicarbonate, by using 13C isotope discrimination and pH drift experiments. We observed a large variation in Rubisco carboxylation kinetics among the selected species, although no correlation was found between either the Michaelis-Menten constant for CO2 (Kc) or Rubisco content per total soluble protein ([Rubisco]/[TSP]) and the ability to use bicarbonate for non-green seaweeds. This study reports intraspecific Rubisco cold adaptation by means of either higher Rubisco carboxylation turnover rate (kcatc) and carboxylase efficiency (kcatc/Kc) at 4 °C or higher [Rubisco]/[TSP] in some of the analyzed species. Our data point to a widespread ability for photosynthetic bicarbonate usage among polar seaweeds, despite the higher affinity of Rubisco for CO2 and higher dissolved CO2 concentration in cold seawater. Moreover, the reported catalytic variation within form ID Rubisco might avert the canonical trade-off previously observed between Kc and kcatc for plant Rubiscos.

Discrimination of three Ephedra species and their geographical origins based on multi-element fingerprinting by inductively coupled plasma mass spectrometry.

Discrimination of species and geographical origins of traditional Chinese medicine (TCM) is essential to prevent adulteration and inferior problems. We studied Ephedra sinica Stapf, Ephedra intermedia Schrenk et C.A.Mey. and Ephedra przewalskii Bge. to investigate the relationship between inorganic element content and these three species and their geographical origins. 38 elemental fingerprints from six major Ephedra-producing regions, namely, Inner Mongolia, Ningxia, Gansu, Shanxi, Shaanxi, and Sinkiang, were determined to evaluate the importance of inorganic elements to three species and their geographical origins. The contents of 15 elements, namely, N, P, K, S, Ca, Mg, Fe, Mn, Na, Cl, Sr, Cu, Zn, B, and Mo, of Ephedra samples were measured using inductively coupled plasma mass spectroscopy. Elemental contents were used as chemical indicators to classify species and origins of Ephedra samples using a radar plot and multivariate data analysis, including hierarchical cluster analysis (HCA), principal component analysis (PCA), and discriminant analysis (DA). Ephedra samples from different species and geographical origins could be differentiated. This study showed that inorganic elemental fingerprint combined with multivariate statistical analysis is a promising tool for distinguishing three Ephedra species and their geographical origins, and this strategy might be an effective method for authenticity discrimination of TCM.

Carbon acquisition characteristics of six microalgal species isolated from a subtropical reservoir: potential implications for species succession.

CO2 levels in freshwater systems can fluctuate widely, potentially influencing photosynthetic rates and growth of phytoplankton. Given the right conditions, this can lead to bloom formation and affect water quality. This study investigated the acquisition of dissolved inorganic carbon (DIC) by six species of microalgae, a cyanobacterium Cylindrospermopsis raciborskii, the diatoms Cyclotella sp., Nitzschia sp., and the green algae Stichococcus sp., Staurastrum sp., and Monoraphidium sp., all isolated from a subtropical reservoir in Australia. Carbon acquisition characteristics, specifically the affinity for DIC, internal pH, and internal DIC concentrations were measured. Affinities for CO2 ( K0.5(CO2) ) ranged between 0.7 and 6 µM CO2 . This was considerably lower than air-equilibrated surface water CO2 concentrations, and below reported affinities for CO2 of RuBisCO suggesting operation of active carbon dioxide concentrating mechanisms (CCMs) in all species. Internal pH was lowest for Cyclotella sp. at 7.19, and highest for Staurastrum sp., at 7.71. At 180 µM external DIC, ratios of internal:external CO2 ranged from 2.5 for Nitzschia sp. to 14 in C. raciborskii. Internal HCO3- concentration showed a linear relationship with surface area to biovolume ratio (SA:Vol). We hypothesized that species with a higher SA:Vol suffer more from diffusive escape of CO2 , thus storage of DIC as bicarbonate is favored in these strains. For C. raciborskii, under stratified summer conditions, its strong CCM, and resilient photosynthetic characteristics may contribute to its bloom forming capacity.

PII-like signaling protein SbtB links cAMP sensing with cyanobacterial inorganic carbon response.

Cyanobacteria are phototrophic prokaryotes that evolved oxygenic photosynthesis ∼2.7 billion y ago and are presently responsible for ∼10% of total global photosynthetic production. To cope with the evolutionary pressure of dropping ambient CO2 concentrations, they evolved a CO2-concentrating mechanism (CCM) to augment intracellular inorganic carbon (Ci) levels for efficient CO2 fixation. However, how cyanobacteria sense the fluctuation in Ci is poorly understood. Here we present biochemical, structural, and physiological insights into SbtB, a unique PII-like signaling protein, which provides new insights into Ci sensing. SbtB is highly conserved in cyanobacteria and is coexpressed with CCM genes. The SbtB protein from the cyanobacterium Synechocystis sp. PCC 6803 bound a variety of adenosine nucleotides, including the second messenger cAMP. Cocrystal structures unraveled the individual binding modes of trimeric SbtB with AMP and cAMP. The nucleotide-binding pocket is located between the subunit clefts of SbtB, perfectly matching the structure of canonical PII proteins. This clearly indicates that proteins of the PII superfamily arose from a common ancestor, whose structurally conserved nucleotide-binding pocket has evolved to sense different adenyl nucleotides for various signaling functions. Moreover, we provide physiological and biochemical evidence for the involvement of SbtB in Ci acclimation. Collectively, our results suggest that SbtB acts as a Ci sensor protein via cAMP binding, highlighting an evolutionarily conserved role for cAMP in signaling the cellular carbon status.

Temperature effects on sinking velocity of different Emiliania huxleyi strains.

The sinking properties of three strains of Emiliania huxleyi in response to temperature changes were examined. We used a recently proposed approach to calculate sinking velocities from coccosphere architecture, which has the advantage to be applicable not only to culture samples, but also to field samples including fossil material. Our data show that temperature in the sub-optimal range impacts sinking velocity of E. huxleyi. This response is widespread among strains isolated in different locations and moreover comparatively predictable, as indicated by the similar slopes of the linear regressions. Sinking velocity was positively correlated to temperature as well as individual cell PIC/POC over the sub-optimum to optimum temperature range in all strains. In the context of climate change our data point to an important influence of global warming on sinking velocities. It has recently been shown that seawater acidification has no effect on sinking velocity of a Mediterranean E. huxleyi strain, while nutrient limitation seems to have a small negative effect on sinking velocity. Given that warming, acidification, and lowered nutrient availability will occur simultaneously under climate change scenarios, the question is what the net effect of different influential factors will be. For example, will the effects of warming and nutrient limitation cancel? This question cannot be answered conclusively but analyses of field samples in addition to laboratory culture studies will improve predictions because in field samples multi-factor influences and even evolutionary changes are not excluded. As mentioned above, the approach of determining sinking rate followed here is applicable to field samples. Future studies could use it to analyse not only seasonal and geographic patterns but also changes in sinking velocity over geological time scales.

Molecular characterization, light-dependent expression, and cellular localization of a host vacuolar-type H+-ATPase (VHA) subunit A in the giant clam, Tridacna squamosa, indicate the involvement of the host VHA in the uptake of inorganic carbon and its supply to the symbiotic zooxanthellae.

The giant clam, Tridacna squamosa, represents a clam-zooxanthellae association. In light, the host clam and the symbiotic zooxanthellae conduct light-enhanced calcification and photosynthesis, respectively. We had cloned the cDNA coding sequence of a Vacuolar-type Proton ATPase (VHA) subunit A, ATP6V1A, from T. squamosa, whereby the VHA is an electrogenic transporter that actively 'pumps' H+ out of the cell. The ATP6V1A of T. squamosa comprised 1866 bp, encoding a protein of 622 amino acids and 69.9 kDa, and had a host-origin. Its gene expression was strong in the ctenidium and the colorful outer mantle, but weak in the whitish inner mantle, corroborating a previous proposition that VHA might have a trivial role in light-enhanced calcification. Light exposure led to significant increases in the gene and protein expression levels of ATP6V1A/ATP6V1A in the ctenidium and the outer mantle. In the ctenidium, the ATP6V1A was localized in the apical epithelia of the filaments and tertiary water channels, indicating that the VHA could participate in the increased excretion of H+ produced during light-enhanced calcification. Additionally, the excreted H+ would augment HCO3- dehydration in the external medium and facilitate the uptake of CO2 by the ctenidium during insolation. In the outer mantle, the ATP6V1A was detected in intracellular vesicles in a type of cells, presumably iridocytes, surrounding the zooxanthellal tubules, and in the apical epithelium of zooxanthellal tubules. Hence, the host VHA could participate in the transfer of inorganic carbon from the hemolymph to the luminal fluid of the tubules by increasing the supply of H+ for the dehydration of HCO3- to CO2 during insolation to benefit the photosynthesizing zooxanthellae.

Fast, asymmetric and nonhomogeneous clearance of SiC nanoaerosol assessed by micro-particle-induced x-ray emission.

AIM: To study the biopersistence of a silicon carbide (SiC) nanoaerosol in rat lungs, as time-dependent clearance and spatial distribution. MATERIALS & METHODS: Sprague-Dawley rats were exposed 6 h/day during 5 days to a SiC nanoaerosol at 4.91 mg SiC/l. SiC biopersistence in rat lungs sections was assessed over 28 days by micro-particle-induced x-ray emission (µPIXE) as 2D maps and by particle-induced x-ray emission (PIXE) for whole-lung quantification. 2D maps were analyzed for SiC spatial distribution as skewness and kurtosis. RESULTS: Half-time clearance was 10.9 ± 0.9 days, agreeing with PIXE measurements. Spatial-temporal analysis of SiC indicated decreased symmetry and homogeneity. CONCLUSION: Fast SiC clearance points that current nanoaerosol exposure may not be enough to trigger lung overload. Spatial distribution shows an asymmetric and nonhomogeneous SiC clearance.

Carbon-concentrating mechanisms in seagrasses.

Seagrasses are unique angiosperms that carry out growth and reproduction submerged in seawater. They occur in at least three families of the Alismatales. All have chloroplasts mainly in the cells of the epidermis. Living in seawater, the supply of inorganic carbon (Ci) to the chloroplasts is diffusion limited, especially under unstirred conditions. Therefore, the supply of CO2 and bicarbonate across the diffusive boundary layer on the outer side of the epidermis is often a limiting factor. Here we discuss the evidence for mechanisms that enhance the uptake of Ci into the epidermal cells. Since bicarbonate is plentiful in seawater, a bicarbonate pump might be expected; however, the evidence for such a pump is not strongly supported. There is evidence for a carbonic anhydrase outside the outer plasmalemma. This, together with evidence for an outward proton pump, suggests the possibility that local acidification leads to enhanced concentrations of CO2 adjacent to the outer tangential epidermal walls, which enhances the uptake of CO2, and this could be followed by a carbon-concentrating mechanism (CCM) in the cytoplasm and/or chloroplasts. The lines of evidence for such an epidermal CCM are discussed, including evidence for special 'transfer cells' in some but not all seagrass leaves in the tangential inner walls of the epidermal cells. It is concluded that seagrasses have a CCM but that the case for concentration of CO2 at the site of Rubisco carboxylation is not proven.

Inorganic carbon availability in benthic diatom communities: photosynthesis and migration.

Diatom-dominated microphytobenthos (MPB) is the main primary producer of many intertidal and shallow subtidal environments, being therefore of critical importance to estuarine and coastal food webs. Owing to tidal cycles, intertidal MPB diatoms are subjected to environmental conditions far more variable than the ones experienced by pelagic diatoms (e.g. light, temperature, salinity, desiccation and nutrient availability). Nevertheless, benthic diatoms evolved adaptation mechanisms to these harsh conditions, including the capacity to move within steep physical and chemical gradients, allowing them to perform photosynthesis efficiently. In this contribution, we will review present knowledge on the effects of dissolved inorganic carbon (DIC) availability on photosynthesis and productivity of diatom-dominated MPB. We present evidence of carbon limitation of photosynthesis in benthic diatom mats and highly productive MPB natural communities. Furthermore, we hypothesize that active vertical migration of epipelic motile diatoms could overcome local depletion of DIC in the photic layer, providing the cells alternately with light and inorganic carbon supply. The few available longer-term experiments on the effects of inorganic carbon enrichment on the productivity of diatom-dominated MPB have yielded inconsistent results. Therefore, further studies are needed to properly assess the response of MPB communities to increased CO2 and ocean acidification related to climate change.This article is part of the themed issue 'The peculiar carbon metabolism in diatoms'.

Identification and characterization of a solute carrier, CIA8, involved in inorganic carbon acclimation in Chlamydomonas reinhardtii.

The supply of inorganic carbon (Ci) at the site of fixation by Rubisco is a key parameter for efficient CO2 fixation in aquatic organisms including the green alga, Chlamydomonas reinhardtii. Chlamydomonas reinhardtii cells, when grown on limiting CO2, have a CO2-concentrating mechanism (CCM) that functions to concentrate CO2 at the site of Rubisco. Proteins thought to be involved in inorganic carbon uptake have been identified and localized to the plasma membrane or chloroplast envelope. However, current CCM models suggest that additional molecular components are involved in Ci uptake. In this study, the gene Cia8 was identified in an insertional mutagenesis screen and characterized. The protein encoded by Cia8 belongs to the sodium bile acid symporter subfamily. Transcript levels for this gene were significantly up-regulated when the cells were grown on low CO2. The cia8 mutant exhibited reduced growth and reduced affinity for Ci when grown in limiting CO2 conditions. Prediction programs localize this protein to the chloroplast. Ci uptake and the photosynthetic rate, particularly at high external pH, were reduced in the mutant. The results are consistent with the model that CIA8 is involved in Ci uptake in C. reinhardtii.

Evaluations of the Carcinogenicity of Carbon Nanotubes, Fluoro-Edinite, and Silicon Carbide by the International Agency for Research on Cancer (IARC).

We reported the evaluations of the carcinogenicity of fluoro-edinite, silicon carbide, and carbon nanotubes performed by IARC working group in October 2014. For carbon nanotubes (CNTs), multi-walled carbon nanotube (MWCNT)-7 was classified as Group 2B, and MWCNTs without MWCNT-7 and single-walled carbon nanotubes (SWCNTs) were classified as not classifiable in terms of their carcinogenicity to humans. There is sufficient evidence of carcinogenicity for MWCNT-7 in experimental animals, limited evidence for other MWCNTs, and inadequate evidence for SWCNTs. The mechanic evidence for CNTs was not strong. Fluoro-edinite was classified as carcinogenic to humans (Group 1) on the basis of sufficient evidence of carcinogenicity to humans and experimental animals. Silicon carbide was classified into silicon carbide fibers and whiskers. Silicon carbide fibers were evaluated as possibly carcinogenic to humans (Group 2B) on the basis of limited evidence of carcinogenicity to humans. Silicon carbide whiskers were evaluated as probably carcinogenic to humans (Group 2A) on the basis of sufficient evidence of carcinogenicity to experimental animals and the similarity of their physicochemical properties to those of asbestos in terms of the mechanism of carcinogenicity. We report the process of progression in meeting and discuss how to determine the evidence and the evaluation of the carcinogenicity of the three materials.

Effect of Inorganic and Organic Carbon Enrichments (DIC and DOC) on the Photosynthesis and Calcification Rates of Two Calcifying Green Algae from a Caribbean Reef Lagoon.

Coral reefs worldwide are affected by increasing dissolved inorganic carbon (DIC) and organic carbon (DOC) concentrations due to ocean acidification (OA) and coastal eutrophication. These two stressors can occur simultaneously, particularly in near-shore reef environments with increasing anthropogenic pressure. However, experimental studies on how elevated DIC and DOC interact are scarce and fundamental to understanding potential synergistic effects and foreseeing future changes in coral reef function. Using an open mesocosm experiment, the present study investigated the impact of elevated DIC (pHNBS: 8.2 and 7.8; pCO2: 377 and 1076 µatm) and DOC (added as 833 µmol L-1 of glucose) on calcification and photosynthesis rates of two common calcifying green algae, Halimeda incrassata and Udotea flabellum, in a shallow reef environment. Our results revealed that under elevated DIC, algal photosynthesis decreased similarly for both species, but calcification was more affected in H. incrassata, which also showed carbonate dissolution rates. Elevated DOC reduced photosynthesis and calcification rates in H. incrassata, while in U. flabellum photosynthesis was unaffected and thalus calcification was severely impaired. The combined treatment showed an antagonistic effect of elevated DIC and DOC on the photosynthesis and calcification rates of H. incrassata, and an additive effect in U. flabellum. We conclude that the dominant sand dweller H. incrassata is more negatively affected by both DIC and DOC enrichments, but that their impact could be mitigated when they occur simultaneously. In contrast, U. flabellum can be less affected in coastal eutrophic waters by elevated DIC, but its contribution to reef carbonate sediment production could be further reduced. Accordingly, while the capacity of environmental eutrophication to exacerbate the impact of OA on algal-derived carbonate sand production seems to be species-specific, significant reductions can be expected under future OA scenarios, with important consequences for beach erosion and coastal sediment dynamics.

Tree-ring δ13C and δ18O, leaf δ13C and wood and leaf N status demonstrate tree growth strategies and predict susceptibility to disturbance.

Understanding how tree growth strategies may influence tree susceptibility to disturbance is an important goal, especially given projected increases in diverse ecological disturbances this century. We use growth responses of tree rings to climate, relationships between tree-ring stable isotopic signatures of carbon (δ(13)C) and oxygen (δ(18)O), wood nitrogen concentration [N], and contemporary leaf [N] and δ(13)C values to assess potential historic drivers of tree photosynthesis in dying and apparently healthy co-occurring northern red oak (Quercus rubra L. (Fagaceae)) during a region-wide oak decline event in Arkansas, USA. Bole growth of both healthy and dying trees responded negatively to drought severity (Palmer Drought Severity Index) and temperature; healthy trees exhibited a positive, but small, response to growing season precipitation. Contrary to expectations, tree-ring δ(13)C did not increase with drought severity. A significantly positive relationship between tree-ring δ(13)C and δ(18)O was evident in dying trees (P < 0.05) but not in healthy trees. Healthy trees' wood exhibited lower [N] than that of dying trees throughout most of their lives (P < 0.05), and we observed a significant, positive relationship (P < 0.05) in healthy trees between contemporary leaf δ(13)C and leaf N (by mass), but not in dying trees. Our work provides evidence that for plants in which strong relationships between δ(13)C and δ(18)O are not evident, δ(13)C may be governed by plant N status. The data further imply that historic photosynthesis in healthy trees was linked to N status and, perhaps, C sink strength to a greater extent than in dying trees, in which tree-ring stable isotopes suggest that historic photosynthesis was governed primarily by stomatal regulation. This, in turn, suggests that assessing the relative dominance of photosynthetic capacity vs stomatal regulation as drivers of trees' C accrual may be a feasible means of predicting tree responses to some disturbance events. Our work demonstrates that a dual isotope, tree-ring approach can be integrated with tree N status to begin to unravel a fundamental question in forest ecology: why do some trees die during a disturbance, while other conspecifics with apparently similar access to resources remain healthy?

Analysis of Active Methylotrophic Communities: When DNA-SIP Meets High-Throughput Technologies.

Methylotrophs are microorganisms ubiquitous in the environment that can metabolize one-carbon (C1) compounds as carbon and/or energy sources. The activity of these prokaryotes impacts biogeochemical cycles within their respective habitats and can determine whether these habitats act as sources or sinks of C1 compounds. Due to the high importance of C1 compounds, not only in biogeochemical cycles, but also for climatic processes, it is vital to understand the contributions of these microorganisms to carbon cycling in different environments. One of the most challenging questions when investigating methylotrophs, but also in environmental microbiology in general, is which species contribute to the environmental processes of interest, or "who does what, where and when?" Metabolic labeling with C1 compounds substituted with (13)C, a technique called stable isotope probing, is a key method to trace carbon fluxes within methylotrophic communities. The incorporation of (13)C into the biomass of active methylotrophs leads to an increase in the molecular mass of their biomolecules. For DNA-based stable isotope probing (DNA-SIP), labeled and unlabeled DNA is separated by isopycnic ultracentrifugation. The ability to specifically analyze DNA of active methylotrophs from a complex background community by high-throughput sequencing techniques, i.e. targeted metagenomics, is the hallmark strength of DNA-SIP for elucidating ecosystem functioning, and a protocol is detailed in this chapter.

Carbon source preference in chemosynthetic hot spring communities.

Rates of dissolved inorganic carbon (DIC), formate, and acetate mineralization and/or assimilation were determined in 13 high-temperature (>73 °C) hot springs in Yellowstone National Park (YNP), Wyoming, in order to evaluate the relative importance of these substrates in supporting microbial metabolism. While 9 of the hot spring communities exhibited rates of DIC assimilation that were greater than those of formate and acetate assimilation, 2 exhibited rates of formate and/or acetate assimilation that exceeded those of DIC assimilation. Overall rates of DIC, formate, and acetate mineralization and assimilation were positively correlated with spring pH but showed little correlation with temperature. Communities sampled from hot springs with similar geochemistries generally exhibited similar rates of substrate transformation, as well as similar community compositions, as revealed by 16S rRNA gene-tagged sequencing. Amendment of microcosms with small (micromolar) amounts of formate suppressed DIC assimilation in short-term (<45-min) incubations, despite the presence of native DIC concentrations that exceeded those of added formate by 2 to 3 orders of magnitude. The concentration of added formate required to suppress DIC assimilation was similar to the affinity constant (K(m)) for formate transformation, as determined by community kinetic assays. These results suggest that dominant chemoautotrophs in high-temperature communities are facultatively autotrophic or mixotrophic, are adapted to fluctuating nutrient availabilities, and are capable of taking advantage of energy-rich organic substrates when they become available.

Assessment of bacterial community structure in nitrifying biofilm under inorganic carbon-sufficient and -limited conditions.

In this work, nitrification and changes in the composition of the total bacterial community under inorganic carbon (IC)-limited conditions, in a nitrifying moving bed biofilm reactor, was investigated. A culture-independent analysis of cloning and sequencing based on the 16S rRNA gene was applied to quantify the bacterial diversity and to determine bacterial taxonomic assignment. IC concentrations had significant effects on the stability of ammonia-oxidation as indicated by the reduction of the nitrogen conversion rate with high NH4(+)-N loadings. The predominance of Nitrosomonas europaea was maintained in spite of changes in the IC concentration. In contrast, heterotrophic bacterial species contributed to a high bacterial diversity, and to a dynamic shift in the bacterial community structure, under IC-limited conditions. In this study, individual functions of heterotrophic bacteria were estimated based on taxonomic information. Possible key roles of coexisting heterotrophic bacteria are the assimilation of organic compounds of extracellular polymeric substances produced by nitrifiers, and biofilm formation by providing a filamentous structure and aggregation properties.

Redox changes accompanying inorganic carbon limitation in Synechocystis sp. PCC 6803.

Inorganic carbon (Ci) is the major sink for photosynthetic reductant in organisms capable of oxygenic photosynthesis. In the absence of abundant Ci, the cyanobacterium Synechocystis sp. strain PCC6803 expresses a high affinity Ci acquisition system, the CO2-concentrating mechanisms (CCM), controlled by the transcriptional regulator CcmR and the metabolites NADP+ and α-ketoglutarate, which act as co-repressors of CcmR by modulating its DNA binding. The CCM thus responds to internal cellular redox changes during the transition from Ci-replete to Ci-limited conditions. However, the actual changes in the metabolic state of the NADPH/NADP+ system that occur during the transition to Ci-limited conditions remain ill-defined. Analysis of changes in the redox state of cells experiencing Ci limitation reveals systematic changes associated with physiological adjustments and a trend towards the quinone and NADP pools becoming highly reduced. A rapid and persistent increase in F0 was observed in cells reaching the Ci-limited state, as was the induction of photoprotective fluorescence quenching. Systematic changes in the fluorescence induction transients were also observed. As with Chl fluorescence, a transient reduction of the NADPH pool ('M' peak), is assigned to State 2→State 1 transition associated with increased electron flow to NADP+. This was followed by a characteristic decline, which was abolished by Ci limitation or inhibition of the Calvin-Benson-Bassham (CBB) cycle and is thus assigned to the activation of the CBB cycle. The results are consistent with the proposed regulation of the CCM and provide new information on the nature of the Chl and NADPH fluorescence induction curves.

The effect of inorganic carbon on microbial interactions in a biofilm nitritation-anammox process.

The overarching goal of this study was to determine the role of inorganic carbon (IC) in influencing the microbial ecology, performance and nitrogen turnover by individual microbial communities of a biofilm based combined nitritation-anammox process. IC limitation was transiently imposed by reducing the IC input from 350% to 40% of the stoichiometric requirement for 40 days. The principal impact observed during IC limitation was the overgrowth of nitrite oxidizing bacteria (NOB) at the expense of anaerobic ammonia oxidizing bacteria (AMX). On the other hand, the concentrations of ammonia oxidizing bacteria (AOB) were relatively stable during the imposition of and recovery from IC limitation. The resulting dominance of NOB, in terms of their concentration and contribution to nitrite consumption over AMX, resulted, in turn, in a decrease in overall nitrogen removal from 78 ± 2.0% before IC limitation to 46 ± 2.9% during IC limitation. Upon recovery back to non-limiting IC input, it took an inordinately long time (about 57*HRT) for the N-removal to recover back to pre-limitation conditions. Even after recovery, NOB were still persistent in the biofilm and could not be washed out to pre-limitation concentrations. The emission of nitrous oxide (N2O) and nitric oxide (NO), likely from AOB, transiently increased in concert with transient increases in ammonia and hydroxylamine concentrations during the period of IC limitation. Therefore, an unintended consequence of IC limitation in nitritation-anammox systems can be an increase in their greenhouse gas footprint, in addition to compromised process performance. Most emphasis to date on nitritation and anammox studies has been on the nitrogen cycle. The results of this study demonstrate that the differing strategies used by AOB, NOB and AMX to compete for their preferred assimilative carbon source can also significantly influence the microbial ecology, performance and carbon footprint of such processes.