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Investigation of cellular metabolic activity with stable-isotope probing (SIP) implies the admittance of an isotope tracer into the metabolic pathway. Incubation with several isotope-markers (multi-isotope tracing) is required to trace nutrient metabolization and elucidate inter-cellular interactions in complex hosts and environmental communities. To cope with the lability of cell nutrition, deuterium in heavy 2 H 2 16 O water is employed as a substrate-independent general tracer of metabolic activity. However, the spatially-resolved deuterium tracing is hampered by detection limits due to its relatively low ionization yield and mass-interference issues. In the present work, we comprehensively assess the quantitation of deuterium incorporation into biomass employing the outstanding capabilities of nanoscale Secondary Ion Mass Spectrometry facilitating quantitative analysis of metabolic activity with single-cell or subcellular resolution. The effect of ion-probe-induced material relocation on the acquired pattern in 2 H enrichment has been considered. Analytical expressions are suggested for the restoration of the deuterium fraction from the unresolved C 2 2 H–C 2 1 H 2 mass-interference. Application of the suggested principle of equal relative assimilation and the multi-isotope tracing with the 2 H-marker on a phototrophic symbiotic consortium paves the way to sensing the metabolic interplay among cells, recognition of homeostatic and shifted nutrition, checking for completeness of isotope-labelling and elucidation of nonlabelled substrate contribution.

Wastewater generation is a worldwide problem, and its treatment is an important practice for maintaining public health and environmental protection. Oxygenic photogranules (OPGs) are a relatively novel type of biogranules that have the potential to substitute the conventional activated sludge (AS) process due to the production of in situ oxygen, better physical properties such as settling velocity and density, as well as carbon and nutrient removal efficiencies. The formation of the granules is attributed to many factors, among which the most influential are light intensity, ammonium nitrogen concentration, and the presence of filamentous cyanobacteria that, along with heterotrophic microorganisms situated in the granule’s core, create a self-sustainable system that combines denitrification, carbon removal, and oxygen production. Hydrostatic and hydrodynamic cultivations are two ways that allow for obtaining OPGs. These two cultivation methods lead to the formation of various types of granules which differ in both structures as well as physical properties. This review article aims to aggregate the available literature information regarding the methods of cultivation of OPGs, their formation mechanisms, and factors that influence the cultivation as well as an overview of studies that were conducted thus far concerning this type of biogranules. Additionally, further research directions are proposed in the article.

The quality of microalgae-bacteria biomass as an organic soil fertilizer may depend on the biomass’s microbial composition, morphology, and growth history. This study aims to characterize the molecular complexity and bioaccessibility of organic matter, nitrogen, and phosphorus from microalgae-bacteria aggregates with different morphologies (flocs and granules) grown under nutrient-abundant and starvation conditions. A biochemical fractionation method was used based on sequential chemical extraction and fluorescence spectroscopy. Microalgae-bacteria aggregates were cultured and collected from photobioreactors using contrasting growth conditions to generate (i) loose flocs, (ii) consolidated flocs, (iii) smooth granules, and (iv) filamentous granules. The organic matter, nitrogen, and phosphorus from consolidated flocs were mostly extractable, accounting for up to 94% of their total content. In contrast, the organic matter from loose flocs was up to 50% non-extractable. The extractability of loose flocs was improved under starvation conditions. All microalgae-bacteria aggregates showed a low structural complexity, corresponding to an abundance of simple microbial-related constituents like tyrosine and tryptophane. Differences between the gradients of bioaccessibility for each microalgae-bacteria structure were related to the abundance of microorganisms and their metabolic products. The findings of this study have implications for the development of sustainable and environment-friendly organic fertilizers. Graphical Abstract

Recently, the use of phototrophs for wastewater treatment has been revisited because of new approaches to separate them from effluent streams. One manifestation uses oxygenic photogranules (OPGs) which are dense, easily-settleable granular biofilms of cyanobacteria, which surrounding populations of heterotrophs, autotrophs, and microalgae. OPGs can remove COD and nitrogenous compounds without external aeration. To better grow and maintain biomass in the proposed wastewater process, this study seeks to understand the factors that contribute to successful granulation. Availability of initial inorganic nitrogen, particularly ammonium, was associated with successful cultivation of OPGs. In the first days of granulation, a decrease in ammonium coupled with an increase in a cyanobacterial-specific 16S rRNA gene, may suggest that ammonium was assimilated in cyanobacteria offering a competitive environment for growth. Though both successful and unsuccessful OPG formation demonstrated a shift from non-phototrophic bacterial dominated communities on day 0 to cyanobacterial dominated communities on day 42, the successful community had a greater relative abundance (46%) of OTUs associated with genera Oscillatoria and Geitlernema than the unsuccessful community (27%), supporting that filamentous cyanobacteria are essential for successful OPG formation. A greater concentration of chlorophyll b in the unsuccessful OPG formation suggested a greater abundance of algal species. This study offers indicators of granulation success, notably availability of inorganic nitrogen and chlorophyll a and b concentrations for monitoring the health and growth of biomass for a potential OPG process.

Here, a syntrophic process was developed to produce polyhydroxy-β-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.