Filamentous Microorganisms Research Articles

METHODOLOGY FOR QUANTITATIVE ASSESSMENT OF THE FILAMENTOUS INDEX IN TECHNOLOGIES OF BIOLOGICAL WASTEWATER TREATMENT

For the quantitative control of the filamentous index in biological treatment facilities, a computerized methodology has been developed within the presented study. Its use enhances the reliability and techno-genic safety of operating biological treatment facilities. Problems related to filamentous microorganisms, which can hinder effective wastewater treatment, are considered. Sludge bulking occurs when the intensive growth of these microorganisms complicates the separation of activated sludge from treated wastewater, leading to various operational issues. The study emphasizes the importance of microscopic analysis in identifying and addressing problems associated with filamentous organisms. The use of microscopic analysis as a crucial tool for managing activated sludge issues is discussed. The classification system developed by D. Eikelboom, which identifies 21 types of filamentous organisms based on visual characteristics, is described in detail. The filamentous index, a key parameter for monitoring the quality of activated sludge, is examined in detail, along with methods to minimize subjective evaluation factors using computer technologies. The primary goal of the study is to develop a computerized methodology for the rapid quantitative determination of the filamentous index, and area of filamentous microorganisms in activated sludge. The ImageJ software was used for processing microphotographs of sludge, enabling precise measurements of geometric characteristics. The analysis includes processing images from Eikelboom's work to establish reference values for the filamentous index. Microsoft Excel software was used for theoretical calculations and statistical analysis of experimental data. The results of the research, which confirm the effectiveness of the proposed methodologies, are presented. Keywords: activated sludge, sludge bulking, filamentous index, quantitative analysis, treatment efficiency.

Distribution and response of electroactive microorganisms to freshwater river pollution

Electroactive microorganisms (EAMs) play a vital role in biogeochemical cycles by facilitating extracellular electron transfer. They demonstrate remarkable adaptability to river sediments that are characterized by pollution and poor water quality, significantly contributing to the sustainability of river ecosystems. However, the distribution and diversity of EAMs remain poorly understood. In this study, 16S rRNA gene high-throughput sequencing and real-time fluorescence quantitative PCR were used to assess EAMs in 160 samples collected from eight rivers within the Pearl River Delta of Southern China. The results indicated that specialized EAMs communities in polluted sediments exhibited variations in response to water quality and sediment depth. Compared to clean sediment, polluted sediments showed a 4.5% increase in the relative abundances of EAMs communities (59 genera), with 45- and 17-times higher abundances of Geobacter and cable bacteria. Additionally, the abundance of cable bacteria decreased with increasing sediment depth in polluted sediments, while the abundance of L. varians GY32 exhibited an opposite trend. Finally, the abundances of Geobacter, cable bacteria, and L. varians GY32 were positively correlated with the abundance of filamentous microorganisms (FMs) across all samples, with stronger interactions in polluted sediments. These findings suggest that EAMs demonstrate heightened sensitivity to polluted environments, particularly at the genus (species) level, and exhibit strong adaptability to conditions characterized by high levels of acid volatile sulfide, low dissolved oxygen, and elevated nitrate nitrogen. Therefore, environmental factors could be manipulated to optimize the growth and efficiency of EAMs for environmental engineering and natural restoration applications.

Microbial Ecology of an Arctic Travertine Geothermal Spring: Implications for Biosignature Preservation and Astrobiology.

Jotun springs in Svalbard, Norway, is a rare warm environment in the Arctic that actively forms travertine. In this study, we assessed the microbial ecology of Jotun's active (aquatic) spring and dry spring transects. We evaluated the microbial preservation potential and mode, as well as the astrobiological relevance of the travertines to marginal carbonates mapped at Jezero Crater on Mars (the Mars 2020 landing site). Our results revealed that microbial communities exhibited spatial dynamics controlled by temperature, fluid availability, and geochemistry. Amorphous carbonates and silica precipitated within biofilm and on the surface of filamentous microorganisms. The water discharged at the source is warm, with near neutral pH, and undersaturated in silica. Hence, silicification possibly occurred through cooling, dehydration, and partially by a microbial presence or activities that promote silica precipitation. CO2 degassing and possible microbial contributions induced calcite precipitation and travertine formation. Jotun revealed that warm systems that are not very productive in carbonate formation may still produce significant carbonate buildups and provide settings favorable for fossilization through silicification and calcification. Our findings suggest that the potential for amorphous silica precipitation may be essential for Jezero Crater's marginal carbonates because it significantly increases the preservation potential of putative martian organisms.

New insights into algal-bacterial sludge granulation based on the tightly-bound extracellular polymeric substances regulation in response to N-Methylpyrrolidone

Algal-bacterial granular sludge (ABGS) system is promising in wastewater treatment for its potential in energy-neutrality and carbon-neutrality. However, traditional cultivation of ABGS poses significant challenges attributable to its long start-up period and high energy consumption. Extracellular polymeric substances (EPS), which could be stimulated as a self-defense strategy in cells under toxic contaminants stress, has been considered to contribute to the ABGS granulation process. In this study, photogranulation of ABGS by EPS regulation in response to varying loading rates of N-Methylpyrrolidone (NMP) was investigated for the first time. The results indicated the formation of ABGS with a maximum average diameter of ∼3.3 mm and an exceptionally low SVI5 value of 67 ± 2 mL g−1 under an NMP loading rate of 125 mg L−1 d−1, thereby demonstrating outstanding settleability. Besides, almost complete removal of 300 mg L−1 NMP could be achieved at hydraulic retention time of 48 h, accompanied by chemical oxygen demand (COD) and total nitrogen (TN) removal efficiencies higher than 90 % and 70 %, respectively. Moreover, possible degradation pathway and metabolism mechanism in the ABGS system for enhanced removal of NMP and nitrogen were proposed. In this ABGS system, the mycelium with network structure constituted by filamentous microorganisms was a prerequisite for photogranulation, instead of necessarily leading to granulation. Stress of 100–150 mg L−1 d−1 NMP loading rate stimulated tightly-bound EPS (TB-EPS) variation, resulting in rapid photogranulation. The crucial role of TB-EPS was revealed with the involved mechanisms being clarified. This study provides a novel insight into ABGS development based on the TB-EPS regulation by NMP, which is significant for achieving the manipulation of photogranules.

Influence of aeration modes and DO on simultaneous nitrification and denitrification in treatment of hypersaline high-strength nitrogen wastewater using sequencing batch biofilm reactor (SBBR)

Sequencing batch biofilm reactor (SBBR) has the potential to treat hypersaline high-strength nitrogen wastewater by simultaneous nitrification-denitrification (SND). Dissolved oxygen (DO) and aeration modes are major factors affecting pollutant removal. Low DO (0.35–3.5 mg/L) and alternative anoxic/aerobic (A/O) mode are commonly used for municipal wastewater treatment, however, the appropriate DO concentration and operation mode are still unknown under hypersaline environment because of the restricted oxygen transfer in denser extracellular polymeric substances (EPS) barrier and the decreased carbon source consumption during the anoxic phase. Herein, two SBBRs (R1, fully aerobic mode; R2, A/O mode) were used for the treatment of hypersaline high-strength nitrogen wastewater (200 mg/L NH4+-N, COD/N of 3 and 3% salinity). The results showed that the relatively low DO (2 mg/L) could not realize effective nitrification, while high DO (4.5 mg/L) evidently increased nitrification efficiency by enhancing oxygen transfer in denser biofilm that was stimulated by high salinity. A stable SND was reached 16 days faster with a ∼10% increase of TN removal under A/O mode. Mechanism analysis found that denser biofilm with coccus and bacillus were present in A/O mode instead of filamentous microorganisms, with the secretion of more EPS. Corynebacterium and Halomonas were the dominant genera in both SBBRs, and HN-AD process might assist partial nitrification-denitrification (PND) for highly efficient TN removal in biofilm systems. By using the appropriate operation mode and parameters, the average NH4+-N and TN removal efficiency could respectively reach 100% and 70.8% under the NLR of 0.2 kg N·m−3·d−1 (COD/N of 3), which was the highest among the published works using SND-based SBBRs in treatment of saline high-strength ammonia nitrogen (low COD/N) wastewater. This study provided new insights in biofilm under hypersaline stress and provided a solution for the treatment of hypersaline high-strength nitrogen (low COD/N) water.

Impact of Combined Electrolysis and Activated Sludge Process on Municipal Wastewater Treatment

Electrochemical methods for the treatment of municipal and industrial wastewater are used either independently or in conjunction with biological methods for pretreatment or posttreatment of biologically treated wastewater. In our work, the combination of these processes was studied, where pre-electrolysis was used to produce dissolved iron before the activation process. Electrolysis was also directly introduced into the activation using either iron or carbon electrodes. The surface of one iron electrode was 32.2 cm2, voltage at the electrodes was 21 V, and current was 270 mA. The surface of one carbon electrode was 7.54 cm2, current was 82.5 mA, and voltage at the electrodes was 21 V. Laboratory research on synthetic municipal wastewater treatment using a combination of electrolysis and activation processes showed that the use of iron electrodes increases the efficiency of phosphorus removal compared to its precipitation with iron salts. Electrolysis has shown a positive effect on the sedimentation properties of sludge and the destruction of filamentous microorganisms. Even though it negatively affected the respiration rates of activated sludge and the denitrification efficiency, it did not have a negative impact on the nitrification activity of sludge.

Host and nonhost bacteria support bacteriophage dissemination along mycelia and abiotic dispersal networks.

Bacteriophages play a crucial role in shaping bacterial communities, yet the mechanisms by which nonmotile bacteriophages interact with their hosts remain poorly understood. This knowledge gap is especially pronounced in structured environments like soil, where spatial constraints and air-filled zones hinder aqueous diffusion. In soil, hyphae of filamentous microorganisms form a network of 'fungal highways' (FHs) that facilitate the dispersal of other microorganisms. We propose that FHs also promote bacteriophage dissemination. Viral particles can diffuse in liquid films surrounding hyphae or be transported by infectable (host) or uninfectable (nonhost) bacterial carriers coexisting on FH networks. To test this, two bacteriophages that infect Pseudomonas putida DSM291 (host) but not KT2440 (nonhost) were used. In the absence of carriers, bacteriophages showed limited diffusion on 3D-printed abiotic networks, but diffusion was significantly improved in Pythium ultimum-formed FHs when the number of connecting hyphae exceeded 20. Transport by both host and nonhost carriers enhanced bacteriophage dissemination. Host carriers were five times more effective in transporting bacteriophages, particularly in FHs with over 30 connecting hyphae. This study enhances our understanding of bacteriophage dissemination in nonsaturated environments like soils, highlighting the importance of biotic networks and bacterial hosts in facilitating this process.

FRESHWATER STROMATOLITES FROM AN EARLY PERMIAN WETLAND (MANEBACH, THURINGIAN-FOREST BASIN, GERMANY): STRUCTURE, DEVELOPMENT, AND PALEOENVIRONMENTAL CONTEXT

Abstract Fossil stromatolites enclosing structurally preserved land plant remains have rarely been documented and studied in detail. Permineralized woody Tylodendron sp. conifer axes (slender stems, branches) from a lacustrine sedimentary sequence in the lower Permian fossil Lagerstätte of Manebach (Thuringian-Forest Basin, central Germany) are frequently surrounded by stromatolites that consist of successive, usually asymmetrical microbial layers. The stromatolites show various growth forms ranging from laminar to palisadic. They developed in stagnant water from microbial overgrowth dominated by slender, unbranched sessile cyanobacterial filaments aligned vertically into tufts or turf-like stands. Interspersed among the filaments were other filamentous and coccoid microorganisms. Preservation of the Tylodendron axes can be exquisite and sometimes even includes extraxylary tissues containing remains of fungi, suggesting that stromatolite formation began soon after the axes had entered the water and were perhaps even conducive to their preservation. Structurally similar fossil microbialitic structures from elsewhere likewise demonstrate that they were effective in preserving plant morphology. The Manebach stromatolites and the plant remains they contain contribute to a more accurate understanding of the complex biological processes in late Paleozoic lake ecosystems.

Quantification and modeling of macroparticle-induced mechanical stress for varying shake flask cultivation conditions.

In biotechnological processes, filamentous microorganisms are known for their broad product spectrum and complex cellular morphology. Product formation and cellular morphology are often closely linked, requiring a well-defined level of mechanical stress to achieve high product concentrations. Macroparticles were added to shake flask cultures of the filamentous actinomycete Lentzea aerocolonigenes to find these optimal cultivation conditions. However, there is currently no model concept for the dependence of the strength and frequency of the bead-induced stress on the process parameters. Therefore, shake flask simulations were performed for combinations of bead size, bead concentration, bead density and shaking frequency. Contact analysis showed that the highest shear stresses were caused by bead-bottom contacts. Based on this, a newly generated characteristic parameter, the stress area ratio (SAR), was defined, which relates the bead wall shear and normal stresses to the total shear area. Comparison of the SAR with previous cultivation results revealed an optimum pattern for product concentration and mean product-to-biomass related yield coefficient. Thus, this model is a suitable tool for future optimization, comparison and scaling up of shear-sensitive microorganism cultivation. Finally, the simulation results were validated using high-speed recordings of the bead motion on the bottom of the shake flask.

Effects of the Coculture Initiation Method on the Production of Secondary Metabolites in Bioreactor Cocultures of Penicillium rubens and Streptomyces rimosus.

Bioreactor cocultures involving Penicillium rubens and Streptomyces rimosus were investigated with regard to secondary metabolite production, morphological development, dissolved oxygen levels, and carbon substrate utilization. The production profiles of 22 secondary metabolites were analyzed, including penicillin G and oxytetracycline. Three inoculation approaches were tested, i.e., the simultaneous inoculation of P. rubens with S. rimosus and the inoculation of S. rimosus delayed by 24 or 48 h relative to P. rubens. The delayed inoculation of S. rimosus into the P. rubens culture did not prevent the actinomycete from proliferating and displaying its biosynthetic repertoire. Although a period of prolonged adaptation was needed, S. rimosus exhibited growth and the production of secondary metabolites regardless of the chosen delay period (24 or 48 h). This promising method of coculture initiation resulted in increased levels of metabolites tentatively identified as rimocidin B, 2-methylthio-cis-zeatin, chrysogine, benzylpenicilloic acid, and preaustinoid D relative to the values recorded for the monocultures. This study demonstrates the usefulness of the delayed inoculation approach in uncovering the metabolic landscape of filamentous microorganisms and altering the levels of secondary metabolites.

Landfill leachate treatment with a full-scale membrane bioreactor: impact of leachate characteristics on filamentous bacteria.

Bulking and foaming are extreme filamentous bacterial growths that present serious challenges for the biological leachate treatment process. The current study evaluates the performance of long-term full-scale membrane bioreactor (MBR) treating landfill leachate, specifically focusing on filamentous bacteria overgrowth in the bioreactors. The influence of the variation in leachate structure and operational conditions on floc morphology and filamentous bacteria overgrowth were analyzed for 11months of operation of the full-scale MBR system. The average chemical oxygen demand (COD) and NH4-N removal efficiencies of the system were 87.8 ± 4% and 99.5 ± 0.7%. However, incomplete denitrification was observed when the F/M ratio was low. The high C/N ratio was observed to enhance the frequency of small flocs. Furthermore, a poor to medium diversity of the microbial community was observed. Haliscomenobacter hydrossis, Microthrix parvicella, and Type 021N were found as the most numerous filamentous organisms. Paramecium spp., Euplotes spp., and Aspidisca spp. were found in small quantities. The limited concentration of PO4-P in the leachate compared to high COD and NH4-N concentrations most probably caused phosphate deprivation and increased abundance of identified filamentous microorganisms. This work is the first study in Türkiye that investigates the bulking and foaming problem in full-scale MBR that treats landfill leachate. Hence, it may provide some pioneering perspectives into landfill leachate remediation by monitoring the hybrid biological system.

Granular biomass technology for providing drinking water: microbial versatility and nitrate performance in response to carbon source

The aerobic granular biomass technology was optimized for treating nitrate-polluted groundwater based on the biological denitrification processes in order to provide drinking water. Reactors inoculated with granular biomass were operated at progressively lower C/N rate using acetate and methanol to encourage heterotrophic denitrification, in order to meet the recommended requirements described by European Drinking Water Framework Directive. The granulation and long-term stability of granular biomass under low C/N were successful for all stages, demonstrated compactness of granules and absence of filamentous microorganisms. The nitrate removal was similar in methanol- and acetate-fed reactors, occurring in both cases nitrate removal ratios > 80%, and fact allows the selection of one of both depending groundwater polluted case. Also, feeding reactors with 2 C/N ratio showed nitrate removal values of ≥ 95%, treating highly polluted groundwater (100 mg·L−1). The microbial diversity was higher in the methanol-fed reactor with representative phylotypes as Flavobacterium, Cytophagaceae, NS9 marine group, while species richness was higher in the acetate-fed reactor, which was mainly represented by Flavobacterium genus. Statistical analyses revealed the higher resilience of bacterial population on granules fed with acetate, showing more resistance under drop C/N ratio. Oscillating pollution in groundwater during seasonal periods should be treated using acetate as carbon source for denitrification carried out by granular biomass, while stable pollution concentrations over time allow the use of methanol as a carbon source since the greater microbial diversity allows the elimination of other contaminants present in groundwater.

Pleural effusion-related Nocardia otitidiscaviarum, Anaplasma platys and Ehrlichia canis coinfection in a dog.

The coinfections by some microorganisms have been related to severe diseases in humans and animals, where immunosuppressive agents favor opportunistic behavior of other pathogens. A 4-month-old, female mixed-breed dog with a two-week history of inappetence, prostration, emaciation, and respiratory distress was admitted at a veterinary hospital in Brazil. Tachycardia, pale mucous membranes, severe respiratory distress, and a large number of ticks (Rhipicephalus sanguineus s.l.) in different body regions were observed at clinical examination. Hematological examination of dog showed leukocytosis, neutrophilia, mild anemia, and thrombocytopenia, whereas unremarkable values in biochemical tests. Thoracic radiography revealed a pleural effusion image. Blood and the pleural fluid (purulent aspect) samples were subjected to qPCR (16S rRNA and dsb genes) and sequencing, which identified Ehrlichia canis and Anaplasma platys coinfection. An aggregate of coccoid-to-branching or long filamentous microorganisms, surrounded by pyogranulomatous inflammatory reaction was seen at the cytology of the pleural fluid. Bacteriological culture of pleural effusion showed colonies compatible with the genus Nocardia, which revealed gram-positive filamentous organisms with a tendency of fragmentation and were identified as Nocardia otitidiscaviarum in mass spectrometry (MALDI-TOF MS). Therapy of N. otitidiscaviarum isolate using levofloxacin (supported by a previous in vitro susceptibility testing) and doxycycline for E. canis and A. platys resulted in complete resolution of the clinical picture. Here, we report for the first time a triple coinfection by Nocardia otitidiscaviarum, A. platys, and E. canis in a dog with pleural effusion, where debilitating or immunosuppressive conditions induced by A. platys and E. canis coinfection probably contributed to the opportunistic behavior of N. otitidiscaviarum.

Pilot-scale study and biochemical verification of salt-tolerant catalyst Fe-Bi@γ-Al2O3 for catalytic ozonation of high-salinity wastewater

In this study, Fe-Bi@ γ-Al2O3 was used as a catalyst to construct a heterogeneous catalytic ozonation system for the treatment of high-salinity organic wastewater and its biochemical verification was studied. The optimum operating conditions and degradation mechanism of Fe-Bi@ γ-Al2O3 catalytic ozonation of high salinity wastewater were systematically investigated. The biodegradability of wastewater before and after treatment was also studied. Under the optimum operating conditions of ozone dosage of 160 mg/L, catalyst filling rate of 15%, ratio of height to diameter of 15, hydraulic retention time of 210 min and recycle ratio of 500%, the removal rate of COD is 45.13%. The mechanical strength of Fe-Bi@ γ-Al2O3 catalyst is almost unchanged after being reused for 20 times. The results of Ultraviolet and visible spectrophotometry (UV-Vis), 3D (Three-Dimensional)-fluorescence and Gas Chromatography-Mass Spectrometer (GC-MS)showed that humus, fulvic acid and undissolved microbial organic matter were degraded. In the biochemical verification experiment, after the high salinity wastewater was used to impact the bio-reactor, the mixed liquor suspended solids (MLSS) was basically stable at 9597 mg/L, 8856 mg/L, 9414 mg/L and 8741 mg/L, and the mixed liquor volatile suspended solids (MLVSS) was basically stable at 7042 mg/L, 6940 mg/L, 6949 mg/L and 6519 mg/L. The dissolved oxygen (DO) in the four reactors were 2.9 mg/L, 3.48 mg/L, 3.6 mg/L and 2.92 mg/L respectively, and the sludge Volume Index (SVI30) were 93 mL/g, 98 mL/g, 93 mL/g and 91 mL/g respectively. After two cycles (20d) of 30% high salinity wastewater, the chemical oxygen demand (COD) removal rates of the four reactors were 78%, 84%, 86% and 91%, respectively. When the reaction time is 40 min, the BOD5/COD (B/C) value of the high-salinity wastewater reaches 0.91, and the biodegradability of the wastewater is improved significantly. Scanning electron microscope (SEM) showed that the granular sludge in the reactor had compact structure and smooth surface, and there were a lot of fungi and filamentous microorganisms on the sludge surface.in that condition of salinity stress, the microbial community in the activate sludge changed to a great extent, and Proteobacteria and Bacteroidetes became the main phylum. The Fe-Bi@ γγ-Al2O3 catalyst has good catalytic performance and can be used for deep treatment of high-salt organic wastewater.

Filamentous electroactive microorganisms promote mass transfer and sulfate reduction in sediment microbial electrochemical systems

Electron transfer and mass transfer are critical processes for efficient sediment remediation using sediment microbial electrochemical systems (SMESs). Increasing reports show that filamentous electroactive microorganisms (f-EAMs) that widely exist in heterogeneous sediments play essential roles in biogeochemical cycles and sediment remediation, significantly impacting process efficiencies. However, the knowledge of the mechanism of f-EAMs in biogeochemical cycles and sediment remediation is still limited. This study investigated the spatiotemporal effect of a unicellular Gram-positive f-EAM, Lysinibacillus varians GY32, on the sulfur cycle in SMESs. The results showed that adding L. varians GY32 into sediments could significantly decrease the diffusion resistances of the sediments by up to 49.4% and reduce the porewater sulfate concentrations by up to 46.4%, which validated that L. varians GY32 contributed to enhancing the mass transfer and sulfate reduction in sediments. Meanwhile, the vertical difference in diffusion resistances and porewater sulfate concentrations was reduced. The dynamics of electrochemical and physicochemical properties of sediments coincided well with the enrichment of filamentous microorganisms, sulfate-reducing bacteria, EAMs (especially f-EAMs), and syntrophic bacteria, as well as the reinforcement of the community interactions that took the above microorganisms as core members. Our findings provide novel insights for understanding the underlying mechanism of f-EAMs in biogeochemical cycles and sediment remediation.

A modified Agrobacterium-mediated transformation for two oomycete pathogens.

Oomycetes are a group of filamentous microorganisms that include some of the biggest threats to food security and natural ecosystems. However, much of the molecular basis of the pathogenesis and the development in these organisms remains to be learned, largely due to shortage of efficient genetic manipulation methods. In this study, we developed modified transformation methods for two important oomycete species, Phytophthora infestans and Plasmopara viticola, that bring destructive damage in agricultural production. As part of the study, we established an improved Agrobacterium-mediated transformation (AMT) method by prokaryotic expression in Agrobacterium tumefaciens of AtVIP1 (VirE2-interacting protein 1), an Arabidopsis bZIP gene required for AMT but absent in oomycetes genomes. Using the new method, we achieved an increment in transformation efficiency in two P. infestans strains. We further obtained a positive GFP transformant of P. viticola using the modified AMT method. By combining this method with the CRISPR/Cas12a genome editing system, we successfully performed targeted mutagenesis and generated loss-of-function mutations in two P. infestans genes. We edited a MADS-box transcription factor-encoding gene and found that a homozygous mutation in MADS-box results in poor sporulation and significantly reduced virulence. Meanwhile, a single-copy avirulence effector-encoding gene Avr8 in P. infestans was targeted and the edited transformants were virulent on potato carrying the cognate resistance gene R8, suggesting that loss of Avr8 led to successful evasion of the host immune response by the pathogen. In summary, this study reports on a modified genetic transformation and genome editing system, providing a potential tool for accelerating molecular genetic studies not only in oomycetes, but also other microorganisms.

Description of new single-chamber continuous-flow reactors of aerobic granular sludge: Technical and biological study

Aerobic granular sludge reactors usually operate in sequential batch mode, although this configuration limits the treatment of large volumes of wastewater, and they require a storage system. To implement this technology at full-scale, it would be necessary to design a simple and compact continuous-flow bioreactor able to treat larger volumes of wastewater. In this study, four aerobic granular sludge single-chamber continuous-flow reactors (R1, R2, R3 and R4) were designed and operated at the lab scale to evaluate and select a bioreactor configuration that achieves high organic matter removal performance while maintaining a stable granulation for long-term operation, promoting the washout of filamentous microorganisms. Results confirmed that the bioreactor including a lateral decanter (R1), was able to work in a steady state without loss of granular biomass and reached 95% of organic matter removal performance. Its granules had excellent compaction, with settling velocity values above 100 m·h−1. The R1 bioreactor also allowed rapid biomass adaptation and therefore a fast start-up (11 days). Results of this preliminary study at the lab scale suggested that the new and simple bioreactor design could be promising for its implementation at full scale. For that, future research is required to optimise the current model and to determine the most suitable operational parameters to treat domestic wastewater at full scale.

Phase-specific transcriptional patterns of the oomycete pathogen Phytophthora sojae unravel genes essential for asexual development and pathogenic processes.

Oomycetes are filamentous microorganisms easily mistaken as fungi but vastly differ in physiology, biochemistry, and genetics. This commonly-held misconception lead to a reduced effectiveness by using conventional fungicides to control oomycetes, thus it demands the identification of novel functional genes as target for precisely design oomycetes-specific microbicide. The present study initially analyzed the available transcriptome data of the model oomycete pathogen, Phytophthora sojae, and constructed an expression matrix of 10,953 genes across the stages of asexual development and host infection. Hierarchical clustering, specificity, and diversity analyses revealed a more pronounced transcriptional plasticity during the stages of asexual development than that in host infection, which drew our attention by particularly focusing on transcripts in asexual development stage to eventually clustered them into 6 phase-specific expression modules. Three of which respectively possessing a serine/threonine phosphatase (PP2C) expressed during the mycelial and sporangium stages, a histidine kinase (HK) expressed during the zoospore and cyst stages, and a bZIP transcription factor (bZIP32) exclusive to the cyst germination stage were selected for down-stream functional validation. In this way, we demonstrated that PP2C, HK, and bZIP32 play significant roles in P. sojae asexual development and virulence. Thus, these findings provide a foundation for further gene functional annotation in oomycetes and crop disease management.

Intensification of bioprocesses with filamentous microorganisms

Abstract Many industrial biotechnological processes use filamentous microorganisms to produce platform chemicals, proteins, enzymes and natural products. Product formation is directly linked to their cellular morphology ranging from dispersed mycelia over loose clumps to compact pellets. Therefore, the adjustment and control of the filamentous cellular morphology pose major challenges for bioprocess engineering. Depending on the filamentous strain and desired product, optimal morphological shapes for achieving high product concentrations vary. However, there are currently no overarching strain- or product-related correlations to improve process understanding of filamentous production systems. The present book chapter summarizes the extensive work conducted in recent years in the field of improving product formation and thus intensifying biotechnological processes with filamentous microorganisms. The goal is to provide prospective scientists with an extensive overview of this scientifically diverse, highly interesting field of study. In the course of this, multiple examples and ideas shall facilitate the combination of their acquired expertise with promising areas of future research. Therefore, this overview describes the interdependence between filamentous cellular morphology and product formation. Moreover, the currently most frequently used experimental techniques for morphological structure elucidation will be discussed in detail. Developed strategies of morphology engineering to increase product formation by tailoring and controlling cellular morphology and thus to intensify processes with filamentous microorganisms will be comprehensively presented and discussed.

Fungal drops: a novel approach for macro- and microscopic analyses of fungal mycelial growth.

This study presents an inexpensive approach for the macro- and microscopic observation of fungal mycelial growth. The 'fungal drops' method allows to investigate the development of a mycelial network in filamentous microorganisms at the colony and hyphal scales. A heterogeneous environment is created by depositing 15-20µl drops on a hydrophobic surface at a fixed distance. This system is akin to a two-dimensional (2D) soil-like structure in which aqueous-pockets are intermixed with air-filled pores. The fungus (spores or mycelia) is inoculated into one of the drops, from which hyphal growth and exploration take place. Hyphal structures are assessed at different scales using stereoscopic and microscopic imaging. The former allows to evaluate the local response of regions within the colony (modular behaviour), while the latter can be used for fractal dimension analyses to describe the hyphal network architecture. The method was tested with several species to underpin the transferability to multiple species. In addition, two sets of experiments were carried out to demonstrate its use in fungal biology. First, mycelial reorganization of Fusarium oxysporum was assessed as a response to patches containing different nutrient concentrations. Second, the effect of interactions with the soil bacterium Pseudomonas putida on habitat colonization by the same fungus was assessed. This method appeared as fast and accessible, allowed for a high level of replication, and complements more complex experimental platforms. Coupled with image analysis, the fungal drops method provides new insights into the study of fungal modularity both macroscopically and at a single-hypha level.