Mapping the biological activities of filamentous oxygenic photogranules.

Oxygenic photogranules have been suggested as alternatives to activated sludge in wastewater treatment. Challenging for modeling photogranule-based processes is the heterogeneity of photogranule morphologies, resulting in different activities by photogranule type. The measurement of microscale-activities of filamentous photogranules is particularly difficult because of their labile interfaces. We present here an experimental and modeling approach to quantify phototrophic O2 production, heterotrophic O2 consumption, and O2 diffusion in filamentous photogranules. We used planar optodes for the acquisition of spatio-temporal oxygen distributions combined with two-dimensional mathematical modeling. Light penetration into the photogranule was the factor controlling photogranule activities. The spatial distribution of heterotrophs and phototrophs had less impact. The photosynthetic response of filaments to light was detectable within seconds, emphasizing the need to analyze dynamics of light exposure of individual photogranules in photobioreactors. Studying other recurring photogranule morphologies will eventually enable the description of photogranule-based processes as the interplay of interacting photogranule populations.

Growth Progression of Oxygenic Photogranules and Its Impact on Bioactivity for Aeration-Free Wastewater Treatment.

Oxygenic photogranules (OPGs), spherical aggregates comprised of phototrophic and nonphototrophic microorganisms, treat wastewater without aeration, which currently incurs the highest energy demand in wastewater treatment. In wastewater-treatment reactors, photogranules grow in number as well as in size. Currently, it is unknown how the photogranules grow in size and how the growth impacts their properties and performance in wastewater treatment. Here, we present that the photogranules' growth occurs with changes in phototrophic community and granular morphology. We observed that as the photogranules grow larger, filamentous cyanobacteria become enriched while other phototrophic microbes diminish significantly. The photogranules greater than 3 mm in diameter showed the development of a layered structure in which a concentric filamentous cyanobacterial layer encloses noncyanobacterial aggregates. We observed that the growth of photogranules significantly impacts their capability of producing oxygen, the key element in OPG wastewater treatment. Among seven size classes investigated in this study, photogranules in the 0.5-1 mm size group showed the highest specific oxygen production rate (SOPR), 21.9 ± 1.3 mg O2/g VSS-h, approximately 75% greater than the SOPR of mixed photogranular biomass. We discuss engineering the OPG process based on photogranules' size, promoting the stability of the granular process and enhancing efficiency for self-aerating wastewater treatment.

The Oxygenic Photogranule Process for Aeration-Free Wastewater Treatment.

This study presents the oxygenic photogranule (OPG) process, a light-driven process for wastewater treatment, developed based on photogranulation of filamentous cyanobacteria, nonphototrophic bacteria, and microalgae. Unlike other biogranular processes requiring airlift or upflow-based mixing, the OPG process was operated in stirred-tank reactors without aeration. Reactors were seeded with hydrostatically grown photogranules and operated in a sequencing-batch mode for five months to treat wastewater. The new reactor biomass propagated with progression of photogranulation under periodic light/dark cycles. Due to effective biomass separation from water, the system was operated with short settling time (10 min) with effective decoupling of hydraulic and solids retention times (0.75 d vs 21-42 d). During quasi-steady state, the diameter of the OPGs ranged between 0.1 and 4.5 mm. The reactors produced effluents with average total chemical oxygen demand less than 30 mg/L. Nitrogen removal (28-71%) was achieved by bioassimilation and nitrification/denitrification pathways. Oxygen needed for the oxidation of organic matter and nitrification was produced by OPGs at a rate of 12.6 ± 2.4 mg O2/g biomass-h. The OPG system presents a new biogranule process, which can potentially use simple mixing and natural light to treat wastewater.

Vertebrate bacterial gut diversity: size also matters.

BACKGROUND: One of the central issues in microbial ecology is to understand the parameters that drive diversity. Among these parameters, size has often been considered to be the main driver in many different ecosystems. Surprisingly, the influence of size on gut microbial diversity has not yet been investigated, and so far in studies reported in the literature only the influences of age, diet, phylogeny and digestive tract structures have been considered. This study explicitly challenges the underexplored relationship connecting gut volume and bacterial diversity. RESULTS: The bacterial diversity of 189 faeces produced by 71 vertebrate species covering a body mass range of 5.6 log. The animals comprised mammals, birds and reptiles. The diversity was evaluated based on the Simpson Diversity Index extracted from 16S rDNA gene fingerprinting patterns. Diversity presented an increase along with animal body mass following a power law with a slope z of 0.338 ± 0.027, whatever the age, phylogeny, diet or digestive tract structure. CONCLUSIONS: The results presented here suggest that gut volume cannot be neglected as a major driver of gut microbial diversity. The characteristics of the gut microbiota follow general principles of biogeography that arise in many ecological systems.

Only Simpson diversity can be estimated accurately from microbial community fingerprints.

Lalande et al. (Microb. Ecol. 66(3):647-658, 2013) introduced a promising approach to quantify microbial diversity from fingerprinting profiles. Their analysis is based on extrapolating the abundance of the phylotypes detectable in a fingerprint towards the rare phylotypes of the community. By considering a set of reconstructed communities, Lalande et al. obtained a range of estimates for phylotype richness, Shannon diversity and Simpson diversity. They reported narrow ranges indicating accurate estimation, especially for Shannon and Simpson diversities. Here, we show that a much larger set of reconstructed communities than the one considered by Lalande et al. is consistent with the fingerprint. We find that the estimates for phylotype richness and Shannon diversity vary over orders of magnitude, but that the estimates for Simpson diversity are restricted to a narrow range (around 10 %). We conclude that only Simpson diversity can be estimated accurately from fingerprints.

High methane potential of oxygenic photogranules decreases after starvation.

Oxygenic photogranules (OPG) are granular biofilms that can treat wastewater without external aeration, making it an advantage over activated sludge. Excess of OPG biomass can serve as energy source through anaerobic digestion. Two sequencing batch photoreactors were operated over 400 days to grow OPG. Biochemical methane potentials (BMP) were obtained from near-infrared spectroscopy. OPGs had an average BMP of 356 mL CH4·gVS-1, much higher than typical BMP from activated sludge. A partial least squares analysis could relate BMP with reactor operating conditions, like light regime, load or biomass concentration. Since organic load was the most influential parameter on BMP, three starvation experiments were set up. An average decrease of BMP by 18.4 % was observed. However, the unexpected growth of biomass during starvation resulted in a higher total methane volume. In conclusion, starvation reduces the BMP of OPGs but anaerobic digestion of OPG biomass remains a promising route for biomass valorization.

Two-stage alkaline-enzymatic pretreatments to enhance biohydrogen production from sunflower stalks.

Because of their rich composition in carbohydrates, lignocellulosic residues represent an interesting source of biomass to produce biohydrogen by dark fermentation. Nevertheless, pretreatments should be applied to enhance the solubilization of holocelluloses and increase their further conversion into biohydrogen. The aim of this study was to investigate the effect of thermo-alkaline pretreatment alone and combined with enzymatic hydrolysis to enhance biohydrogen production from sunflower stalks. A low increase of hydrogen potentials from 2.3 ± 0.9 to 4.4 ± 2.6 and 20.6 ± 5.6 mL of H2 g(-1) of volatile solids (VS) was observed with raw sunflower stalks and after thermo-alkaline pretreatment at 55 °C, 24 h, and 4% NaOH and 170 °C, 1 h, and 4% NaOH, respectively. Enzymatic pretreatment alone showed an enhancement of the biohydrogen yields to 30.4 mL of H2 g(-1) of initial VS, whereas it led to 49 and 59.5 mL of H2 g(-1) of initial VS when combined with alkaline pretreatment at 55 and 170 °C, respectively. Interestingly, a diauxic effect was observed with sequential consumption of sugars by the mixed cultures during dark fermentation. Glucose was first consumed, and once glucose was completely exhausted, xylose was used by the microorganisms, mainly related to Clostridium species.

Initial type and abundance of cyanobacteria determine morphotype development of phototrophic ecosystems.

Phototrophic aggregates containing filamentous cyanobacteria occur naturally, for example, as cryoconite on glaciers and microbialites in fresh or marine waters, but their formation is not fully understood. Laboratory models are now available to reproduce aggregation, that is, the formation of different morphotypes like hemispheroids, microbial mats or sphere-like aggregates we call photogranules. In the model, activated sludge as starting matrix is transformed into aggregates enclosed by a phototrophic layer of growing cyanobacteria. These cyanobacteria were either enriched from the matrix or we added them intentionally. We hypothesize that the resulting morphotype depends on the type and concentration of the added cyanobacteria. When cyanobacteria from mature photogranules were added to activated sludge, photogranulation was not observed, but microbial mats were formed. Photogranulation of sludge could be promoted when adding sufficient quantities of cyanobacterial strains that form clumps when grown as isolates. The cyanobacteria putatively responsible for photogranulation were undetectable or only present in low abundance in the final communities of photogranules, which were always dominated by mat-forming cyanobacteria. We suggest that, in a temporal succession, the ecosystem engineer initiating photogranulation eventually disappears, leaving behind its structural legacy. We conclude that understanding phototrophic aggregate formation requires considering the initial succession stages of the ecosystem development.

Use of additives to improve collective biogas plant performances: A comprehensive review.

Nowadays, anaerobic digestion (AD) is being increasingly encouraged to increase the production of biogas and thus of biomethane. Due to the high diversity among feedstocks used, the variability of operating parameters and the size of collective biogas plants, different incidents and limitations may occur (e.g., inhibitions, foaming, complex rheology). To improve performance and overcome these limitations, several additives can be used. This literature review aims to summarize the effects of the addition of various additives in co-digestion continuous or semi-continuous reactors to fit as much as possible with collective biogas plant challenges. The addition of (i) microbial strains or consortia, (ii) enzymes and (iii) inorganic additives (trace elements, carbon-based materials) in digester is analyzed and discussed. Several challenges associated with the use of additives for AD process at collective biogas plant scale requiring further research work are highlighted: elucidation of mechanisms, dosage and combination of additives, environmental assessment, economic feasibility, etc.

Polyhydroxybutyrate Production from Methane and Carbon Dioxide by a Syntrophic Consortium of Methanotrophs with Oxygenic Photogranules without an External Oxygen Supply.

Here, a syntrophic process was developed to produce polyhydroxy-ß-butyrate (PHB) from a gas stream containing CH4 and CO2 without an external oxygen supply using a combination of methanotrophs with the community of oxygenic photogranules (OPGs). The co-culture features of Methylomonas sp. DH-1 and Methylosinus trichosporium OB3b were evaluated under carbon-rich and carbon-lean conditions. The critical role of O2 in the syntrophy was confirmed through the sequencing of 16S rRNA gene fragments. Based on their carbon consumption rates and the adaptation to a poor environment, M. trichosporium OB3b with OPGs was selected for methane conversion and PHB production. Nitrogen limitation stimulated PHB accumulation in the methanotroph but hindered the growth of the syntrophic consortium. At 2.9 mM of the nitrogen source, 1.13 g/L of biomass and 83.0 mg/L of PHB could be obtained from simulated biogas. These results demonstrate that syntrophy has the potential to convert greenhouse gases into valuable products efficiently.

Screening and Application of Ligninolytic Microbial Consortia to Enhance Aerobic Degradation of Solid Digestate.

Recirculation of solid digestate through digesters has been demonstrated to be a potential simple strategy to increase continuous stirred-tank reactor biogas plant efficiency. This study extended this earlier work and investigated solid digestate post-treatment using liquid isolated ligninolytic aerobic consortia in order to increase methane recovery during the recirculation. Based on sampling in several natural environments, an enrichment and selection method was implemented using a Lab-scale Automated and Multiplexed (an)Aerobic Chemostat system to generate ligninolytic aerobic consortia. Then, obtained consortia were further cultivated under liquid form in bottles. Chitinophagia bacteria and Sordariomycetes fungi were the two dominant classes of microorganisms enriched through these steps. Finally, these consortia where mixed with the solid digestate before a short-term aerobic post-treatment. However, consortia addition did not increase the efficiency of aerobic post-treatment of solid digestate and lower methane yields were obtained in comparison to the untreated control. The main reason identified is the respiration of easily degradable fractions (e.g., sugars, proteins, amorphous cellulose) by the selected consortia. Thus, this paper highlights the difficulties of constraining microbial consortia to sole ligninolytic activities on complex feedstock, such as solid digestate, that does not only contain lignocellulosic structures.

Development and application of an enzymatic and cell flotation treatment for the recovery of viable microbial cells from environmental matrices such as anaerobic sludge.

Efficient dissociation of microorganisms from their aggregate matrix is required to study the microorganisms without interaction with their native environment (e.g., biofilms, flocs, granules, etc.) and to assess their community composition through the application of molecular or microscopy techniques. To this end, we combined enzymatic treatments and a cell extraction by density gradient to efficiently recover anaerobic microorganisms from urban wastewater treatment plant sludge. The enzymes employed (amylase, cellulase, DNase, and pectinase) as a pretreatment softly disintegrated the extrapolymeric substances (EPS) interlocked with the microorganisms. The potential damaging effects of the applied procedure on bacterial and archaeal communities were assessed by studying the variations in density (using quantitative PCR), diversity (using capillary electrophoresis single-strand conformation polymorphism fingerprinting [CE-SSCP]), and activity (using a standard anaerobic activity test) of the extracted microorganisms. The protocol preserved the general capacity of the microbial community to produce methane under anaerobic conditions and its diversity; particularly the archaeal community was not affected in terms of either density or structure. This cell extraction procedure from the matrix materials offers interesting perspectives for metabolic, microscopic, and molecular assays of microbial communities present in complex matrices constituted by bioaggregates or biofilms.

Engineered methanotrophic syntrophy in photogranule communities removes dissolved methane.

The anaerobic treatment of wastewater leads to the loss of dissolved methane in the effluent of the treatment plant, especially when operated at low temperatures. The emission of this greenhouse gas may reduce or even offset the environmental gain from energy recovery through anaerobic treatment. We demonstrate here the removal and elimination of these comparably small methane concentrations using an ecologically engineered methanotrophic community harbored in oxygenic photogranules. We constructed a syntrophy between methanotrophs enriched from activated sludge and cyanobacteria residing in photogranules and maintained it over a two-month period in a continuously operated reactor. The novel community removed dissolved methane during stable reactor operation by on average 84.8±7.4% (±standard deviation) with an average effluent concentration of dissolved methane of 4.9±3.7 mg CH4∙l-1. The average methane removal rate was 26 mg CH4∙l-1∙d-1, with an observed combined biomass yield of 2.4 g VSS∙g CH4 -1. The overall COD balance closed at around 91%. Small photogranules removed methane more efficiently than larger photogranule, likely because of a more favorable surface to volume ratio of the biomass. MiSeq amplicon sequencing of 16S and 23S rRNA revealed a potential syntrophic chain between methanotrophs, non-methanotrophic methylotrophs and filamentous cyanobacteria. The community composition between individual photogranules varied considerably, suggesting cross-feeding between photogranules of different community composition. Methanotrophic photogranules may be a viable option for dissolved methane removal as anaerobic effluent post-treatment.

Wastewater treatment using oxygenic photogranule-based process has lower environmental impact than conventional activated sludge process.

The Life Cycle Assessment (LCA) methodology was applied to assess the environmental feasibility of a novel wastewater treatment technology based on oxygenic photogranules (OPG) biomass in comparison to a conventional activated sludge (CAS) system. LCA using laboratory scale experimental data allowed for eco-design of the process during the early stage of process development at laboratory scale. Electricity consumption related to artificial lighting, the fate of the generated biomass (renewable energy and replacement of mineral fertilizer), and the nitrogen flows in the OPG system were identified as major contributors to the potential environmental impact of the OPG treatment system. These factors require optimization in order to reduce the environmental impact of the overall OPG system. Nonetheless, the environmental impact of a non-optimized OPG scenario was generally lower than for a CAS reference system. With an optimization of the artificial lighting system, an energy neutral treatment system may be within reach.

Simple Time-lapse Imaging for Quantifying the Hydrostatic Production of Oxygenic Photogranules.

Oxygenic photogranules (OPGs) are dense, three-dimensional aggregates containing a syntrophic, light-driven microbial community. Their temporal and spatial development interests microbial ecologists working at the bioprocess engineering interface, as this knowledge can be used to optimize biotechnological applications, such as wastewater treatment and biomass valorization. The method presented here enables the high-throughput quantification of photogranulation. OPGs are produced from a loose sludge-like microbial matrix in hydrostatic batch cultures exposed to light. This matrix transforms into a consolidated, roughly spherical aggregate over time. Photogranulation is quantified by time-lapse imaging coupled to automated image analysis. This allows studying the development of many OPGs simultaneously and in a fully automated way to systematically test what factors drive photogranulation. The protocol can also be used to quantify other types of (a)biotic aggregation.

Novel Outlook in Microbial Ecology: Nonmutualistic Interspecies Electron Transfer.

Recent advances in microbial electrochemical technologies have revealed the existence of numerous and highly diverse microorganisms able to exchange electrons with electrodes. This diversity could reflect the capacity of microorganisms to release and/or retrieve electrons with each other in natural environments. So far, this interspecies electron transfer has been studied with a special focus on syntrophy and was successfully demonstrated for several couples of species. In this article we argue that electron exchange between microbes exists beyond syntrophy or mutualism and could also promote competitive and even parasitic behaviour. Based on three interesting case studies identified from the literature, we also highlight that such nonmutualistic interactions could be widespread and of particular significance for the survival of pathogens or the shaping of complex microbial communities.

Absolute quantitation of microbes using 16S rRNA gene metabarcoding: A rapid normalization of relative abundances by quantitative PCR targeting a 16S rRNA gene spike-in standard.

Metabarcoding of the 16S rRNA gene is commonly used to characterize microbial communities, by estimating the relative abundance of microbes. Here, we present a method to retrieve the concentrations of the 16S rRNA gene per gram of any environmental sample using a synthetic standard in minuscule amounts (100 ppm to 1% of the 16S rRNA sequences) that is added to the sample before DNA extraction and quantified by two quantitative polymerase chain reaction (qPCR) reactions. This allows normalizing by the initial microbial density, taking into account the DNA recovery yield. We quantified the internal standard and the total load of 16S rRNA genes by qPCR. The qPCR for the latter uses the exact same primers as those used for Illumina sequencing of the V3-V4 hypervariable regions of the 16S rRNA gene to increase accuracy. We are able to calculate the absolute concentration of the species per gram of sample, taking into account the DNA recovery yield. This is crucial for an accurate estimate as the yield varied between 40% and 84%. This method avoids sacrificing a high proportion of the sequencing effort to quantify the internal standard. If sacrificing a part of the sequencing effort to the internal standard is acceptable, we however recommend that the internal standard accounts for 30% of the environmental 16S rRNA genes to avoid the PCR bias associated with rare phylotypes. The method proposed here was tested on a feces sample but can be applied more broadly on any environmental sample. This method offers a real improvement of metabarcoding of microbial communities since it makes the method quantitative with limited efforts.

Multiplexed chemostat system for quantification of biodiversity and ecosystem functioning in anaerobic digestion.

Continuous cultures in chemostats have proven their value in microbiology, microbial ecology, systems biology and bioprocess engineering, among others. In these systems, microbial growth and ecosystem performance can be quantified under stable and defined environmental conditions. This is essential when linking microbial diversity to ecosystem function. Here, a new system to test this link in anaerobic, methanogenic microbial communities is introduced. Rigorously replicated experiments or a suitable experimental design typically require operating several chemostats in parallel. However, this is labor intensive, especially when measuring biogas production. Commercial solutions for multiplying reactors performing continuous anaerobic digestion exist but are expensive and use comparably large reactor volumes, requiring the preparation of substantial amounts of media. Here, a flexible system of Lab-scale Automated and Multiplexed Anaerobic Chemostat system (LAMACs) with a working volume of 200 mL is introduced. Sterile feeding, biomass wasting and pressure monitoring are automated. One module containing six reactors fits the typical dimensions of a lab bench. Thanks to automation, time required for reactor operation and maintenance are reduced compared to traditional lab-scale systems. Several modules can be used together, and so far the parallel operation of 30 reactors was demonstrated. The chemostats are autoclavable. Parameters like reactor volume, flow rates and operating temperature can be freely set. The robustness of the system was tested in a two-month long experiment in which three inocula in four replicates, i.e., twelve continuous digesters were monitored. Statistically significant differences in the biogas production between inocula were observed. In anaerobic digestion, biogas production and consequently pressure development in a closed environment is a proxy for ecosystem performance. The precision of the pressure measurement is thus crucial. The measured maximum and minimum rates of gas production could be determined at the same precision. The LAMACs is a tool that enables us to put in practice the often-demanded need for replication and rigorous testing in microbial ecology as well as bioprocess engineering.

A Single Community Dominates Structure and Function of a Mixture of Multiple Methanogenic Communities.

The ecology of microbes frequently involves the mixing of entire communities (community coalescence), for example, flooding events, host excretion, and soil tillage [1, 2], yet the consequences of this process for community structure and function are poorly understood [3-7]. Recent theory suggests that a community, due to coevolution between constituent species, may act as a partially cohesive unit [8-11], resulting in one community dominating after community coalescence. This dominant community is predicted to be the one that uses resources most efficiently when grown in isolation [11]. We experimentally tested these predictions using methanogenic communities, for which efficient resource use, quantified by methane production, requires coevolved cross-feeding interactions between species [12]. After propagation in laboratory-scale anaerobic digesters, community composition (determined from 16S rRNA sequencing) and methane production of mixtures of communities closely resembled that of the single most productive community grown in isolation. Analysis of each community's contribution toward the final mixture suggests that certain combinations of taxa within a community might be co-selected as a result of coevolved interactions. As a corollary of these findings, we also show that methane production increased with the number of inoculated communities. These findings are relevant to the understanding of the ecological dynamics of natural microbial communities, as well as demonstrating a simple method of predictably enhancing microbial community function in biotechnology, health, and agriculture [13].

The importance of filamentous cyanobacteria in the development of oxygenic photogranules.

Microorganisms often respond to their environment by growing as densely packed communities in biofilms, flocs or granules. One major advantage of life in these aggregates is the retention of its community in an ecosystem despite flowing water. We describe here a novel type of granule dominated by filamentous and motile cyanobacteria of the order Oscillatoriales. These bacteria form a mat-like photoactive outer layer around an otherwise unconsolidated core. The spatial organization of the phototrophic layer resembles microbial mats growing on sediments but is spherical. We describe the production of these oxygenic photogranules under static batch conditions, as well as in turbulently mixed bioreactors. Photogranulation defies typically postulated requirements for granulation in biotechnology, i.e., the need for hydrodynamic shear and selective washout. Photogranulation as described here is a robust phenomenon with respect to inoculum characteristics and environmental parameters like carbon sources. A bioprocess using oxygenic photogranules is an attractive candidate for energy-positive wastewater treatment as it biologically couples CO2 and O2 fluxes. As a result, the external supply of oxygen may become obsolete and otherwise released CO2 is fixed by photosynthesis for the production of an organic-rich biofeedstock as a renewable energy source.