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Island biogeography on a micro-scale: Larger anammox granules not only harbor higher species diversity but also support more functional diversity
Hui Chen, Tao Liu, Jie Li, Likai Mao, Jun Ye, Xiaoyu Han, Mike S. M. Jetten, and 1 more
https://doi.org/10.21203/rs.3.rs-18517/v1
posted 25 Mar, 2020
Background: According to the theory of island biogeography, there is a strong relationship between the species diversity and the isolated area. However, it is unclear whether the ecological distribution of microorganisms follows this island biogeography pattern at micro-scale. Here, we use microbial granules harvested from a partial nitritation and anammox (PN/A) system as a model to test if the microbial and functional diversity follow the island biogeography. We collected and divided these granules into five discrete size-fractions (<0.2, 0.2–0.5, 0.5–0.8, 0.8–1.0 and >1.0 mm).
Results: By comparing the composition and functional attribute of five pools of the size-fractionated granules by 16S rRNA gene amplicon, metagenomic and metatranscriptomic sequencing, larger granules were shown not only to harbor higher microbial diversity, but also to support more diverse functions than smaller granules. De novo co-assembly and binning of metagenomic reads yielded 22 near-complete genomes of dominant microorganisms, which allowed us to infer an ecological model of the microbial ecosystem in anammox-based granules. This genome-based ecological model indicates that nitrifying organisms in smaller granules feed nitrite to anammox bacteria in larger granules.
Conclusion: Our findings substantiate that microbial communities in PN/A granules follow a species-volume relationship, suggesting the generality of the theory of island biogeography on microscopic scale.
General Microbiology
island biogeography
anammox bacteria
microbial community structure
metatranscriptomics
For centuries, ecologists have studied how the diversity and functional traits of plant and animal communities vary across biomes, and have developed a number of ecological theories that interpret some of the observed general patterns. The theory of island biogeography is one of classic tenets and has been well tested at macro-level, where species richness (e.g., animals and plants) is a function of island characteristics-notably the island size and remoteness (accessibility of islands to colonizing organisms) [1, 2]. Generally, larger island houses more species diversity and possesses larger absolute population size. However, despite important advances in evolutionary and conservation biology, the question regarding whether larger island harbors more microbial diversity and functional diversity is yet to be verified at a microscopic scale. Currently, there is much dispute regarding whether microorganisms have distinct biogeographies [3, 4], or whether all microorganisms have a cosmopolitan distribution, as presumed by the cosmopolitan theory of Baas-Becking that 'everything is everywhere' and the prevailing environmental conditions select [5]. According to the traditional cosmopolitan theory, the microorganisms are universally dispersed without impediment which would make biogeographic patterning unlikely due to fewer constraints of distribution and high population densities [4, 6]. In contrast, recent studies using molecular techniques suggested that microbial diversity in natural ecosystems follows biogeographical patterns that are similar to those observed for plants and animals [7-9], where an increase in the number of taxa is observed with increasing sample area. For example, Bell et al. [7] observed that the bacterial diversity (taxon richness) in water-filled treeholes, taken as islands, tended to increase with water volume (island size), which can be explained by the theory of island biogeography.
In contrast to the studies in natural ecosystems [7-9], we have just begun exploring similar questions for microbial communities in engineered ecosystems, where microbial communities are assembled by function, and render ecosystem service for the benefit of mankind. With increasing reliance on engineered ecosystems (e.g., wastewater treatment and bioremediation), the understanding of the microbial ecology has become essential for improving the efficiency and robustness of engineered systems [10, 11]. For instance, the one-stage partial nitritation and anammox (PN/A), which combines aerobic ammonia oxidation with anaerobic ammonium oxidation (anammox), is a microbially mediated technology for treating ammonium-containing wastewater [12]. In the PN/A process, ammonia-oxidizing bacteria (AOB) oxidize about half of ammonium to nitrite while consuming the available but limiting oxygen, and anammox bacteria (AMX) convert the generated nitrite and the other half of ammonium into dinitrogen gas in the anoxic environment, with the production of some nitrate (Reaction: NH4+ + 1.32NO2- → 1.02N2 + 2.03H2O + 0.26NO3-) [13] . Compared to the conventional two-stage nitrification/denitrification, the PN/A process exhibits several advantages, including no need for carbon source, less oxygen demand, less sludge production and less N2O emission (a potent greenhouse gas) [14]. Granulation is an important strategy to retain sufficient biomass in the reactor, especially when dealing with AMX which grow slowly (with a doubling time ranges from 3 to 20 days) [15, 16]. Granule-associated bioreactors are intriguing systems for the consideration of biogeographic patterns, as granules provide well-replicated and clearly defined habitats for testing ecological theory. Granules in these bioreactors, like isolated oceanic land masses, are discrete and island-like habitats, which could be considered as "microscopic islands". Therefore, we hypothesized granules with different sizes represent microscopic islands with different island areas, thus affecting microbial diversity and functional diversity [17, 18].
Culture-independent molecular techniques have been used to characterize microbial communities in granule-based PN/A systems [19, 20]. These studies have provided insights of the taxonomic composition (in particular key nitrogen cycling microorganisms), but the interactions and differences of microbial and functional diversity among and within granules with different sizes remain not well explored. Metagenomic/metatranscriptomic sequencing, the direct sequencing of the collective genomes in a given sample, enables to gain a more comprehensive understanding of microbial communities and a more detailed distribution of the networked metabolic interactions among anammox granules [21]. For example, previous studies combining metagenome and/or metatranscriptome have provided insights into the interactions of anammox and heterotrophic bacteria [22, 23], as well as suggested a clear variation in N cycling genes among different microbial aggregates (e.g., suspended biomass, biofilm, and granule) [24, 25]. However, all of these studies were conducted based on the premise that the granules present in a uniform size. In reality, the granules have a heterogenetic size distribution, and microbial diversity and functional diversity of PN/A granules might vary in complex patterns in response to granule size [26]. Thus, it is required to consider the granule size distribution in order to gain a more comprehensive view of changes of functional capabilities across size-fractions, and the extent to which microbial metabolisms are partitioned between size-fractionated granule communities. In this way, the microbial populations can be analysed at multiple scales within and among granules, which function as microbial "archipelagos".
The current study was designed to study the microbial biogeography by using a pilot-scale granule-based PN/A reactor as a model system. We hypothesized that the microbial communities found in the large size-fractions might be taxonomically and functionally distinct compared to those found in smaller fractions. This was predicted based on the fact that, from a macro-scale view, the biodiversity of island is affected by the island size, where larger island provides a wider variety of habitats, so species will diversify to fill up the available habitats [1]. Granules from a pilot-scale PN/A reactor were collected and separated to five size-fractions (<0.2, 0.2–0.5, 0.5–0.8, 0.8–1.0 and >1.0 mm). We combined 16S rRNA gene amplicon, metagenomic and metatranscriptomic sequencing to investigate the differentiation of microbial diversity and functional diversity among the size-fractions. Moreover, based on the assembled draft genomes and mathematic modeling, we sought to delineate an ecological model of the microbial and metabolic interactions within and between size-fractions in the PN/A reactor by using multiple granule sizes instead of the use of a uniform size. We demonstrate that microbes inhabiting in the PN/A granules exhibit species-volume relationship. Our findings gave insight into the island biogeographical theory of microorganisms in water engineered ecosystems at a microscopic scale.
Microbial community overview of size-fractionated PN/A granules
The pilot-scale reactor had been in operation continuously for more than 500 days before the sampling. The influent was rich in ammonium (1724 ± 846 mg N L-1) and contained residual organic carbon (336 ± 116 mg COD L-1) (Table S1). The reactor was oxygen limited via continuous aeration resulting in dissolved oxygen DO values of 0.5-1.0 mg L-1. Stable PN/A performance was achieved in terms of an average total nitrogen removal rate of 0.82 kg N m-3 d-1 and efficiency of 87%.
The microbial community structure of the PN/A granule samples was characterized using the combination of 16S rRNA gene sequencing and metagenomic sequencing based on Illumina technology. Taxonomic analyses based on 16S rRNA gene sequencing showed that, microbial communities in granules with different sizes were dominated by members of Proteobacteria (24~46%), Chlorobi (14~34%), Bacteroidetes (11~20%), Chloroflexi (8~11%) and Planctomycetes (0.5~10%) (Fig. 1). These bacterial phyla are known to be relatively abundant and ubiquitous in PN/A granules [20, 22, 23] and these five phyla accounted for at least 87% of all sequencing reads in granules of all size-fractions in this study. In addition, it was observed that the relative abundance of these members changed with increasing of granule size. Proteobacteria, accounted for 46% of all sequences in <0.2 mm size fraction, while decreasing to 27% in >1.0 mm size-fraction. Similarly, Bacteriodetes were consistently enriched in the smaller size-fractions, where they constituted of 20% all sequences in 0.2-0.5mm size-fraction (versus 11% in >1.0mm size-fraction). In contrast, clades of Planctomycetes and Chloroflexi were consistently enriched in the larger size-fractions. Sequences matching WS6 and Acidobateria represented relatively minor components of the total amplicon sequences (1.6±1% and 1.3±1%, respectively), but showed more abundances in large size-fractions. A previous study has predicted a fermentative and denitrifying role in the anaerobic granule core for WS6 and Acidobacteria, respectively [22]. It was surprising that each size-fraction also contained different bacterial communities, with a range of 2.3-7% of unique OTUs in each size-fraction (Fig. S2), supporting the fact that they harbor distinct habitats. For example, Zoogloea was only present in the <0.2 mm size-fraction, whereas Opitutus was only observed in the >1.0 mm size-fraction.
Although similar taxonomic distributions were observed in metagenomic datasets, some differences were found in terms of species abundance, such as a slightly higher representation of Chloroflexi, Planctomycetes and Acidobacteria in metagenomic sequencing compared to 16S rRNA gene sequencing. Also, some phyla were less represented (e.g., Bacteriodetes and Chlorobi) in metagenome than in 16S rRNA gene sequencing datasets. Such differences could be partly explained by biases derived from the PCR process in amplicon sequencing [27]. Additionally, 16S rRNA gene recovered from the metagenomics data can span the entire length of the gene, whereas the PCR-based amplicon approach only targets specific region. The differences in accuracy of taxonomic assignments with various regions of the 16S rRNA gene may lead to the observed discrepancies with the two approaches [28].
Metagenome co-assembly and binning yielded 22 high-quality (> 80% completeness and <4% contamination rates) MAGs (Table 2). The phylogenetic inference (Fig. 2) showed that these 22 MAGs spanned 8 phyla, including Proteobacteria, Planctomycetes, Bacteriodetes, Chloroflexi, Verrucomicrobia, Acidobacteria, Chlorobi, and Omnitrophica. The recovered genomes in the PN/A granules mostly resemble those of previously detected organisms in other PN/A system [22]. Among the recovered genomes were characterized taxa known to be involved in the canonical PN/A process: Nitrosomonas (AOB) encoded the key enzyme for ammonia oxidation (amo) (Fig. S3); and Candidatus Kuenenia (AMX) contained the core genes for hydrazine metabolism (hzs and hdh) and the gene cluster for nitrite reduction (nir), all essential for the anammox process. In addition, a few of organisms (e.g., PTB1-4, and CFX1-3) were capable of nitrate/nitrite respiration, which was evident from the genes encoding nitrate/nitrite reductase (e.g., nar and nir). Among them, only PTB3 and PTB4 encoded a complete denitrification pathway from nitrate to dinitrogen gas (Fig. S3), suggesting their potential roles in removing organic carbon and nitrate.
Larger size fractions harbor more diverse communities and sustain more redundant functions
In order to investigate the difference of the species diversity among size-fractions, we calculated alpha-diversity indices (including ACE and Chao1 for richness, and Shannon H and Simpson D for diversity) based on the 16S rRNA gene sequencing results (Table 1). Granule size explained the increasing number of OTUs (species) in larger size-fraction, ranging from 356 (<0.2 mm) to 434 (>1.0 mm). Similarly, the microbial richness, as indicated by the estimators ACE and Chao1, generally showing an increasing trend along with the granule size (except for 0.2-0.5mm size-fraction being the lowest). In addition to granule size, microbial richness could also be affected by biotic factors such as competition and facilitation, and abiotic factors such as nutrient availability and disturbance frequency [29]. In terms of microbial diversity, Shannon H rose from 3.19 in <0.2 mm size-fraction to 3.83 in >1.0 mm size-fraction; whereas Simpson D showed the lowest value of 0.0735 for 0.8-1.0 mm size-fraction and the highest of 0.1347 for <0.2 mm size-fraction (overall showing a downward trend). Both the increasing Shannon H and decreasing Simpson D in larger granules indicate that larger size-fraction contain higher microbial diversity in this PN/A system.
According to the theory of island biogeography, there is a strong species-area relationship at macro-scale. As shown in Fig. 3A, taxonomic diversity contained within each size-fractions increased with increasing granule size, showing an approximately linear growth. The granules with >1.0 mm size-fraction present the most diverse community, whereas the <0.2 mm size-fraction had the lowest diversity. Considering the granule have a three-dimensional habitat distribution, which means microorganisms can grow on the inner layer of granules, rather than only growing on the surface of an island. Thus, we test whether the ecological distribution of microorganisms in granules follows a species-volume relationship (Fig. 3B). A sharp increase of the taxonomic diversity was observed in granules with volume smaller than 0.38 mm3 (<1.0 mm size-fractions), while there was a slow increase in taxonomic diversity in granules larger than 0.38 mm3 (>1.0 mm size-fraction). The elevated diversity in larger granule has been shown to coincide with previous studies of PN/A granules in lab-scale reactors [19, 20]. Taxa are usually adapted to a particular habitat, and large granules contain more distinct layered habitats due to the mass transport limitation of nutrient [30-33]. Therefore, large granules could support the survival of more species.
Functional diversity has been used to reveal the diversity and distribution of functional traits (or functional genes) across communities [34, 35]. Here we used Shannon's diversity index to indicate functional diversity [35], estimated from the diversity of the protein-coding genes categories identified from annotated metagenomic reads. Similar to the distribution of taxonomic diversity among size-fractions, the communities found in small size-fractions typically had lower level of functional diversity (Fig. 3C and D). This is likely a result of that large granule having more anaerobic habitats suitable for the growth of anaerobes, therefore expressing a broader array of genes associated with anaerobic pathways (e.g., methanophenazine hydrogenation and dipicolinate synthesis). With granule size larger than 1.0 mm, interestingly, no increase in the functional diversity was observed despite increasing species richness along with the increasing granule size. It is widely assumed that community diversity could be decoupled with functioning [36, 37]. With a certain microbial diversity, additional species would not bring significant additional functional benefits [38]. Multiple microbial taxon can carry out the same process within an ecosystem, thus resulting in the functional redundancy, where the system can perform just as well with fewer species. Considering both functional diversity and the taxonomic diversity, we found that the overall diversity of functional gene categories found in a given sample was, to some degree, predictable from taxonomic diversity of the microbial communities. It should be noted that the samples collected in this study were from one steady-state PN/A system. Future investigations on more granule-based systems might be required to confirm our observations of this work and would be better for a more comprehensive comparison.
Nitrogen conversion microorganisms and metabolic pathways in different size-fractionated granules
A PN/A bioreactor is a nitrogen-driven ecosystem, and its stable performance relies on achieving an appropriate balance between the activities of the principal microbial protagonists therein, including AMX and AOB, while suppressing or washing out nitrite-oxidizing bacteria (NOB). Here, we specifically analyzed the populations involved in nitrogen cycle of the five size fractions based on both 16S rRNA gene and metagenomic sequences. Anammox bacteria were proportionally enriched in larger size-fractions (Table S2). Sequencing matching anammox Candidatus Kuenenia with a similarity of 99.6% (classified as Planctomycetes in Fig. 1) peaked in relative abundance (9.4% of total) in >1.0 mm size-fraction, while decreased to 5.3% in 0.5-0.8 mm size-fraction and were nearly negligible in <0.2 mm size fraction (0.3%). Their elevated abundances in the larger size fractions are understandable since Candidatus Kuenenia is inhibited by oxygen [39], which favors more anaerobic space in larger granules due to a longer oxygen permeation depth [32, 33]. Conversely, the relative abundance of the sequences matching a typical AOB, i.e. Nitrosomonas (classified as Proteobacteria in Fig. 1) showed an opposite trend, peaking at 33.8% of the total <0.2mm size-fraction amplicons, compared with 5.7-6.4% in >0.8mm fractions. Since smaller granules have higher surface/volume ratio, which provide larger aerobic volume fraction [40], the enrichment of Nitrosomonas in small granules is consistent with these bacteria being aerobes adapted for the efficient use of oxygen. The presence of Nitrospira (one of the typical NOB), which could negatively affect nitrogen removal efficiency by competing nitrite with anammox bacteria, was negligible with an abundance of 0.01-0.3% across all size-fractions. Additionally, a typical heterotrophic denitrifier, Denitratisoma (classified as Proteobacteria in Fig. 1), was also observed in all the granules, which can utilize the organic carbon for nitrate/nitrite removal. The relative abundance of this organic carbon-scavenger increased from 3.8% (<0.2 mm-granules) to 8.6% (0.8-1.0 mm-granules), because larger granules harbour more anaerobic zones supporting their growth by taking up nitrite/nitrate as the electron acceptors.
Functional screening of the metagenomic and metatranscriptomic datasets revealed genes and transcripts involved in nitrogen metabolisms across size-fractions, which were correlated with the presence of the functional microorganisms (Fig. 4 and Table S2). In both the gene-abundance and gene-expression levels, the key nitrogen-related genes, were detected in all granules with different sizes. Specifically, the core genes and transcripts involved in hydrazine metabolism (hzs and hdh), which are all essential for anammox process, were expressed in larger granules, suggesting that larger size fractions predominantly performed the anammox process. In contrast, nitrification is the predominant nitrogen metabolism in the smaller granules, where the key genes and transcripts encoded for ammonia oxidation enzymes (amo and hao) were more abundant. Additionally, genes that encode catabolic enzyme specific for denitrification, including napA, nar, nirK, nor and nosZ, were low in abundance and expression compared with those involved in anammox and nitrification process. The counts of genes and transcripts generally show an increasing trend along with granule size, whereas 0.8-1.0 mm granules have the highest transcript hits.
Based on the established mathematical model, the microbial stratification inside the granules was also investigated (Fig. S4). Overall, anammox bacteria were shown to dominate the larger granules (0.6-1.2 mm) while AOB were predicted to be present in the smaller granules (0.1-0.3 mm. The model showed that the NOB were out selected in all granule sizes in the competition with anammox and heterotrophic denitrifiers. These model predictions are in accordance with metagenomic and metatranscriptomic results. The good match between the experimental and simulation results supported the relationship between microbial stratification and granule size. Specifically, in 0.1-0.3 mm granules, AOB occupy the granule surface where oxygen and ammonium are sufficiently present, while heterotrophs dominate the inner layer. In the granules with radius of 0.6-1.2 mm, anammox bacteria gradually develop at the inner side of the granule, with the relative distribution of AOB and heterotrophic bacteria unchanged at outer layer. Anammox bacteria are outcompeted by heterotrophs at outer shell of the granule mainly due to the inhibition of oxygen at the granule surface. The predicted microbial stratification inside biofilm (Fig. S4) remains to be experimentally verified by using advanced methods (e.g., 3D-FISH and microsensor).
Overall, the fraction-specific distribution was evident for key nitrogen-involving taxa, genes and transcripts, as well as the microbial stratification inside biofilm matrix. These patterns suggest that the small granule plays a dominant role in ammonium oxidation that is critically dependent on access to sufficient oxygen, and the removal of the produced nitrite will mostly rely on the anammox and heterotrophic denitrification in large granule. However, as large granules provide habitats for both aerobes and anaerobes, it is capable of complete nitrogen removal, but the availability of nitrite will restrict the overall nitrogen removal rate. Additionally, the stratified biofilm was determined by the availability and inhibition effect of specific substrates (e.g., nitrite and oxygen). Variable granule size distribution may drive heterogeneity in nitrogen removal performance in PN/A process.
Granular aggregate as a microscopic model system for island biogeography
Island biogeography theory is one of the most influential paradigms in ecology, which is central to several disciplines (e.g., conservation ecology and metapopulation theory) and continues to provide insights into research of island-like systems [41-44]. For example, Ali, Wang, Salam, Hari, Pronk, Van Loosdrecht, Saikaly [44] observed that the species sorting was significantly higher in flocs and small granules than large granules in aerobic granular sludge system, while putative functional populations were enriched with an increase in microbial aggregates size. Here, we adopted the theory of island biogeography as a conceptual framework for understanding the microbial ecology of microbes within a granule-based PN/A bioreactor. Granules, as insular habitats, provide favorable microscopic habitats for microorganisms. Viewing granules as islands in a bioreactor of potential colonists, increasing taxonomic diversity in larger size-fractions was shown to be congruent with the theory of island biogeography as the microbial richness was largely dependent on the size of the island [1] and larger islands house more taxa [7] (Fig. 3).
The observed increase in microbial diversity can be partially explained by the increased micro-habitats diversity and substrate-transmission complexity that associated with a larger granule. Firstly, larger granule provides better habitat differentiation and the enhanced heterogeneity, which foster more functional populations. Specifically, the large granules do have an anaerobic layer at the inner core that accommodates populations of other physiological types of bacteria. Secondly, each of the granule houses a micro-ecosystem that derives its nutrients and energy from wastewater. The availability of nutrients/substrate that may favor different bacterial species to colonize and perform differently. In PN/A reactor, the availability of oxygen, to a large extent, differentiates the microbial communities of small granules from large granules. The substrate (e.g., oxygen) drawdown from the outer layer to the inner core of granules significantly restructures the microbial ecosystem in granules, resulting in the differences in the abundances and the activities of aerophilic or anaerobic microbes. Large granules offer more favourable habitat for anaerobic bacteria (e.g., heterotrophic bacteria) that are capable of anaerobically decomposing organic matter within the anoxic core of the granules. Aerobic COD degradation in small granules and on the surface of large granules, together with anaerobic COD degradation in the core of large granules contributed to the COD removal in this PN/A system (as indicated by the COD removal efficiency in Table S1).
It is worth to note that PN/A granules are recirculated in the bioreactor, which make the scenario of the interaction and overview of microbial ecology in granules more complicated. Taking the bioreactors as a whole, where a group of granules closely scattered in the bioreactor, numerous of granules form an archipelago and there were reciprocal interactions between these islands. Under such circumstance, a higher immigration rate of species is expected, which might reduce the species differences between size-fractions.
Overall, granules function as islands for housing microorganisms, in which larger granules not only harbor higher microbial diversity, but also support more functional diversity. The theory of island biogeography is instructive to understand the microbial ecology associated with granule. A better understanding of the microbial ecology in PN/A reactor could guide us to better design and operate bioreactors to foster the development of specific microbial communities that can support the desired functional processes [11].
Integrating nitrogen cycling microorganism and activities in granules
As indicated by the different expression of aerobic and anaerobic ammonium oxidation genes in size-fractionated anammox granules (Fig. 4), an ecological model based on the granule PN/A system with size distribution was delineated in Fig. 5. The small-size granules are dominated by AOB and heterotrophs, which catalyze aerobic oxidation of ammonium to nitrite and aerobic oxidation of organic carbon to CO2. On the other hand, both aerobic and anaerobic microorganisms (AOB, anammox and heterotrophs) are observed in large-size granules, in which ammonium, produced nitrite and nitrate are removed to dinitrogen gas. It is also worth to note that NOB are rarely detected in all granules, thus the nitrate involved is mainly produced from anammox reaction and partially consumed by heterotrophic denitrification [15].
In small granules, AOB contribute to ammonium oxidation while heterotrophs contribute to COD oxidation. As the oxygen permeates along the entire small granules (0.1-0.3 mm) (Fig. S4), the anaerobic processes (anammox and heterotrophic denitrification) are both inhibited in small granules. Thus, the produced nitrite will escape from small granules and transfer to the larger granules. In large granules (0.6-1.2 mm), the oxygen permeation from surface ceases after 300-400 μm, and the anaerobic layer can expand. In the anaerobic layer, both anammox and heterotrophic denitrification work together to remove nitrite either transferred from small granules or from the AOB growing at outer layer of the large granule itself. It is worth to note that the kinetics of anammox and heterotrophic denitrifiers selected were slightly higher than the average values [45]. Therefore, NOB are out-selected during the competition for nitrite with AMX and heterotrophic denitrifiers in the present model. In the real reactor, the out-selection of NOB could be due to the high free nitrous acids (FNA) and free ammonia (FA) concentrations under sidestream condition [46].
To quantify the nitrogen flows among granules, mass balance was analyzed based on the simulation results and the calculated biomass volume of each granule size. The granules with radius of 0.1 mm, only contributed 4.2% to nitrite production which can be removed inside the same granules while the majority (95.8%) will be transferred to the larger granules with radius of 0.6 mm (14.8%), 0.9 mm (46.5%) and 1.2 mm (34.5%). Similarly, about 14.4%, 45.3% and 33.7% of the produced nitrite in granules with radius of 0.3 mm will be transferred to 0.6 mm, 0.9 mm and 1.2 mm granules, respectively (Fig. 5). This cross-feeding of nitrite from small to large granules indicates the mutualism and competition in PN/A system occur not only inside granules, but also among granules with different sizes [47]. However, this cross-feeding of substrate is unidirectional (Fig. 5). The granules with radius of 0.6-1.2 mm could offer both aerobic and anaerobic conditions, which can foster AOB, anammox bacteria and heterotrophic bacteria at different micro-habitats (Fig. S4). By utilizing the ammonium, oxygen and organic carbon from the feeding, and the nitrite transferred from the smaller granules, these large granules can develop a stratified microbial community to utilize all these inputs by themselves with a balance. Thus, there is no substrate transferred from the large granules to the small granules. However, these results do not suggest that larger granules are better than smaller granules. Indeed, it was observed that the N2O production increased significantly in larger granules due to incomplete denitrification [48]. The optimal granule size in practice is determined based on the balance between nitrogen removal performance, greenhouse gas emission (N2O) and also the operational cost (maintaining large granules requires more energy consumption), which warrants further studies.
Although the microbial ecology of anammox granules was studied in previous studies [22, 23, 25, 49, 50], the size-fractioned anammox granule system proposed here delineates the interactions among granules with different sizes from micro-scale view for the first time. In a recent study, the cross-feeding among microorganisms was investigated based on metagenomics analysis [50]. However, the present study touched another dimension to demonstrate the interactions between granules based on more comprehensive data (metagenomics and metatranscriptomics). The cross-feeding model proposed here offers an explanation for the granule size distribution impacts on PN/A performance.
Granules in a pilot-scale PN/A reactor were collected and divided into five discrete size-fractions (<0.2, 0.2–0.5, 0.5–0.8, 0.8–1.0 and >1.0 mm). By comparing the composition and functional attributes of five size-fractionated granules by 16S rRNA gene amplicon, metagenomic and metatranscriptomic sequencing, larger granules were shown not only to harbor higher microbial diversity, but also to support more diverse functions than smaller granules. 22 near-complete genomes of dominant microorganisms were obtained using de novo co-assembly and binning of metagenomic reads. In addition, an ecological model was developed based on microbial characteristics and mathematical model, which indicated that nitrifying organisms in smaller granules feed nitrite to anammox bacteria in larger granules. Our findings substantiate that microbial communities in PN/A granules follow a species-volume relationship, suggesting the generality of the theory of island biogeography on microscopic scale.
Granular samples were collected from a pilot-scale sequencing batch reactor (SBR) performing one-stage PN/A process, with a working volume of 60 m3. The reactor was completely mixed with sludge (granules) homogeneously distributed. The reactor was fed with the effluent from a primary clarifier of a full-scale WWTP (Beijing, China). Ammonium carbonate was added to the real wastewater to obtain an average ammonium concentration of 1700 mg N/L. The mean concentrations of chemical oxygen demand (COD) and phosphorus were 340 mg/L and 6 mg P/L, respectively. Dissolved oxygen (DO) was maintained at 0.5–1.0 mg/L via an aeration control system. After long-term operation, this reactor achieved stable autotrophic nitrogen removal mediated by AOB and anammox bacteria (operational performance as shown in Table S1).
This pilot-scale reactor had been operated continuously for more than 500 days before the sampling. Since the granules were homogeneously distributed in the complete-mixing reactor, to collect representative samples, granular sludge (150 mL) was harvested from the middle of reactor. While single-granule based investigation is beneficial for the study [51], it is time- and cost-consuming. Therefore, based on the granular size, the sampled sludge was further fractionated into five groups by sequential sieving through a series of sieves (0.2, 0.5, 0.8 and 1.0mm mesh) in a short time (<1 min). Five subsamples with different granular size P1 (<0.2mm), P2 (0.2-0.5mm), P3 (0.5-0.8mm), P4 (0.8-1.0mm) and P5 (>1.0mm) were obtained (Fig. S1) and were instantly flash frozen in liquid nitrogen before storing at -80◦C for later DNA and RNA extraction. The relative abundances of each size-fraction were described in Table S3.
Nucleic-acid extraction and sequencing
Total genomic DNA and RNA were extracted from each of the five subsamples using the FastDNA SPIN Kit for Soil (MP Biomedicals, Santa Ana, CA, USA) and the Trizol kit (Invitrogen, Carlsbad, CA, USA), respectively, according to the manufacturer's protocols. The purity and concentration of the extracted DNA and RNA was detected using agarose gel electrophoresis an Ultra-micro spectrophotometer NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). For DNA library construction, DNA was fragmented to an average size of approximately 300 bp by ultrasonication (Covaris M220 series, Woburn, MA, USA) and the fragmented DNA were end-repaired, A-tailed and ligated to TruSeq adapters using a TruSeqTM DNA sample prep kit (Illumina, SanDiego, CA, USA). Following RNA extraction, total RNA samples are DNase-treated to remove contaminant genomic DNA. Depletion of ribosomal RNA (rRNA) was performed using a Ribo-Zero rRNA Removal Kits (Epicentre, Wisconsin, USA) following the manufacturer's instructions. The rRNA-depleted samples were then input into the TruSeqTM RNA sample prep kit (Illumina, SanDiego, CA, USA) for RNA-seq library preparation. Briefly, the purified RNA was fragmented and then reverse transcribed to cDNA with adaptors attached to the ends. Both DNA and RNA sequencing were performed on an Illumina Hiseq 2000 platform (Illumina, San Diego, CA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China).
An amplicon survey of a portion of 16S rRNA gene was carried out to provide a higher resolution and more in-depth analysis of the composition and diversity of various granule size fractions. Amplification of 16S rRNA genes from genomic DNA were conducted using the barcoded primer pair 338F/806R[52] that targets the V3-V4 regions of the 16S rRNA gene. DNA was amplified in triplicate following the protocol described previously [20], and the triplicate reactions of each sample were composited. Amplicon sequencing was conducted on an Illumina MiSeq platform (Illumina, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China), creating 2 × 250 bp paired-end reads. The resulted sequencing reads were de-multiplexed and quality filtered to remove low quality sequences using QIIME [53] with default parameters. And chimeric sequences were removed with UCHIME [54] with default parameters. Sequence reads were assigned into operational taxonomic units (OTUs) with 97% similarity, taxonomy was assigned by aligning the representative sequences from each OTU against the Silva database [55].
Metagenomic assembly, binning and phylogenetic analysis of recovered draft genomes
Raw paired-end reads from the HiSeq platforms were initially filtered using Trimmomatic v0.36 [56] based on a minimum average quality score of 30, a minimum sequence length of 50 bp. Paired-end reads were then merged using fq2fa program in IDBA-UD v 1.1.3 [57]. All read data were co-assembled, that is, the data sets from five samples were combined and the 169 million reads were assembled de novo together, to obtain the maximum amount of biologically relevant information on contigs with an average coverage about 10. Assembly was performed using the de novo assembler in IDBA-UD v 1.1.3 [57] with pre-correction and otherwise default settings. 135 million reads (79.7% of the data, 18.6 Gbp) were incorporated in the assembled contigs. This assembly resulted in 144,876 contigs larger than 1,000 bp, and a total of 18,312 of these contigs were longer than 5,000 bp.
Assembled contigs greater than 2,500 bp were binned as metagenome-assembled genomes (MAGs) on the basis of abundance and tetranucleotide frequency using MetaBAT [58]. The completeness and contamination of the recovered genomes was assessed using CheckM [59], evaluated from number of marker genes present in genome and from the number of multi-copy marker genes respectively. Further analysis used only high-quality draft genomes, defined as bins that are near complete (>80%) with low contamination (<5%).
For taxonomic analyses based on the metagenomic data sets, we identified and classified the 16S rRNA gene sequences from the metagenome contig files using the communityM tool (https://github.com/dparks1134/CommunityM) with Hidden Markov Models (HMM). The selected contigs were then mapped to the Greengenes database at 97% similarity with the Burrows-Wheeler Aligner (BWA) software, and operational taxonomic unit (OTU) table, showing the relative abundances of the microbial community population [60, 61], was generated.
Phylogenetic inference was conducted to classify the MAGs using genome taxonomy database (GTDB v2.1.15) [62]. Briefly, the MAGs were inferred based on a multiple sequence alignment of 120 single-copy marker proteins identified as being present in ≥90% of bacterial genomes and, when present, single-copy in ≥95% of genomes. The concatenated protein sequences were aligned to Pfam and TIGRfam hidden Markov models(HMMs) using HMMER61 v3.1b2 (http://hmmer.org/) with default parameters and tree was constructed using FastTree 2 v2.1.9 [63] under the WAG+GAMMA model using the approximately maximum likelihood method. This tree was then bootstrapped using genometreetk v0.0.35 (D. H. Parks, unpublished materials, https://github.com/dparks1134 /GenomeTreeTk), calculating bootstrap support from 100 FastTree iterations. The associated taxonomy was derived using NCBI annotations, and was used to decorate the tree using tax2tree [64] and adjusted manually. This tree was visualized in ARB v6.0.6 [65] and exported for further refinements in Adobe Illustrator.
Metatranscriptomic analysis
Quality filtering and merging of metatranscriptome sequences were carried out in the same manner as for metagenome sequences. Metatranscriptomic reads were mapped back to all assembled metagenomic contigs using STAR v020201 [66] following settings of ENCODE Long RNA-Seq processing pipeline (https://github.com/ENCODE-DCC/long-rna-seq-pipeline).
Phylogenetic and functional diversity analysis
To determine the diversity of the microbiome, we calculated the alpha diversity based on the 16S rRNA gene sequencing data sets and plotted it vs. granule volume and granule diameter. The microbial diversity was described by the OTU number, richness and diversity indices (Table 1), which was calculated in QIIME using a rarified data (22834 sequences per sample) that corrects for differences in sequencing depth.
Metagenomic and metatranscriptomic sequences were uploaded to MG-RAST (Rapid Annotation using Subsystems Technology for Metagenomes) for general functional annotation. Sequences were annotated against SEED subsystems databases with an e value of 10−5. Functional diversity were determined by calculating Shannon's diversity index from the level 3 gene categories (of which there were a total of 1107 across the 5 datasets) based on metagenomic annotation.
Analysis of key nitrogen cycle inventory
Analysis of protein-coding genes and transcripts for nitrogen metabolic pathways was performed using Prokka [67]. The targeted key enzymes includes: ammonia monooxygenase (amo), hydroxylamine oxidoreductase (hao), hydrazine synthase (hzs), hydrazine dehydrogenase (hdh), periplasmic nitrate reductase (nap), membrane-bound nitrate reductase (nar), nitrite reductase (nir), cytc dependent nitric oxide reductase (nor), nitrous oxide reductase (nos), pentaheme nitrite reductase (nrf), nitrite oxidoreductase (nxr) and nitrogenase (nif). Read counts were calculated for each predicted open reading frame (ORF) using RSEM v1.2.31 [34] and normalized by sequencing depth and ORF length and expressed as Transcripts per million (TPM) values.
A one-dimensional biofilm model was developed to simulate the bioconversion and microbial structure in the granule-based anammox process in the software AQUASIM 2.1d [68]. The model development was based on our previous study, which investigated the impacts of granule size distribution on one-stage anammox performance [45]. The biological model describes the relationships among six soluble species, i.e., degradable COD (Ss), ammonium (SNH4), nitrite (SNO2), nitrate (SNO3), nitrogen (SN2), dissolved oxygen (SO2), and six particulate species, including AOB (XAOB), NOB (XNOB), anammox bacteria (XAn), heterotrophic denitrifiers (XHB), slowly degradable organic substrate (XS), as well as inert biomass (XI). For all the microorganisms, both growth and decay were included in the model (Table S4). Kinetic control of all the enzymatic reaction rates was described through the Michaelis–Menten equation. More details about the parameter definitions and kinetics values can be found in previous study [45]. The implementation of the granular sludge reactor model with five granule size classes in AQUASIM 2.1d was based on the method proposed by previous studies [45, 69]. The essence of the model considering size distribution is dividing one single reactor into five sub-compartments. Each of them is dominated by the granules with one size. According to the measured granule size distribution (Table S3), five granule sizes (0.1 μm, 0.3 μm, 0.6 μm, 0.9 μm and 1.2 μm) were selected as the representative for each group. These five sub-compartments are connected with each other in order to simulate the conditions in one single reactor (the schematic modelling structure as shown in Fig. S5). Based on the simulated results, the mass balance was conducted to calculate the nitrogen flows among each sub-compartment. The effluent from each sub-compartment was used to compare with the final effluent from the reactor. Based on the established model, the spatial microbial stratification was studied.
Genomic DNA and RNA sequencing data can be found on the MG-RAST (Rapid Annotation using Subsystems Technology for Metagenomes) under Project ID mgp83671. Raw 16S rRNA gene sequencing data can be accessed from NCBI Sequence Read Archive (SRA) database (Accession Number: SRP080297).
This work was financially supported by the Australian Research Council Discovery Early Career Researcher Award (DE 130101401).
JG designed the study. HC, XH and JL prepared samples for 16S rRNA gene, metagenomic and metatranscriptomic sequencing. TL developed the mathematical model. XH performed the sampling, preservation and nucleic acid extractions. HC, JL, LM and JY performed the microbial community analysis and the bioinformatics analysis. HC, TL, MJ, JY and JG wrote the manuscript in consultation with all other authors.
Jianhua Guo would like to thank the support by the Australian Research Council through the Future Fellowship (FT170100196). Hui Chen would like to acknowledge the support of the International Postgraduate Research Scholarship (IPRS) and The University of Queensland Centennial Scholarship (UQCent). MSM Jetten is supported by ERC AG 339880 and OCW/SIAM 024002002.
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Table 1 Numbers of OTUs from 16S rRNA sequencing and alpha diversity indices
Granule size fractions (mm)
OTU richness
OTU diversity
Chao1
Shannon H
Simpson D
430 (404, 471)
3.19 (3.16, 3.22)
0.1347 (0.1311, 0.1382)
>1.0
Table 2 Characteristics of the 22 draft genomes obtained in this study
Bin ID
Draft genome
size (Mbp)
Completeness (%)
Contamination (%)
Number of Scaffolds
Scaffolds (bp)
GC (%)
Predicted
ACD1
Acidobacteria
Planctomycetes
Proteobacteria
BCD1
Bacteroidetes
CFX1
Chloroflexi
CHB1
Chlorobi
Omnitrophica
PTB1
Verrucomicrobia
SupplementaryInformation.docx


Nobody lies. We can know that truth for sure. And if we are ever in doubt of this truth, all we have to do is ask someone if they lie, and we can be quite certain that they will tell us they are always truthful. This is especially true if we were to ask a used car salesman or a politician.
Of course you know I am being facetious. We all lie to one degree or another. The question we should ask, especially of ourself, is how much we lie, and if we try to keep our lie-ability count as low as possible.
I think most of us try to be as honest as possible. We like to think that when we do lie, it is for a good cause, that is, for someone else's benefit. However, if you are like me, the person who ultimately benefits most from my lie is the one telling it, which, of course, is me.
Another problem we run into when we consider lies, in ourselves or others, is just what is a lie? Is a "White lie" a lie? And, if not, just what constitutes a white lie?
The first lie ever recorded in the history books, that is the Bible, is the one told to Eve in the garden of Eden. The lie the serpent told was only a partial lie. He (or it, however you read the Bible) said that the apple would make Eve wise, knowing good and evil. This, as we discover, was not a lie. Eating the fruit did indeed make her wise in this area. Of course she had to play the fool to acquire this wisdom, something we all continue to do day-by-day. Just watch the TV programs if you are in doubt of this statement.
When the serpent said Eve would not surely die, he was lying through his teeth (or fangs, whichever the case may be).
Since the serpent's lie was only partial, albeit disastrous, I wonder where it would fit on a scale of one to a hundred: one being a white lie, and one hundred being black as brimstone.
We lie. But our intentional lies are not the subject of this study. One reason blatant lies are not a consideration here is that if we were to solve the issue of what constitutes a lie, it would do little to sway us from lying, since lying is such an integral part of our nature.
To begin this study, I asked the question: "Your lie, or mine?" Many of the lies we tell are so obvious that even we know we are lying. We hope others can't tell it's a lie, but we know they do, unless they are terribly stupid or gullible. But there are lies we do not know they are lies. We tell untruths many times a day, and are totally unaware that we are doing so. Is an untruth, told in faith, a lie? That is another subject not to be considered here. What is to be considered is if we are willing to cease from telling these lies if we discover them to be so; and if we are willing to look beyond our comfort zone to learn if what we relate to others as truth, is in fact not a lie, or at least not an obvious misconception.
From the time we took our first breath we have had people telling us what is so, and what is not so. Of course our parents were the first of these instructors. Later in our early life we encountered a stream of teachers and sandbox experts who were ready and willing to lead us in their direction. On top of these, and forever present in our lives, we have the media, the advertisers, and the politicians telling us right from wrong. Add to this that for most of us, somewhere in our life, we have had some church, or at least some organization, telling us what is the right road to take for happiness, health, and certainly wealth and popularity.
If you have been in tune at all to what is being touted as "truth," you have discovered that almost everyone sees truth as something other than what everyone else sees as truth. If I accept one truth over another, and relate this truth to others, I have then claimed this truth as my own.
But is this "truth" my own? Is it something I am just relaying to others without giving it any thought, consideration or investigation? If I later discover that what I have been claiming as truth, is in fact a lie, or at least false, do I continue relating it as truth?
In other words, is your "truth" truly your own truth? Or is the truth you hold merely something told to you by another who either has something to gain from you believing as they do, or something they do not regard one way or another?
Bottom line: it would seem to me that there are only two ways to be certain at all that everything we say is the truth, or at least a well-considered untruth. The first of these methods is to studiously evaluate every single thing you hold as true. If this method is utilized, than you had better prepare for a very long, boring life.
The second way to be sure you always tell the truth is by far the most efficient, but highly unlikely to be practiced. This is certainly true of me, since there is hardly a single topic I do not hold a strong opinion on, and am ever ready and willing to expound on what I know nothing about. The efficient method I am referring to is to remain silent at all times.
Ps. You might well ask why I have this subject under the heading: Signs of the Times since lying is certainly not a modern invention. And that is true: but never in history has lying been so prevalent, so accepted, or so easy what with all the electronic devices we have available to spread our lies. One such medium is the written word. And since so much has been said by the experts on truth, I have provided a few comments for your perusal.


text:
Trish F Leger-Author
Clickrishe to edit subtitle
What I'm reading!
The Amber Druid Men~etc...
Excerpt page
My books / Reviews too!
The Amber Druids-
Prequel to the Amber Druid Series
HER DRUID TEMPTATION--Liana and Traven
(out now on Amazon--ebook only!!)
He's always been her temptation…will she finally give in?
Liana Morgan is the consummate business woman, buttoned-up, strait-laced, and very in tune with every aspect of her restaurants. She is closed off from emotional entanglements of any kind, not letting a single distraction slip under her skin.
Now she's in New Orleans, tying up the last few loose ends with a new restaurant, when her one temptation strolls, oh-so-casually, back into her life. Traven Campbell is an AMBER DRUID like her, and the one man she's never been able to completely block from her mind. Every molecule of her 'good girl' persona wants him, and always has, but it's not enough for her…or is it?
Traven Campbell is a player, and proud of it. He's easy-going, laid-back, and an expert bachelor. But even womanizers have jobs. His latest is shadowing a target for the New Orleans P.D. But while on the watch, he's shocked to see Liana, the one woman he's never managed to coerce into bed, WITH his target.
Determined to find answers, Traven confronts Liana, and soon business and pleasure are mixing with the sultry heat of New Orleans. Will Liana finally give in? Will Traven be able to tempt her beyond her invisible moral line? Or will Traven's target call the final shot?
Reviews for HER DRUID TEMPTATION!
From InD'Tale Magazine (4 1/2 stars and a crowned heart!)
"Traven Campbell is a PI on a case to find hard evidence against a well known business man in New Orleans. While investigating, he comes across Liana Morgan, an Amber Druid like himself. She is also one very sexy woman that he has had on his mind for a centuries. Can he make room for this beautiful woman while building his case at the same time?
Liana Morgan is all business with no time for dating, or a social life. But seeing Traven again makes heat spread to every part of her body and leaves her brain in a fog. When Traven tells her the person she is in town to do business with is dangerous, it gives her the perfect excuse to stay close to him.
Amber Druids, dangerous men, an investigation, hostility, and sexy, steamy love scenes are all rolled up in Trish F. Legers "Her Druid Temptation". Paranormals are always fun, especially when the characters have the type of heightened senses that make for a very sensual journey. This story enjoys written scenes, great detail of the here and now, along with romance between two very interesting characters. Although lacking slightly in history between Traven and Liana, it does not take away from the wonderful story that is unveiled before one's very eye. Truly a fun book to warm the soul!"
From Romance Junkies (4 stars!)
"HER DRUID TEMPTATION is an amazingly awesome novella. I just wish it could have lasted a little longer. It is dangerously sensual and the story simply mesmerized me. Author Trish F. Leger could not have written a better teaser for her AMBER DRUID series than HER DRUID TEMPTATION. Traven and Liana are a passionate and the fact that they are Amber Druids only makes things more interesting. Their story is a page-turner. I cannot wait to read the next novel in the AMBER DRUID series, HER DRUID DESIRE. I am sure it will be just as fascinating."
by Pamela Lynn
"Ms. Leger does an excellant job of character development early in this book. "MNChick | 5 reviewers made a similar statement
"I was captivated from the moment I began to read this story and didn't put it down until I was finished. "L. Leger | 3 reviewers made a similar statement
"I know it is just an intro to her Druid Series, but I really wished it would have been longer because it was really good - read in oneday. "Simple | 2 reviewers made a similar statement
HER DRUID DESIRE--Drake and Nadia
Out now!!!! For purchase at Amazon (ebook) Print coming soon!
Blurb for HER DRUID DESIRE
At the tender age of sixteen, Nadia Morales loses both of her parents in a car accident caused by a drunk driver. Growing up as a ward of the state of Texas, she was shy and painfully introverted. Against all odds, Nadia became one of the youngest CEO's in the state of Texas. She learned the hard way that putting her trust in the wrong man, an insider in the business world, sometimes gets you nothing but pain and heartache. But sometimes, karma has a way of straightening out those curves life throws at you.
Meet Drake Thompson-business owner, Wall Street guru, and good ole Texas playboy- he's got the requisite amount of ladies fawning over him to prove it. Since the death of Nadia's parents, he's watched out for her, and always kept a keen eye on her business proceedings. He's never been more proud than when she was named President for the Browning Corporation. He believes his soft spot for her is purely innocent…as that of a family member…but is it?
But each of them is hiding something life altering from the other. Nadia…her obsessive love for a man that treats her like a kid sister, and Drake, well, he's not wholly human. While their secrets marinate in mystery and denials, the pressure builds, culminating in a final revelation of barrier breaking truths.
Will Nadia finally be able to have the man of her dreams, or will Drake's true nature destroy this couple's chance at love?
HER DRUID FANTASY--Ramsey and Vivienne
Out NOW--Ready for Purchase at AMAZON and SOON in Paperback!
"Trish Leger writes with such elegance, such depth that I was transported into the Druid world and didn't want to come back from it. A truly sweeping, sensual romance that had me under its spell from the first page."--EDEN BUTLER, Amazon Bestselling Author of the Seeking Serenity Series
HER DRUID FANTASY--
Can her fantasy survive a life-altering change to become her reality?
Vivienne Rockport has never shied away from commitment or hard work to achieve her goals. She is proud owner and proprietor of a successful, high-end antique shop, Diamond In The Rough. She's dedicated years of her life to establishing her business, and she's done it all on her own. Or has she?
Disappointment sets in when Vivienne discovers that someone with extensive connections may be responsible for her shop's success. The knowledge creates a snowball effect that rolls into an avalanche of hard-hitting truths. Will those truths, along with a coming of age occurrence, be more than Vivienne can bear?
Ramsey McMurray is a typical Wall Street businessman—with a debonair sense of style, devastating charm, and dripping with immeasurable wealth. However, there is more to the man than he reveals to the world. When he learns that a close acquaintance of his is merging into his world of Amber Druids, it puts a strain on an already stressful situation.
Can Vivienne and Ramsey's tumultuous relationship survive the turmoil?
HER DRUID SEDUCTION--Brenan and Julianna

redpajama_set_name: RedPajamaCommonCrawl


text:
Q: ADF: Capture Previous Task Details throws error I am trying to capture the error from failed activity in a variable activity or in web request activity for sending email.
I am using "@activity().error.message" to capture it. It is throwing the attached error.
Any help is appreciated to resolve this. Thank you.

Web Activity dynamic content:

A: Please try to use this expression:
@activity('Activity name').Error.Message

Update
I test this expression and it works fine.

redpajama_set_name: RedPajamaStackExchange


text:
Centres and units
Home » Research » Projects » Aquatic conservation ecology
Aquatic conservation ecology
Chris Fulton
Fulton Group - Marine ecology and conservation
Behavioural, evolutionary and physiological ecology
Here we are working to determine the habitat requirements and environmental tolerances of threatened and endangered species to assist their adaptive management and protection in marine and freshwater ecosystems.
Adaptive management of threatened and endangered species requires a solid understanding of their specific habitat requirements and tolerance to environmental conditions. In this ongoing project, we are using a diversity of approaches to assess how species of conservation concern respond to changes in habitat availability and abiotic conditions. For example, we have used assessments of swimming performance to identify barriers to upstream migration in the endangered Macquarie perch (Starrs et al. 2011), new methods for surveying rare and threatened species such as the Murray River crayfish (Fulton et al. 2012) and uncovered how a variety of flow microhabitats support a diversity of habitat specialist gobies in tropical streams (Donaldson et al. 2013).


text:
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Live Centre
2020 Grid
Home Uncategorized Perez: I need to move to win a World title
Perez: I need to move to win a World title
Date published: August 8 2016
Sergio Perez admits he needs to leave Force India and move further up the grid if he wants a shot at winning a Drivers' Championship title.
Despite team boss Vijay Mallya confirming him for next season, the Mexican driver has insisted that his future is still undecided.
Perez has said he will sit down with his sponsors during the summer break and hopes to arrive in Belgium knowing what the future holds.
The former McLaren man has been linked to Renault and also Williams, and concedes he will have to leave Force India to win a World title.
"It's going to be difficult, realistically," he told GPUpdate.net.
"This team, no matter what happens, will always be in my heart. What Vijay did for me to save my Formula 1 career, and the whole team since I arrived here, is huge.
"[But] I know that to become a World Champion in Formula 1 I need to move further up the grid. Whatever happens, whether the opportunity comes or not, who knows."
On the podium in all three seasons with Force India, including two P3s this year alone, Perez reckons both his growth and his brief stint at McLaren show that he is up to the task.
"I think one of the best things about me is that I've already been in a top team in Formula 1, and I know what it's like working in a top team.
"I think I have learned hugely, how to be a leader of a team and how to work with the big teams, so that's a big difference, the way I motivate people and the way I work with people.
"Obviously I'm not the same guy as I was three years ago. I've grown not only in age, but as a driver and a person, the way I see life and the way I see the sport."
However, the 26-year-old acknowledges that even moving to a top team does not guarantee him success.
He added: "I joined McLaren because it is a big team, but the car wasn't at the level of the team.
"If I ever go to a top team again, no one guarantees me that the car will be a top car, but I have to work around that car and make the best out of that car to prove to that team that I'm a top driver."
Leclerc and Verstappen signed, over to you Lewis…
F1 2021: How is the driver line-up shaping up?
Five early questions for the 2020 Formula 1 season
Formula 1 drivers of the decade – 5-1
Ranking the best F1 seasons of this decade – 5 to 1
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Cholesterol Test Kits is a great device for testing the cholesterol level of your body at home without going to the lap in the hospital or something like that. Here in this article, we introduce you to the top 10 best Cholesterol Test Kits on Amazon in 2019.
This Cholesterol Home Test Kit secures our number 10 spot in this review. The main characteristic that stands out when it comes to this kit is its easiness to use and it only takes minutes to give you the results that you expect. Cholesterol testing is not something you can put away or neglect as it will have dire financial impacts on your future as well on your health. Therefore you can save the trouble of visiting a hospital to get your level of cholesterol checked by buying the accurate cholesterol home test kit.
Don't put yourself in danger by having increased levels of cholesterol as it will eventually you're your life at risk to the level of subjecting heart attacks. Most of the time people aren't comfortable with the common symptoms, which are bound with having higher cholesterol levels and you don't need to worry about keeping them all in your head by simply investing on this cholesterol home kit where you will have control of your life and your health.
This cholesterol kit has gone through rigorous testing in order to maintain high quality, and thus it is revealed that these kits actually help control and lower cholesterol levels minimizing any negative effects. Our customer reviews reveal how it has one of our customers to bring his cholesterol level down to 120 from a high of 280 because of close monitoring thanks to the LipidShield Plus Lower cholesterol home kit. This is a testimony on how it can benefit you, your friends and family in order to lead a long healthy life.
This can be considered a new kit developed Prima to test cholesterol levels of people. Since its introduction, it has been rising in popularity and is a crowd pleaser. This kit uses strips in order to figure out the cholesterol levels of your body. In case you would like to try out the cholesterol strips by Prima you must also have the Prima device which will do wonders for you. The kit contains 25 different strips, which can be used to measure the cholesterol levels and the data for inputting data into the device. According to its customer reviews, you should not think twice before buying a product of this nature.
The Easylife Hemoglobin Test kit secures the number 7 spot on our list of best cholesterol kits. This device is equipped with a comprehensive meter which automatically identifies the strips in order provide you the best readings. The main advantage of using this would be that there is no proper cleaning required when using them. There is a large LCD which there to present the values in an easily readable manner. The LCD has a memory that can recall the last 50 test results.
Cholesterol levels need to be kept at their right levels in order to maintain a healthy life. But unless you go to the hospital every week to get a testing done, you will not have an idea of your cholesterol levels. Therefore CholesTrak provides you with a personal home cholesterol testing kit which gives you the control of your health. By monitoring your cholesterol levels closely on a regular basis, you can easily control your cholesterol levels which will lead to a happy and healthy life.
Heart diseases and strokes are common for people who are diagnosed with cholesterol. It is important to be aware of the repercussions of not taking actions immediately. But its even better if you have a way to control your cholesterol levels without much trouble thereby allowing you to be in charge of your cholesterol charges. Easylife all in one hemoglobin, cholesterol, glucose test kit provides you with a way to test, detect and control your cholesterol levels ahead of time.
By purchasing this cholesterol kit, you can test for cholesterol, glucose as well as hemoglobin. It has the capability to test every single measurement as stated above so that the results also represent each test in order for easier understanding. The kit has the ability hold up to 50 previous test results. You can even use these results to generate your cholesterol level charts on your own.
This kit is manufactured in the United States and it contains a dual monitoring system to provide you with cholesterol readings from the biometer cholesterol strips. The kit contains only 3 strips. Purchase the kit and using it to check your cholesterol levels is a good investment in your future, a choice that you will not come to regret.
This can be considered as one of the most versatile cholesterol kits available for you to purchase and use among all the family members. It comes along with the ability to take effective measurements of triglycerides, HDL and total cholesterol.
I'm sure with so many options out there you are in a dilemma to select which product to buy as your personal home cholesterol kit. Buying this kit will save you plenty of time and money, while it will give you more value as this kit is capable of testing hemoglobin, cholesterol, and glucose. It also has the ability to store the previous 50 results for you to look back.
This is by far the best value cholesterol kit available out there on the market. This product includes a total of 6 Cholesterol test strips, 6 HDL test strips, 6 triglyceride test strips, 18 lancets, 18 capillaries. The cardio check cholesterol analyzer is required but that can have to be bought separately.


Highly Recommend written by mobile Satisfying the humidification function in the air purifier!
Highly Recommend written by mobile I really like it!
Highly Recommend I'm satisfied !!!!!!!!!!!!!!!!!!!!!!!
I give them a manual and I like them very much. I feel like giving them fresh air.
Recommend written by mobile The ship is a nuclear weapon.
No hassle. Quick delivery to Korea.
조용하고 성능 좋습니다. 매뉴얼이 일본어로만 되어 있지만, 사용상 별로 문제될 것은 없고요.
가습 기능도 굉장히 유용합니다. 만족스러운 제품이에요.
I loved it so much that I gave it to my brother. I think it's best for the price.
The product is satisfactory, I should expect to continue to write, but I do not have any more.
Very good. The price is cheap and comes sooner than you think.
I will stick! I left it in my newborn room.
When will clearance be completed?
Can you get it this week?
Your item is out of stock at the shop on March 7th. Currently, the quantity is available this week.
You can view detailed information at the address below.
It comes out of stock.
Please note that the items you have given in the current product page are available for selection.
Where is the replacement 175,300 won?
There is an extra charge for each model.
What is the difference between the G-50 and the H-50?
There is little difference in functionality.
Is the H50 new to the G50?
Is the PM2.5 sensor added to the H50?
I wonder if there is any difference in function and appearance.
What is the time sale price? I have written 10 remnants. How do I apply?
Time sale is limited time special discount, you can purchase at discount time.
This item is discontinued in Japan, and we have one stock left in our warehouse.
However, this product has been opened for inspection at the customs during customs clearance.
It is a new product and has no problem with performance.
If you would like to purchase this product, please contact us.
It is a product that was opened in customs, but not used.
The product, box and packaging status are the same as the new product.
It is available now for $ 226.
If you would like to make a purchase, please let me know the date and time of purchase.
Hello Japan, I bought the same model in less than a month.
It was $ 215 then.
Can I adjust the amount?
100% is not a new product, thank you for your purchase, thank you.
Because of this, exchange and return are difficult, thank you for your understanding.
It is also available at 09:00 am on the 8th of tomorrow.
Please put them in at that time.
We are currently in stock for $ 215.
Please give me a safe delivery.
Next Thursday (2/14) We will ship the product.
I will inform you of the invoice number after shipment.
The product is in a Korean warehouse. Will it ship later?
Please refer to the top answer and correct delivery date again.
Goods can be shipped next Thursday (2/14).
What is the difference between G50 and G40?
Is not the G40 a HEPA filter?
The product that uses HEPA filter is KC-G50.


Welcome to Upper Beach Health and Wellness, a multidisciplinary, hands-on therapeutic centre located in Toronto's east end.
We take a functional approach to supportive and preventative health care, offering various complementary health therapies including chiropractic care, contemporary medical acupuncture, naturopathy, physiotherapy and massage.
We believe in an individualized and more personal approach to family wellness, spending more time with our visitors to assist in rehabilitation, pain & injury, movement & function restoration and especially general wellness. We strive to seek out the root of any problems and not just deal with the pain.
We are available to answer any questions you may have about certain conditions or what treatment options may suit your needs. For more information or to make an appointment, please visit us at: 1937 Gerrard Street East, call (416) 627-5006 or info@upperbeach health.com.


The Spatial Awareness Skills Program (SASP) assesses and teaches the fundamental analysis and organization abilities that enable children to make sense out of arithmetic and, more generally, to address written work and multistep listening and reading comprehension tasks in an efficient, step-by-step fashion. SASP is an updated, expanded version of the visual skills component (Blue Book) of PREP. PREP focused on analysis skills; SASP focuses on analysis and organization skills.
SASP is intended for use by occupational therapists, developmental optometrists, and teachers who work with pre-kindergarten through elementary-school-age LD, ADD, or dyslexic children, individually or in groups. SASP consists of two components: the Test, which enables you to identify and determine the performance (instructional) level of those children who lack adequate spatial awareness skills; and the Curriculum, which provides methods for training those skills.
The Curriculum is divided into two major sections: activities to improve spatial analysis and organizational skills, and activities to teach the words (verbal organizers) that support this process. Many of the activities are accompanied by worksheets that may be given to parents for supplemental use at home.


Is there anyone smart enough to figure out how to compute the price of Bitcoin?
MOVED: Is EzBTC.ca trustworthy and legit?
Why other people afraid about activating segwit through BIP148?
bitcoin changing my ideology from socialism to libertarianism! What about you?
Almost 900 UASF nodes, how much hashing power?
Where do I buy ICO tokens?
Are people able to deposit ETC on Poloniex?


text:
(434) 525-4841 info@forestbaptistchurch.org
Weekly Gatherings
A Word from the Pastor
An Election Week Prayer (Updated: 2020)
by Tyler Scarlett | Nov 2, 2020
In 2016, Pastor Tyler wrote a prayer for election week. Here is a slightly updated version for the 2020 election. We invite all of our FBC family to pray this prayer before bed this evening or as you rise in the morning.
Heavenly Father, You have commanded us in 1 Timothy 2, to pray "for kings and all who are in authority," and so we do just that. We pray for our nation's election and for our soon-to-be President-elect. Immerse each one with your grace.
We confess, O God, Your goodness, Your sovereignty, and Your power over all things – even events like this. We are reminded, that as Daniel said, "It is God who changes the times and the epochs; Who removes kings and establishes them…" Do Your work, we pray. We know that "there is no governing authority except from God, and those which exist are established by" You (Rom 13:1). And, so, Father, act according to Your plan, Your will, and Your purposes.
Father, first of all, we thank you for our nation. Indeed, we are a blessed people. For our abundant freedoms and opportunities, we thank you. May we not take them for granted. Instead, may Your church leverage them for the good of the gospel and the increase of Your kingdom. Thank you for the undeserved favor that You have shown our land.
Forgive us – Your church – O Lord, for not praying for our leaders and nation as we should. Forgive us for fearing men more than God. Remove the stains and smooth the wrinkles of Your bride, the church. While we do not join hands with those who do evil, as fellow citizens we feel the burden of our nation's sinful choices. And so, we mourn the sins of our country – asking for Your pardon, Your grace, and Your lovingkindness. Forgive us for murdering the unborn. Forgive us for normalizing sexual sin. Forgive us for neglecting the needy, poor, and hungry. Have mercy on us, O Lord. And grant us better, more upright, days ahead.
We pray, Father, for a peaceful election day. You have allowed our country, in days past, to calmly transfer power with ballots and not bullets. Allow it once again, we pray. May polling sites be teeming with dignity, mutual respect, kindness, and decency. Help us, as Your ambassadors, to treat the men and women that we meet there with thoughtfulness and the love of Christ. May our civic process be a civil one.
For the voting booths and necessary calculations, we pray for honesty, accuracy, and truthfulness. Proverbs 11:1 tells us that "dishonest scales are an abomination to the LORD." Thwart the plans of any who seek to manipulate or falsify records. We thank you for the blessing of modern technology that makes our results so quickly known. May these useful innovations work as they are intended, totally with precision and be reported correctly.
For our soon-to-be President-elect, grant them wisdom, courage, and integrity as a leader. Proverbs 16:12 reminds us that it is "an abomination for kings to commit wickedness." And so, we pray, for a leader committed to righteousness, truthfulness, and usefulness. Help them to be faithful, kind, and loving.
We thank You, Father, for the common grace gift of family. Protect our new president's home, we pray. Stop anyone who would threaten or seek to do them harm. May the elect-First Family be safe, respected, and treated with goodness. May the White House become a true home for them. Fill it with laughter, encouragement, and love despite the pressures of the job.
In Proverbs 8:15, wisdom declares, "by me kings reign, and rulers decree justice. By me princes rule, and nobles, all how judge rightly." May our President seek wisdom – from those near, from those far, and especially from You above. Grant them sensibility, self-control, faith and discipline.
Make our President a defender of all life, a protector of true marriage, and an advocate for religious liberty across our land. Give them a heart for the needy, a concern for the powerless, a compassion for the weak, and a diplomatic spirit to our neighbors abroad.
Father, Your word says, "Where there is no guidance the people fall, but in an abundance of counselors there is victory." (Prov 11:14) Put before our President-elect the best information, the most intelligent counselors and prudent, experienced advisors. May they may make sound decisions about economic policy, judicial appointments, and just governance.
As Commander-and-Chief, may they be guided by both courage and restraint. May any necessary wars be a tool for lasting peace. Grant them the discernment to know when to put our troops in harm's way and when to bring them safely home.
Your Word is clear that "God opposes the proud." And so we ask that you would grant our President-elect the humility to admit faults, forgive enemies, and change their mind when righteousness and justice require it. Lead them to a firm understanding of the truth of the gospel, a resolute commitment to obey the Word of God, and a passion to promote what accords with Your truth.
By your grace, may our new President be a better person than we anticipate and a better person than we deserve.
In all of these matters, we ultimately pray as our Lord taught us to pray: "Thy kingdom come, Thy will be done on earth as it is in heaven." In the name of our Lord Jesus Christ, we pray. Amen.
About Tyler
Tyler holds a DMin and MDiv from Southeastern Baptist Theological Seminary as well as an MAR from LBTS. Tyler serves as the teaching pastor at FBC,
Popular Encyclopedia of Church History (Contributed Articles on: Charles Spurgeon & Youth With A Mission)
Eye, Eye, Preacher! (July/Aug 2015) Preaching Magazine.
Sermon Whittling: Edit Before You Preach (Sept/Oct 2013) Preaching Magazine. Also here
If It's Greek To You, Make It English to Them (March/April 2013) Preaching Magazine. Also here
Start With a Bang! - Crafting a Memorable Sermon Opener (March/April 2011) Preaching Magazine. Also here
Ten Commandments for Preaching (March/April 2010) Preaching Magazine. Also here
Forest Baptist Church
Services and Times
Wednesday Prayer Meeting
Forest, Virginia 24551
1313 Two Church Lane


text:
{{Infobox person
| name          = William Eldon Warwick
| image         =
| alt           =
| caption       =
| birth_name    =
| birth_date    =
| birth_place   = Birkenhead, UK
| death_date    =
| death_place   =
| nationality   = British
| other_names   =
| occupation    = Master Mariner
| years_active  =
| known_for     = First Master of the Queen Elizabeth 2 (QE2)
| children      =Eldon J WARWICK, Ronald W Warwick and David B Warwick.
| notable_works =
}}
Captain William "Bil" Warwick, CBE was a merchant sailor and the Master of Cunard's Queen Elizabeth 2 (QE2), the first person to hold that position did so for over 25 years.

He was born on 12 November 1912 in Birkenhead, England.

He appeared as a castaway on the BBC Radio programme Desert Island Discs'' on 18 September 1967.

He was made a Commander of the Order of the British Empire (CBE) in 1971.

He died on 27 February 1999. His son, Captain Ronald W Warwick, was a master of the Queen Elizabeth 2 during the time of his death, Ronald W Warwick was Captain of the QE2 for 13 years this was the first time a Father and Son had made it to the rank of captain of the Cunard and the first time a Father and Son had been in command of the same ship as Captain BIL WARWICK was QE2's first Master, Both Captains would become Cunard Commodores as Commmodor RON WARWICK would become the first Master of the Queen Mary 2 the largest Ocean liner in the world.

Eldon  J Warwick would become the Royal Naval Reserve and was the Commodore of the Cunard Line.

References
He had three sons all of whom spent sometime at sea.

1912 births
Place of birth missing
1999 deaths
Place of death missing
Commanders of the Order of the British Empire
Ship captains of the Cunard Line
People from Birkenhead
British Merchant Navy officers

redpajama_set_name: RedPajamaWikipedia


text:
Q: Using custom units with siunitx v3 in captions - is \protect insufficient? I am using my previously discussed custom commands (also provided below), in siunitx. Since version 3, I am no longer able to use them in captions, whereas previously, adding \protect before them always worked.
Definition (in a loaded sty file):
% Define "quantity-product", as an alias in versions prior to 3.0; allows its unconditional use
% From: https://tex.stackexchange.com/a/621037/134641
\@ifpackagelater{siunitx}{2021/05/17}  % main release of 3.0.0
    { } {
        \ExplSyntaxOn
            \keys_define:nn { siunitx }
            { quantity-product .meta:n = { number-unit-product = {#1} } }
        \ExplSyntaxOFf
    }
%--------------------------------------------

\DeclareSIUnit[quantity-product = \;]\basepair{bp}

Usage example:
\begin{figure}
    \caption{Pertains to $\SI{500}{\protect\basepair}$ regions and uncalibrated data.}
\end{figure}

A: siunitx makes unit macros \protected, so they don't explode when used in expansion-only contexts, like in captions, so you can simply use \SI{500}{\basepair} without having to worry about protection (otherwise you'd have to \protect\SI too).  Here's the full example:
\documentclass{article}
\usepackage{siunitx}

\ExplSyntaxOn\makeatletter
\@ifpackagelater{siunitx}{2021/05/17}  % main release of 3.0.0
  { }
  {
    \keys_define:nn { siunitx }
      { quantity-product .meta:n = { number-unit-product = {#1} } }
  }
\ExplSyntaxOff\makeatother

\DeclareSIUnit[quantity-product = \;]\basepair{bp}

\begin{document}
\listoffigures
\begin{figure}
  \caption{Pertains to $\SI{500}{\noexpand\basepair}$ regions and uncalibrated data.}
\end{figure}
\end{document}

When you added \protect you made it break because inside a \caption, \protect is \@unexpandable@protect, so it prevents the expansion of the following token with \noexpand, so you essentially have \SI{500}{\noexpand\basepair} (if you try this, you'll get the same error).  Then, when siunitx goes parsing the unit (at which point every unit macro is defined to have their correct meaning), it doesn't expand \basepair, and then later when typesetting, \basepair is expanded, but then it is back to being \ERROR.
As a rule of thumb, adding \protect is the exception rather than the norm, so only add it if you know that the macro will explode (but then consider making it robust with \MakeRobust).

Note that, in the code:
\@ifpackagelater{siunitx}{2021/05/17}  % main release of 3.0.0
    { } {
        \ExplSyntaxOn
            \keys_define:nn { siunitx }
            { quantity-product .meta:n = { number-unit-product = {#1} } }
        \ExplSyntaxOff
    }

the \ExplSyntaxOn and \ExplSyntaxOff are doing nothing (and I'm surprised you didn't get an "Undefined control sequence \keys" error when running that). There's an explanation at the beginning of this answer, but that code should be:
\ExplSyntaxOn
\@ifpackagelater{siunitx}{2021/05/17}  % main release of 3.0.0
  { }
  {
    \keys_define:nn { siunitx }
      { quantity-product .meta:n = { number-unit-product = {#1} } }
  }
\ExplSyntaxOff

In your case \makeatletter is not necessary because it's in a .sty file (\makeatletter is on by default in packages and classes), but note that in the example above I also added it outside the braces: inside it would have done nothing useful.