Relative Importance of Stochastic Assembly Process of Membrane Biofilm Increased as Biofilm Aged.

The relative importance of different ecological processes controlling biofilm community assembly over time on membranes with different surface characteristics has never been investigated in membrane bioreactors (MBRs). In this study, five ultrafiltration hollow-fiber membranes - having identical nominal pore size (0.1µm) but different hydrophobic or hydrophilic surface characteristics - were operated simultaneously in the same MBR tank with a constant flux of 10 liters per square meter per hour (LMH). In parallel, membrane modules operated without permeate flux (0 LMH) were submerged in the same MBR tank, to investigate the passive microbial adsorption onto different hydrophobic or hydrophilic membranes. Samples from the membrane biofilm were collected after 1, 10, 20, and 30days of continuous filtration. The membrane biofilm microbiome were investigated using 16S rRNA gene amplicon sequencing from DNA and cDNA samples. Similar beta diversity trends were observed for both DNA- and cDNA-based analyses. Beta diversity analyses revealed that the nature of the membrane surface (i.e., hydrophobic vs. hydrophilic) did not seem to have an effect in shaping the bacterial community, and a similar biofilm microbiome evolved for all types of membranes. Similarly, membrane modules operated with and without permeate flux did not significantly influence alpha and beta diversity of the membrane biofilm. Nevertheless, different-aged membrane biofilm samples exhibited significant differences. Proteobacteria was the most dominant phylum in early-stage membrane biofilm after 1 and 10days of filtration. Subsequently, the relative reads abundance of the phyla Bacteroidetes and Firmicutes increased within the membrane biofilm communities after 20 and 30days of filtration, possibly due to successional steps that lead to the formation of a relatively aged biofilm. Our findings indicate distinct membrane biofilm assembly patterns with different-aged biofilm. Ecological null model analyses revealed that the assembly of early-stage biofilm community developed after 1 and 10days of filtration was mainly governed by homogenous selection. As the biofilm aged (days 20 and 30), stochastic processes (e.g., ecological drift) started to become important in shaping the assembly of biofilm community.

Temperature sensitivity of biomass-specific microbial exo-enzyme activities and CO2 efflux is resistant to change across short- and long-term timescales.

Accurate representation of temperature sensitivity (Q10 ) of soil microbial activity across time is critical for projecting soil CO2 efflux. As microorganisms mediate soil carbon (C) loss via exo-enzyme activity and respiration, we explore temperature sensitivities of microbial exo-enzyme activity and respiratory CO2 loss across time and assess mechanisms associated with these potential changes in microbial temperature responses. We collected soils along a latitudinal boreal forest transect with different temperature regimes (long-term timescale) and exposed these soils to laboratory temperature manipulations at 5, 15, and 25°C for 84 days (short-term timescale). We quantified temperature sensitivity of microbial activity per g soil and per g microbial biomass at days 9, 34, 55, and 84, and determined bacterial and fungal community structure before the incubation and at days 9 and 84. All biomass-specific rates exhibited temperature sensitivities resistant to change across short- and long-term timescales (mean Q10  = 2.77 ± 0.25, 2.63 ± 0.26, 1.78 ± 0.26, 2.27 ± 0.25, 3.28 ± 0.44, 2.89 ± 0.55 for ß-glucosidase, N-acetyl-ß-d-glucosaminidase, leucine amino peptidase, acid phosphatase, cellobiohydrolase, and CO2 efflux, respectively). In contrast, temperature sensitivity of soil mass-specific rates exhibited either resilience (the Q10 value changed and returned to the original value over time) or resistance to change. Regardless of the microbial flux responses, bacterial and fungal community structure was susceptible to change with temperature, significantly differing with short- and long-term exposure to different temperature regimes. Our results highlight that temperature responses of microbial resource allocation to exo-enzyme production and associated respiratory CO2 loss per unit biomass can remain invariant across time, and thus, that vulnerability of soil organic C stocks to rising temperatures may persist in the long term. Furthermore, resistant temperature sensitivities of biomass-specific rates in spite of different community structures imply decoupling of community constituents and the temperature responses of soil microbial activities.

Pre-Conditioning Stem Cells in a Biomimetic Environment for Enhanced Cardiac Tissue Repair: In Vitro and In Vivo Analysis.

INTRODUCTION: Stem cell-based therapies represent a valid approach to restore cardiac function due to their beneficial effect in reducing scar area formation and promoting angiogenesis. However, their translation into the clinic is limited by the poor differentiation and inability to secrete sufficient therapeutic factors. To address this issue, several strategies such as genetic modification and biophysical preconditioning have been used to enhance the efficacy of stem cells for cardiac tissue repair. METHODS: In this study, a biomimetic approach was used to mimic the natural mechanical stimulation of the myocardium tissue. Specifically, human adipose-derived stem cells (hASCs) were cultured on a thin gelatin methacrylamide (GelMA) hydrogel disc and placed on top of a beating cardiomyocyte layer. qPCR studies and metatranscriptomic analysis of hASCs gene expression were investigated to confirm the correlation between mechanical stimuli and cardiomyogenic differentiation. In vivo intramyocardial delivery of pre-conditioned hASCs was carried out to evaluate their efficacy to restore cardiac function in mice hearts post-myocardial infarction. RESULTS: The cyclic strain generated by cardiomyocytes significantly upregulated the expression of both mechanotransduction and cardiomyogenic genes in hASCs as compared to the static control group. The inherent angiogenic secretion profile of hASCs was not hindered by the mechanical stimulation provided by the designed biomimetic system. Finally, in vivo analysis confirmed the regenerative potential of the pre-conditioned hASCs by displaying a significant improvement in cardiac function and enhanced angiogenesis in the peri-infarct region. CONCLUSION: Overall, these findings indicate that cyclic strain provided by the designed biomimetic system is an essential stimulant for hASCs cardiomyogenic differentiation, and therefore can be a potential solution to improve stem-cell based efficacy for cardiovascular repair.

Temporal Microbial Community Dynamics in Microbial Electrolysis Cells - Influence of Acetate and Propionate Concentration.

Microbial electrolysis cells (MECs) are widely considered as a next generation wastewater treatment system. However, fundamental insight on the temporal dynamics of microbial communities associated with MEC performance under different organic types with varied loading concentrations is still unknown, nevertheless this knowledge is essential for optimizing this technology for real-scale applications. Here, the temporal dynamics of anodic microbial communities associated with MEC performance was examined at low (0.5 g COD/L) and high (4 g COD/L) concentrations of acetate or propionate, which are important intermediates of fermentation of municipal wastewaters and sludge. The results showed that acetate-fed reactors exhibited higher performance in terms of maximum current density (I: 4.25 ± 0.23 A/m2), coulombic efficiency (CE: 95 ± 8%), and substrate degradation rate (98.8 ± 1.2%) than propionate-fed reactors (I: 2.7 ± 0.28 A/m2; CE: 68 ± 9.5%; substrate degradation rate: 84 ± 13%) irrespective of the concentrations tested. Despite of the repeated sampling of the anodic biofilm over time, the high-concentration reactors demonstrated lower and stable performance in terms of current density (I: 1.1 ± 0.14 to 4.2 ± 0.21 A/m2), coulombic efficiency (CE: 44 ± 4.1 to 103 ± 7.2%) and substrate degradation rate (64.9 ± 6.3 to 99.7 ± 0.5%), while the low-concentration reactors produced higher and dynamic performance (I: 1.1 ± 0.12 to 4.6 ± 0.1 A/m2; CE: 52 ± 2.5 to 105 ± 2.7%; substrate degradation rate: 87.2 ± 0.2 to 99.9 ± 0.06%) with the different substrates tested. Correlating reactor's performance with temporal dynamics of microbial communities showed that relatively similar anodic microbial community composition but with varying relative abundances was observed in all the reactors despite differences in the substrate and concentrations tested. Particularly, Geobacter was the predominant bacteria on the anode biofilm of all MECs over time suggesting its possible role in maintaining functional stability of MECs fed with low and high concentrations of acetate and propionate. Taken together, these results provide new insights on the microbial community dynamics and its correlation to performance in MECs fed with different concentrations of acetate and propionate, which are important volatile fatty acids in wastewater.

Membrane biofilm communities in full-scale membrane bioreactors are not randomly assembled and consist of a core microbiome.

Finding efficient biofouling control strategies requires a better understanding of the microbial ecology of membrane biofilm communities in membrane bioreactors (MBRs). Studies that characterized the membrane biofilm communities in lab-and pilot-scale MBRs are numerous, yet similar studies in full-scale MBRs are limited. Also, most of these studies have characterized the mature biofilm communities with very few studies addressing early biofilm communities. In this study, five full-scale MBRs located in Seattle (Washington, U.S.A.) were selected to address two questions concerning membrane biofilm communities (early and mature): (i) Is the assembly of biofilm communities (early and mature) the result of random immigration of species from the source community (i.e. activated sludge)? and (ii) Is there a core membrane biofilm community in full-scale MBRs? Membrane biofilm (early and mature) and activated sludge (AS) samples were collected from the five MBRs, and 16S rRNA gene sequencing was applied to investigate the bacterial communities of AS and membrane biofilms (early and mature). Alpha and beta diversity measures revealed clear differences in the bacterial community structure between the AS and biofilm (early and mature) samples in the five full-scale MBRs. These differences were mainly due to the presence of large number of unique but rare operational taxonomic units (∼13% of total reads in each MBR) in each sample. In contrast, a high percentage (∼87% of total reads in each MBR) of sequence reads was shared between AS and biofilm samples in each MBR, and these shared sequence reads mainly belong to the dominant taxa in these samples. Despite the large fraction of shared sequence reads between AS and biofilm samples, simulated biofilm communities from random sampling of the respective AS community revealed that biofilm communities differed significantly from the random assemblages (P < 0.001 for each MBR), indicating that the biofilm communities (early and mature) are unlikely to represent a random sample of the AS community. In addition to the presence of unique operational taxonomic units in each biofilm sample (early or mature), comparative analysis of operational taxonomic units and genera revealed the presence of a core biofilm community in the five full-scale MBRs. These findings provided insight into the membrane biofilm communities in full-scale MBRs. More comparative studies are needed in the future to elucidate the factors shaping the core and unique biofilm communities in full-scale MBRs.

Metatranscriptomics reveals the molecular mechanism of large granule formation in granular anammox reactor.

Granules enriched with anammox bacteria are essential in enhancing the treatment of ammonia-rich wastewater, but little is known about how anammox bacteria grow and multiply inside granules. Here, we combined metatranscriptomics, quantitative PCR and 16S rRNA gene sequencing to study the changes in community composition, metabolic gene content and gene expression in a granular anammox reactor with the objective of understanding the molecular mechanism of anammox growth and multiplication that led to formation of large granules. Size distribution analysis revealed the spatial distribution of granules in which large granules having higher abundance of anammox bacteria (genus Brocadia) dominated the bottom biomass. Metatranscriptomics analysis detected all the essential transcripts for anammox metabolism. During the later stage of reactor operation, higher expression of ammonia and nitrite transport proteins and key metabolic enzymes mainly in the bottom large granules facilitated anammox bacteria activity. The high activity resulted in higher growth and multiplication of anammox bacteria and expanded the size of the granules. This conceptual model for large granule formation proposed here may assist in the future design of anammox processes for mainstream wastewater treatment.

Diversity and dynamics of dominant and rare bacterial taxa in replicate sequencing batch reactors operated under different solids retention time.

In this study, 16S rRNA gene pyrosequencing was applied in order to provide a better insight on the diversity and dynamics of total, dominant, and rare bacterial taxa in replicate lab-scale sequencing batch reactors (SBRs) operated at different solids retention time (SRT). Rank-abundance curves showed few dominant operational taxonomic units (OTUs) and a long tail of rare OTUs in all reactors. Results revealed that there was no detectable effect of SRT (2 vs. 10 days) on Shannon diversity index and OTU richness of both dominant and rare taxa. Nonmetric multidimensional scaling analysis showed that the total, dominant, and rare bacterial taxa were highly dynamic during the entire period of stable reactor performance. Also, the rare taxa were more dynamic than the dominant taxa despite expected low invasion rates because of the use of sterile synthetic media.

Temporal and spatial stability of ammonia-oxidizing archaea and bacteria in aquarium biofilters.

Nitrifying biofilters are used in aquaria and aquaculture systems to prevent accumulation of ammonia by promoting rapid conversion to nitrate via nitrite. Ammonia-oxidizing archaea (AOA), as opposed to ammonia-oxidizing bacteria (AOB), were recently identified as the dominant ammonia oxidizers in most freshwater aquaria. This study investigated biofilms from fixed-bed aquarium biofilters to assess the temporal and spatial dynamics of AOA and AOB abundance and diversity. Over a period of four months, ammonia-oxidizing microorganisms from six freshwater and one marine aquarium were investigated at 4-5 time points. Nitrogen balances for three freshwater aquaria showed that active nitrification by aquarium biofilters accounted for ≥ 81-86% of total nitrogen conversion in the aquaria. Quantitative PCR (qPCR) for bacterial and thaumarchaeal ammonia monooxygenase (amoA) genes demonstrated that AOA were numerically dominant over AOB in all six freshwater aquaria tested, and contributed all detectable amoA genes in three aquarium biofilters. In the marine aquarium, however, AOB outnumbered AOA by three to five orders of magnitude based on amoA gene abundances. A comparison of AOA abundance in three carrier materials (fine sponge, rough sponge and sintered glass or ceramic rings) of two three-media freshwater biofilters revealed preferential growth of AOA on fine sponge. Denaturing gel gradient electrophoresis (DGGE) of thaumarchaeal 16S rRNA genes indicated that community composition within a given biofilter was stable across media types. In addition, DGGE of all aquarium biofilters revealed low AOA diversity, with few bands, which were stable over time. Nonmetric multidimensional scaling (NMDS) based on denaturing gradient gel electrophoresis (DGGE) fingerprints of thaumarchaeal 16S rRNA genes placed freshwater and marine aquaria communities in separate clusters. These results indicate that AOA are the dominant ammonia-oxidizing microorganisms in freshwater aquarium biofilters, and that AOA community composition within a given aquarium is stable over time and across biofilter support material types.

Stable performance of non-aerated two-stage partial nitritation/anammox (PANAM) with minimal process control.

Partial nitritation/anammox (PANAM) technologies have rapidly developed over the last decade, but still considerable amounts of energy are required for active aeration. In this study, a non-aerated two-stage PANAM process was investigated. In the first-stage upflow fixed-film bioreactor, nitratation could not be prevented at ammonium loading rates up to 186 mg N l(-1) d(-1) and low influent dissolved oxygen (0.1 mg O(2) l(-1)). Yet, increasing the loading rate to 416 and 747 mg N l(-1) d(-1) by decreasing the hydraulic retention time to 8 and 5 h, respectively, resulted in partial nitritation with the desired nitrite to ammonium nitrogen ratio for the subsequent anammox stage (0.71-1.05). The second-stage anammox reactor was established with a synthetic feeding based on ammonium and nitrite. After establishing anammox at low biomass content (0.5 g VSS l(-1)), the anammox influent was switched to partial nitritation effluent at a loading rate of 71 mg N l(-1) d(-1), of which 78% was removed at the stoichiometrically expected nitrite to ammonium consumption ratios (1.19) and nitrate production to ammonium consumption ratio (0.24). The combined PANAM reactors were operated for 3 months at a stable performance. Overall, PANAM appeals economically, saving about 50% of the energy costs, as well as technically, given straightforward operational principles.

Successful hydraulic strategies to start up OLAND sequencing batch reactors at lab scale.

Oxygen-limited autotrophic nitrification/denitrification (OLAND) is a one-stage combination of partial nitritation and anammox, which can have a challenging process start-up. In this study, start-up strategies were tested for sequencing batch reactors (SBR), varying hydraulic parameters, i.e. volumetric exchange ratio (VER) and feeding regime, and salinity. Two sequential tests with two parallel SBR were performed, and stable removal rates > 0.4 g N l(-1) day(-1) with minimal nitrite and nitrate accumulation were considered a successful start-up. SBR A and B were operated at 50% VER with 3 g NaCl l(-1) in the influent, and the influent was fed over 8% and 82% of the cycle time respectively. SBR B started up in 24 days, but SBR A achieved no start-up in 39 days. SBR C and D were fed over 65% of the cycle time at 25% VER, and salt was added only to the influent of SBR D (5 g NaCl l(-1)). Start-up of both SBR C and D was successful in 9 and 32 days respectively. Reactor D developed a higher proportion of small aggregates (0.10-0.25 mm), with a high nitritation to anammox rate ratio, likely the cause of the observed nitrite accumulation. The latter was overcome by temporarily including an anoxic period at the end of the reaction phase. All systems achieved granulation and similar biomass-specific nitrogen removal rates (141-220 mg N g(-1) VSS day(-1)). FISH revealed a close juxtapositioning of aerobic and anoxic ammonium-oxidizing bacteria (AerAOB and AnAOB), also in small aggregates. DGGE showed that AerAOB communities had a lower evenness than Planctomycetes communities. A higher richness of the latter seemed to be correlated with better reactor performance. Overall, the fast start-up of SBR B, C and D suggests that stable hydraulic conditions are beneficial for OLAND while increased salinity at the tested levels is not needed for good reactor performance.

Stability and microbial community structure of a partial nitrifying fixed-film bioreactor in long run.

A partial nitrification system was investigated for 471 days under DO varying concentrations for assessing its stability and population dynamics. Within 130 days of operation at feed DO concentration of 1.0±0.1 mg/L, more than 85% of nitrite was accumulated. Efficiency deteriorated when the feed DO concentration was increased to 4.2±0.3 mg/L. Nitrite accumulation could not be re-established on decreasing feed DO to 1.0±0.1 mg/L. Even at DO concentration of<0.05 mg/L, nitrate production was observed; a condition termed as anoxic nitrification. NOB was detected in the biomass even under this condition by Fluorescence in-situ hybridization (FISH) analysis. Through 16S rRNA gene sequencing a major fraction of unknown bacterial sequences closely resembling haloalkalophilic bacteria of marine origin were detected. The study indicated that these bacterial species might play a role in anoxic nitrification and that NOB could survive extreme low DO condition.

Alkalinity and dissolved oxygen as controlling parameters for ammonia removal through partial nitritation and ANAMMOX in a single-stage bioreactor.

The oxidation of ammonia to dinitrogen through partial nitritation and anaerobic ammonium oxidation (ANAMMOX) in a single-stage bioreactor is based on suppressing the nitratation process. The single-stage process operated on a laboratory-scale fixed film bioreactor achieved ammonia removal of 0.7 kg NH4-N/(m(3) day) at 4 h hydraulic retention time (HRT) by controlling the nitratation process through a 'three-way control mechanism' comprising control of electron donor (nitrite), electron acceptor (oxygen) and carbon source (bicarbonate). The control of alkalinity and dissolved oxygen (DO) concentrations in feed to maintain an alkalinity to ammonia ratio of less than 8 and DO loading of less than 0.06 mg O/(mg N day), respectively, was necessary for inhibiting nitratation and enhancing partial nitritation and ANAMMOX. Therefore, feed alkalinity along with DO concentrations are critical controlling parameters in a single-stage biological process for nitrogen removal.

Start-up and stabilization of an Anammox process from a non-acclimatized sludge in CSTR.

Development of an Anammox (anaerobic ammonium oxidation) process using non-acclimatized sludge requires a long start-up period owing to the very slow growth rate of Anammox bacteria. This article addresses the issue of achieving a shorter start-up period for Anammox activity in a well-mixed continuously stirred tank reactor (CSTR) using non-acclimatized anaerobic sludge. Proper selection of enrichment conditions and low stirring speed of 30 +/- 5 rpm resulted in a shorter start-up period (82 days). Activity tests revealed the microbial community structure of Anammox micro-granules. Ammonia-oxidizing bacteria (AOB) were found on the surface and on the outer most layers of granules while nitrite-oxidizing bacteria (NOB) and Anammox bacteria were present inside. Fine-tuning of influent NO2(-)/NH4+ ratio allowed Anammox activity to be maintained when mixed microbial populations were present. The maximum nitrogen removal rate achieved in the system was 0.216 kg N/(m(3) day) with a maximum specific nitrogen removal rate of 0.434 g N/(g VSS day). During the study period, Anammox activity was not inhibited by pH changes and free ammonia toxicity.