Quantitative Morphological Analysis of Filamentous Microorganisms in Cocultures and Monocultures: Aspergillus terreus and Streptomyces rimosus Warfare in Bioreactors

The aim of this study was to quantitatively characterize the morphology of the filamentous microorganisms Aspergillus terreus ATCC 20542 and Streptomyces rimosus ATCC 10970, cocultivated in stirred tank bioreactors, and to characterize their mutual influence with the use of quantitative image analysis. Three distinct coculture initiation strategies were applied: preculture versus preculture, spores versus spores and preculture versus preculture with time delay for one of the species. Bioreactor cocultures were accompanied by parallel monoculture controls. The results recorded for the mono- and cocultures were compared in order to investigate the effect of cocultivation on the morphological evolution of A. terreus and S. rimosus. Morphology-related observations were also confronted with the analysis of secondary metabolism. The morphology of the two studied filamentous species strictly depended on the applied coculture initiation strategy. In the cocultures initiated by the simultaneous inoculation, S. rimosus gained domination or advance over A. terreus. The latter microorganism dominated only in these experiments in which S. rimosus was introduced with a delay.

Improving the hydrobiological analysis of activated sludge at the Minsk wastewater treatment facilities

На основании накопленного опыта эксплуатации сооружений Минской очистной станции и обзора источников информации определена схема гидробиологического анализа активного ила, приведена форма регистрации результатов проведенного анализа. Для количественного учета нитчатых микроорганизмов разработана собственная пятибалльная система. Для нитчатых микроорганизмов, выявленных в активном иле, на основании анализа литературных данных определены условия, благоприятствующие их массовому развитию. В течение длительного периода времени, включающего разные сезоны года, проведена апробация усовершенствованной методики для мониторинга состояния иловой суспензии очистных сооружений двух площадок станции: классических четырехкоридорных аэротенков-вытеснителей первой площадки и биореакторов с каскадной денитрификацией второй площадки. Определены представители нитчатых микроорганизмов, постоянно обитающие в очистных сооружениях, выявлены доминирующие виды. Проведено сопоставление результатов анализа нитчатых форм и седиментационных характеристик активного ила. Сделаны заключения об особенностях биоценоза аэротенков первой площадки и биореакторов второй площадки, идентичности по видовому составу сообществ нитчатых бактерий, влиянии состава сточных вод и условий их очистки (нагрузка на ил, наличие зон с разным уровнем аэрации) на свойства иловой суспензии. Based on the accumulated experience in operating the Minsk Wastewater Treatment Facilities and a review of information sources, a scheme for the hydrobiological analysis of activated sludge was determined, and a form for recording the results of the analysis was provided. For the quantitative account of filamentous microorganisms, an original five-point system was developed. Based on the literature data analysis, favorable conditions for the mass growth of filamentous microorganisms found in activated sludge were determined. Over a long period of time, including different seasons of the year, the improved methodology was tested for monitoring the state of the sludge suspension at two sites of the treatment facilities: traditional four-line continuous-flow aeration tanks at the first site and bioreactors with cascade denitrification at the second site. The species of filamentous microorganisms, permanently living in wastewater treatment structures, have been identified, and the dominant species have been determined. The results of the analysis of filamentous forms and sedimentation characteristics of activated sludge are compared. Conclusions were made about the features of the biocenosis in the aeration tanks at the first site and in the bioreactors at the second site; about the identity of the species composition of the filamentous bacteria communities, the effect of the wastewater composition and treatment conditions (sludge load, occurrence of zones with different levels of aeration) on the properties of the sludge suspension.

“Microbial Wars” in a Stirred Tank Bioreactor: Investigating the Co-Cultures of Streptomyces rimosus and Aspergillus terreus, Filamentous Microorganisms Equipped With a Rich Arsenal of Secondary Metabolites

Microbial co-cultivation is an approach frequently used for the induction of secondary metabolic pathways and the discovery of novel molecules. The studies of this kind are typically focused on the chemical and ecological aspects of inter-species interactions rather than on the bioprocess characterization. In the present work, the co-cultivation of two textbook producers of secondary metabolites, namely Aspergillus terreus (a filamentous fungus used for the manufacturing of lovastatin, a cholesterol-lowering drug) and Streptomyces rimosus (an actinobacterial producer of an antibiotic oxytetracycline) in a 5.5-L stirred tank bioreactor was investigated in the context of metabolic production, utilization of carbon substrates and dissolved oxygen levels. The cultivation runs differed in terms of the applied co-culture initiation strategy and the composition of growth medium. All the experiments were performed in three bioreactors running in parallel (corresponding to a co-culture and two respective monoculture controls). The analysis based upon mass spectrometry and liquid chromatography revealed a broad spectrum of more than 40 secondary metabolites, including the molecules identified as the oxidized derivatives of rimocidin and milbemycin that were observed solely under the conditions of co-cultivation. S. rimosus showed a tendency to dominate over A. terreus, except for the runs where S. rimosus was inoculated into the already developed bioreactor cultures of A. terreus. Despite being dominated, the less aggressive strain still had an observable influence on the production of secondary metabolites and the utilization of substrates in co-culture. The monitoring of dissolved oxygen levels was evaluated as a fast approach of identifying the dominant microorganism during the co-cultivation process.

A bioprocess perspective on the production of secondary metabolites by Streptomyces in submerged co-cultures

Filamentous microorganisms are potent sources of bioactive secondary metabolites, the molecules formed in response to complex environmental signals. The chemical diversity encoded in microbial genomes is only partially revealed by following the standard microbiological approaches. Mimicking the natural stimuli through laboratory co-cultivation is one of the most effective methods of awakening the formation of high-value metabolic products. Whereas the biosynthetic outcomes of co-cultures are reviewed extensively, the bioprocess aspects of such efforts are often overlooked. The aim of the present review is to discuss the submerged co-cultivation strategies used for triggering and enhancing secondary metabolites production in Streptomyces, a heavily investigated bacterial genus exhibiting an impressive repertoire of secondary metabolites, including a vast array of antibiotics. The previously published studies on influencing the biosynthetic capabilities of Streptomyces through co-cultivation are comparatively analyzed in the bioprocess perspective, mainly with the focus on the approaches of co-culture initiation, the experimental setup, the design of experimental controls and the ways of influencing the outcomes of co-cultivation processes. These topics are discussed in the general context of secondary metabolites production in submerged microbial co-cultures by referring to the Streptomyces-related studies as illustrative examples.

Effect of Operating Conditions on Membrane Fouling in Pilot-Scale MBRs; Filaments Growth, Diminishing Dissolved Oxygen and Recirculation Rate of the Activated Sludge

This is the first study that examines the effect of operating conditions on fouling of Membrane Bio-Reactors (MBRs), which treat municipal wastewater in field conditions, with specific regard to the controlled development of filamentous microorganisms (or filaments). The novelty of the present work is extended to minimize the dissolved oxygen (DO) in recirculated activated sludge for improving the process of denitrification. For this purpose, two pilot-scale MBRs were constructed and operated in parallel: i) Filament-MBR, where an attempt was made to regulate the growth of filaments by adjustment of DO, the Food-to-Microorganisms (F/M) ratio and temperature, and ii) Control-MBR, where a gentle stirring tank was employed for the purpose of zeroing the DO in the recycled sludge. Results showed that low temperature (< 15 °C) slightly increased the number of filaments in the Filament-MBR which, in turn, decreased the Trans-Membrane Pressure (TMP). As the Soluble Microbial Products (SMP) and the colloids are considered to be the basic foulants of membranes in MBR systems, specific attention was directed to keep their concentration at low values in the mixed liquor. The low F/M ratio in the aeration tanks which preceded the membrane tank was achieved to keep the SMP proteins and carbohydrates at very low values in the mixed liquor, i.e., less than 6 mg/L. Moreover, as a result of the low recirculation rate (2.6∙Qin), good aggregation of the produced excess sludge was achieved, and low concentration of colloids with a size ≤50 nm (nearly the membranes’ pore size used for filtration/separation) was measured, accounted for maximum 15% of the total colloids. Additionally, the increase in filamentous population at the Filament-MBR contributed to the further reduction of colloids in the mixed liquor at 7.9%, contributing beneficially to the reduction of TMP and of membrane fouling. The diminishing of DO in the recirculated sludge improved denitrification, and resulted in lower concentrations of Ν-NO3− and TN in the effluent of the Control-MBR. Furthermore, the recirculation rate of Qr = 2.6∙Qin, in comparison with Qr = 4.3∙Qin, resulted in improved performance regarding the removal of N-NH4+. Finally, high organics removal and ammonium nitrification was observed in the effluent of both pilots, since COD and Ν-ΝΗ4+ concentrations were generally in the range of 10–25 mg/L and < 0.1 mg/L, respectively.

Low Global Diversity of Candidatus Microthrix, a Troublesome Filamentous Organism in Full-Scale WWTPs

Candidatus Microthrix is one of the most common bulking filamentous microorganisms found in activated sludge wastewater treatment plants (WWTPs) across the globe. One species, Ca. M. parvicella, is frequently observed, but global genus diversity, as well as important aspects of its ecology and physiology, are still unknown. Here, we use the MiDAS ecosystem-specific 16S rRNA gene database in combination with amplicon sequencing of Danish and global WWTPs to investigate Ca. Microthrix spp. diversity, distribution, and factors affecting their global presence. Only two species were abundant across the world confirming low diversity of the genus: the dominant Ca. M. parvicella and an unknown species typically present along with Ca. M. parvicella, although usually in lower abundances. Both species were mostly found in Europe at low-to-moderate temperatures and their growth was favored in municipal WWTPs with advanced process designs. As no isolate is available for the novel species, we propose the name “Candidatus Microthrix subdominans.” Ten high-quality metagenome-assembled genomes recovered from Danish WWTPs, including 6 representing the novel Ca. M. subdominans, demonstrated high genetic similarity between the two species with a likely preference for lipids, a putative capability to reduce nitrate and nitrite, and the potential to store lipids and poly-P. Ca. M. subdominans had a potentially more versatile metabolism including additional sugar transporters, higher oxygen tolerance, and the potential to use carbon monoxide as energy source. Newly designed fluorescence in situ hybridization probes revealed similar filamentous morphology for both species. Raman microspectroscopy was used to quantify the in situ levels of intracellular poly-P. Despite the observed similarities in their physiology (both by genomes and in situ), the two species showed different seasonal dynamics in Danish WWTPs through a 13-years survey, possibly indicating occupation of slightly different niches. The genomic information provides the basis for future research into in situ gene expression and regulation, while the new FISH probes provide a useful tool for further characterization in situ. This study is an important step toward understanding the ecology of Ca. Microthrix in WWTPs, which may eventually lead to optimization of control strategies for its growth in this ecosystem.

Microalgal-Bacterial Granular Sludge Process in Non-Aerated Municipal Wastewater Treatment under Natural Day-Night Conditions: Performance and Microbial Community

The microalgal-bacterial granular sludge (MBGS) process is expected to meet the future requirements of municipal wastewater treatment technology for decontamination, energy consumption, carbon emission and resource recovery. However, little research on the performance of the MBGS process in outdoor treatment was reported. This study investigated the performance of the MBGS system in treating municipal wastewater under natural alternate day and night conditions in late autumn. The results showed that the average removal efficiencies of Chemical oxygen demand (COD), NH4+-N and PO43−-P on daytime before cooling (stage I, day 1−4) could reach 59.9% ± 6.8%, 78.1% ± 7.9% and 61.5% ± 4.5%, respectively, while the corresponding average removal efficiencies at night were 47.6% ± 8.0%, 56.5% ± 17.9% and 74.2% ± 7.6%, respectively. Due to the dramatic changes in environmental temperature and light intensity, the microbial biomass and system stability was affected with fluctuation in COD and PO43−-P removal. In addition, the relative abundance of filamentous microorganisms (i.e., Clostridia and Anaerolineae) decreased, while Chlorella maintained a dominant position in the eukaryotic community (i.e., relative abundance > 99%). This study can provide a theoretical basis and technical support for the further engineering application of the MBGS process.

Microbial-driven Impact of the Topmost Bed Sediment Layer on Aquatic Phosphate Fluxes

&lt;p&gt;There is a longstanding principle that the uppermost layer of aquatic sediment is the primary regulator of nutrient loads in the bottom water zone, pertaining to the fact that it is significantly biological in nature and thus the site of a myriad of biota-associated processes. Nevertheless, although this principle is seemingly obvious, there is unusually scant literature corroborating the impact of the uppermost sediment layer on water column nutrient fluxes, in particular soluble reactive phosphorus (SRP). It has also been theorized that in certain environments, large bacteria play a major role in phosphorus cycling in the sediment. This challenges the prevailing dogma that the control of bottom water phosphate (PO&lt;sub&gt;4&lt;/sub&gt;&lt;sup&gt;3-&lt;/sup&gt;) is mainly attributed to the SRP flux contribution from iron (Fe) oxide-bound P in sediment or remineralisation under anoxia and warming conditions respectively. In this study, elevated temperature as well as anoxic incubation treatments were set up to demonstrate that in response to an increased level of PO&lt;sub&gt;4&lt;/sub&gt;&lt;sup&gt;3-&lt;/sup&gt; being released under stressful conditions, the topmost bed sediment layer (TBSL) has an unmistakable impact on P sequestration and stabilisation of the bottom water PO&lt;sub&gt;4&lt;/sub&gt;&lt;sup&gt;3-&lt;/sup&gt; fluxes. Likewise, we also show that large filamentous microorganisms residing in the TBSL were seemingly active in polyphosphate (polyP) accumulation during these stress-inducing conditions. This therefore strongly points to a new and important biological sink for the SRP flux at the benthic layer of an aquatic environment.&lt;/p&gt;

Metabarcoding on both environmental DNA and RNA highlights differences between fungal communities sampled in different habitats

In recent years, metabarcoding has become a key tool to describe microbial communities from natural and artificial environments. Thanks to its high throughput nature, metabarcoding efficiently explores microbial biodiversity under different conditions. It can be performed on environmental (e)DNA to describe so-called total microbial community, or from environmental (e)RNA to describe active microbial community. As opposed to total microbial communities, active ones exclude dead or dormant organisms. For what concerns Fungi, which are mostly filamentous microorganisms, the relationship between DNA-based (total) and RNA-based (active) communities is unclear. In the present study, we evaluated the consequences of performing metabarcoding on both soil and wood-extracted eDNA and eRNA to delineate molecular operational taxonomic units (MOTUs) and differentiate fungal communities according to the environment they originate from. DNA and RNA-based communities differed not only in their taxonomic composition, but also in the relative abundances of several functional guilds. From a taxonomic perspective, we showed that several higher taxa are globally more represented in either “active” or “total” microbial communities. We also observed that delineation of MOTUs based on their co-occurrence among DNA and RNA sequences highlighted differences between the studied habitats that were overlooked when all MOTUs were considered, including those identified exclusively by eDNA sequences. We conclude that metabarcoding on eRNA provides original functional information on the specific roles of several taxonomic or functional groups that would not have been revealed using eDNA alone.