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Key points Numerous biofilm forming PGPR reside in grass rhizospheres from arid grasslands. Drought tolerance of wheat is enhanced by bacterial inoculations. Wheat variety and the level of drought stress modify the plant’s response to the bacteria.

The Micrarchaeota lineage includes poorly characterized archaea with reduced genomes that likely depend on host interactions for survival. Here, the authors report a stable co-culture of a member of the Micrarchaeota and its host, and use multi-omic and physiological analyses to shed light on this symbiosis.

Environmental biofilms represent a complex mixture of different microorganisms. Their identity is usually analyzed by means of nucleic acid-based techniques. However, these biofilms are also composed of a highly complex extracellular matrix produced by the microbes within a particular biofilm system. The biochemical identity of this extracellular matrix remains in many cases an intractable part of biofilms and bioaggregates. Consequently, there is a need for an approach that will give access to the fully hydrated structure of the extracellular matrix or at least a major part of it. A crucial compound of the matrix identified as carbohydrate-based polymers represents major structural and functional constituents. These glycoconjugates can be characterized by using fluorescently-labeled lectins in combination with confocal laser scanning microscopy. The lectin approach is defined previously, as fluorescence lectin barcoding (FLBC) and fluorescence lectin-binding analysis (FLBA), where FLBC is equal to the screening of a particular sample with all the commercially available lectins and FLBA is the actual analysis of the matrix throughout an experiment with a selected panel of lectins. As the application of immune-based techniques in environmental biofilm systems is impossible, the lectin approach is currently the only option for probing lectin-specific glycoconjugates in complex biofilms and bioaggregates. From all the commercially available lectins tested, the lectins such as AAL, HAA, WGA, ConA, IAA, HPA, and LEA showed the highest binding efficiency. Furthermore, 20 of the overall lectins tested showed an intermediate signal intensity, nevertheless very useful for the assessment of matrix glycoconjugates. With the data compiled, we shall virtually shed more light on the dark matter of the extracellular matrix and their 3-dimensional distribution in environmental biofilm systems. The results will be helpful in future studies with a focus on the extracellular matrix glycoconjugates present in environmental microbial communities.

Abstract Sessile microorganisms were described as early as the seventeenth century. However, the term biofilm arose only in the 1960s in wastewater treatment research and was adopted later in marine fouling and in medical and dental microbiology. The sessile mode of microbial life was gradually recognized to be predominant on Earth, and the term biofilm became established for the growth of microorganisms in aggregates, frequently associated with interfaces, although many, if not the majority, of them not being continuous “films” in the strict sense. In this sessile form of life, microorganisms live in close proximity in a matrix of extracellular polymeric substances (EPS). They share emerging properties, clearly distinct from solitary free floating planktonic microbial cells. Common characteristics include the formation of synergistic microconsortia, using the EPS matrix as an external digestion system, the formation of gradients and high biodiversity over microscopically small distances, resource capture and retention, facilitated gene exchange as well as intercellular communication, and enhanced tolerance to antimicrobials. Thus, biofilms belong to the class of collective systems in biology, like forests, beehives, or coral reefs, although the term film addresses only one form of the various manifestations of microbial aggregates. The uncertainty of this term is discussed, and it is acknowledged that it will not likely be replaced soon, but it is recommended to understand these communities in the broader sense of microbial aggregates.

Abstract Background Current knowledge of the urinary tract microbiome is limited to urine analysis and analysis of biofilms formed on Foley catheters. Bacterial biofilms on ureteral stents have rarely been investigated, and no cultivation-independent data are available on the microbiome of the encrustations on the stents. Results The typical encrustations of organic and inorganic urine-derived material, including microbial biofilms formed during 3–6 weeks on ureteral stents in patients treated for kidney and ureteral stones, and without reported urinary tract infection at the time of stent insertion, were analysed. Next-generation sequencing of the 16S rRNA gene V3–V4 region revealed presence of different urotypes, distinct bacterial communities. Analysis of bacterial load was performed by combining quantification of 16S rRNA gene copy numbers by qPCR with microscopy and cultivation-dependent analysis methods, which revealed that ureteral stent biofilms mostly contain low numbers of bacteria. Fluorescence microscopy indicates the presence of extracellular DNA. Bacteria identified in biofilms by microscopy had mostly morphogenic similarities to gram-positive bacteria, in few cases to Lactobacillus and Corynebacterium, while sequencing showed many additional bacterial genera. Weddellite crystals were absent in biofilms of patients with Enterobacterales and Corynebacterium-dominated microbiomes. Conclusions This study provides novel insights into the bacterial burden in ureteral stent encrustations and the urinary tract microbiome. Short-term (3–6 weeks) ureteral stenting is associated with a low load of viable and visible bacteria in ureteral stent encrustations, which may be different from long-term stenting. Patients could be classified according to different urotypes, some of which were dominated by potentially pathogenic species. Facultative pathogens however appear to be a common feature in patients without clinically manifested urinary tract infection. Trial registration ClinicalTrials.gov, NCT02845726. Registered on 30 June 2016—retrospectively registered.

Abstract Background By altering their habitats, engineering species can improve their own fitness. However, the effect of this strategy on the fitness of coexisting species or on the structure of the respective food web is poorly understood. In this study, bacteria and bacterivorous nematodes with short (Caenorhabditis elegans) and long (Plectus acuminatus) life cycles were exposed to the mucus secreted by the freshwater flatworm Polycelis tenuis. The growth, reproduction, and feeding preferences of the nematodes in the presence/absence of the mucus were then determined. In addition, confocal laser scanning microscopy (CLSM) was used to examine the structural footprint of the mucus and the mucus colonization dynamics of bacteria and protozoans. Results Mucus exposure resulted in a greater reproductive output in P. acuminatus than in C. elegans. In a cafeteria experiment, both nematode species were attracted by bacteria-rich patches and were not deterred by mucus. CLSM showed that the flatworms spread a layer of polysaccharide-rich mucus ca. 15 µm thick from their tails. Subsequent colonization of the mucus by bacteria and protozoans resulted in an architecture that progressively resembled a complex biofilm. The presence of protozoans reduced nematode reproduction, presumably due to competition for their bacterial food supply. Conclusion Animal secretions such as mucus may have broader, community-level consequences and contribute to fueling microbial food webs.

Biooxidation of reduced inorganic sulfur compounds (RISCs) by thermoacidophiles is of particular interest for the biomining industry and for environmental issues, e.g., formation of acid mine drainage (AMD). Up to now, interfacial interactions of acidophiles with elemental sulfur as well as the mechanisms of sulfur oxidation by acidophiles, especially thermoacidophiles, are not yet fully clear. This work focused on how a crenarchaeal isolate Acidianus sp. DSM 29099 interacts with elemental sulfur. Analysis by Confocal laser scanning microscopy (CLSM) and Atomic force microscopy (AFM) in combination with Epifluorescence microscopy (EFM) shows that biofilms on elemental sulfur are characterized by single colonies and a monolayer in first stage and later on 3-D structures with a diameter of up to 100 μm. The analysis of extracellular polymeric substances (EPS) by a non-destructive lectin approach (fluorescence lectin-barcoding analysis) using several fluorochromes shows that intial attachment was featured by footprints rich in biofilm cells that were embedded in an EPS matrix consisting of various glycoconjugates. Wet chemistry data indicate that carbohydrates, proteins, lipids and uronic acids are the main components. Attenuated reflectance (ATR)-Fourier transformation infrared spectroscopy (FTIR) and high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) indicate glucose and mannose as the main monosaccharides in EPS polysaccharides. EPS composition as well as sugar types in EPS vary according to substrate (sulfur or tetrathionate) and lifestyle (biofilms and planktonic cells). This study provides information on the building blocks/make up as well as dynamics of biofilms of thermoacidophilic archaea in extremely acidic environments.

Due to the current dependence on biocidal antifouling coatings for biofouling control, there is a continuing international challenge to develop more environmentally acceptable antifouling systems. Fluctuating the pH values on paint surfaces is one of these approaches. We developed an antifouling test device to investigate algal biofilms on conductive paints by using a flume with electrochemically working test panels and subsequent confocal laser scanning microscopy (CLSM) of biofilms. By employing a pole reversal of direct current, fluctuating pH values on the paint surface were generated. As a consequence of the resulting pH stress, colonization of the paint surface by diatoms decreased substantially. The density of biofilm algae decreased with increasing pH fluctuations. However, breaks between electrochemical treatments should not exceed one hour. Overall, we established an experimental setup for testing the antifouling capabilities of electrodes based on conductive paints, which could be used for further development of these varnishes.

Although methanol has frequently been used as an inexpensive supplementary carbon source to support treatment processes, knowledge of the resultant microbial biofilms, their 3D architecture, microenvironments, exopolymer chemistry and populations remains limited. We supplied methanol as a supplementary carbon source to biofilms developing in rotating annular reactors. Analysis of circulation waters (1.0 l d−1) indicated that dissolved organic carbon was reduced by 25%, NO3-nitrogen by 95%, and total phosphorus by 70%. Analyses of populations using culture based techniques and fluorescence in situ hybridization indicated enrichment of nitrifiers, denitrifiers, and methylotrophic bacteria relative to reference biofilms not receiving methanol. The biofilms that developed were up to 4,000 μm thick. Staining with fluor conjugated lectins in combination with nucleic acid stains, revealed the presence of discrete bacterial cells inside complex globular polymeric structures. These structures were in turn surrounded by an interstitial polymer containing a variety of bacterial cell types. The globular structures bound FITC-conjugated lectins, from Canavalia ensiformis and Ulex europeaus. The FITC-lectin of Phaseolus vulgaris bound the surface of the globular structures and more generally within the matrix. Chemical analyses of the polymer paralleled the results of lectin analyses indicating that the dominant neutral sugars were glucose, galactose, mannose, rhamnose, with fucose and ribose as minor constituents. Amino sugars were not detected. Dual channel imaging with pH sensitive probes indicated that pH gradients from pH 4 to 7 occurred across the globular microcolonies. Critically for the maintenance of aerobic conditions throughout the thick biofilm it was extensively penetrated by a fine fissure network revealed by the location of fluorescent latex microbeads as detected by confocal laser scanning microscopy. Microelectrode studies confirmed the absence of any detectable Eh gradients within the biofilm. However, mobility of various size-fractionated fluorescent probes indicated that the basal region was only penetrated by the lowest molecular weight probes with a hydrated radius of 2.2 nm or less. These observations indicate the selection of a unique, thick (>4,000 μm) microbial community in which a self-organized architecture promotes the maintenance of optimal conditions and metabolism throughout the biofilm community.

Anaerobic granulation at elevated salinities has been discussed in several analytical and engineering based studies. They report either enhanced or decreased efficiencies in relation to different Na+ levels. To evaluate this discrepancy, we focused on the microbial and structural dynamics of granules formed in two upflow anaerobic sludge blanket (UASB) reactors treating synthetic wastewater at low (5 g/L Na+) and high (20 g/L Na+) salinity conditions. Granules were successfully formed in both conditions, but at high salinity, the start-up inoculum quickly formed larger granules having a thicker gel layer in comparison to granules developed at low salinity. Granules retained high concentrations of sodium without any negative effect on biomass activity and structure. 16S rRNA gene analysis and Fluorescence in Situ Hybridization (FISH) identified the acetotrophic Methanosaeta harundinacea as the dominant microorganism at both salinities. Fluorescence lectin bar coding (FLBC) screening highlighted a significant shift in the glycoconjugate pattern between granules grown at 5 and 20 g/L of Na+, and the presence of different extracellular domains. The excretion of a Mannose-rich cloud-like glycoconjugate matrix, which seems to form a protective layer for some methanogenic cells clusters, was found to be the main distinctive feature of the microbial community grown at high salinity conditions.

The extracellular matrix is a poorly studied, yet important component of dental biofilms. Fluorescence lectin-binding analysis (FLBA) is a powerful tool to characterize glycoconjugates in the biofilm matrix. This study aimed to systematically investigate the ability of 75 fluorescently labeled lectins to visualize and quantify extracellular glycoconjugates in dental biofilms. Lectin binding was screened on pooled supragingival biofilm samples collected from 76 subjects using confocal microscopy. FLBA was then performed with 10 selected lectins on biofilms grown in situ for 48 h in the absence of sucrose. For five lectins that proved particularly suitable, stained biovolumes were quantified and correlated to the bacterial composition of the biofilms. Additionally, combinations of up to three differently labeled lectins were tested. Of the 10 lectins, five bound particularly well in 48-h-biofilms: Aleuria aurantia (AAL), Calystega sepiem (Calsepa), Lycopersicon esculentum (LEA), Morniga-G (MNA-G) and Helix pomatia (HPA). No significant correlation between the binding of specific lectins and bacterial composition was found. Fluorescently labeled lectins enable the visualization of glycoconjugates in the dental biofilm matrix. The characterization and quantification of glycoconjugates in dental biofilms require a combination of several lectins. For 48-h-biofilms grown in absence of sucrose, AAL, Calsepa, HPA, LEA, and MNA-G are recommendable.

Microbial biofilm systems are defined as interface-associated microorganisms embedded into a self-produced matrix. The extracellular matrix represents a continuous challenge in terms of characterization and analysis. The tools applied in more detailed studies comprise extraction/chemical analysis, molecular characterization, and visualisation using various techniques. Imaging by laser microscopy became a standard tool for biofilm analysis, and, in combination with fluorescently labelled lectins, the glycoconjugates of the matrix can be assessed. By employing this approach a wide range of pure culture biofilms from different habitats were examined using the commercially available lectins. From the results, a binary barcode pattern of lectin binding can be generated. Furthermore, the results can be fine-tuned and transferred into a heat map according to signal intensity. The lectin barcode approach is suggested as a useful tool for investigating the biofilm matrix characteristics and dynamics at various levels, e.g. bacterial cell surfaces, adhesive footprints, individual microcolonies, and the gross biofilm or bio-aggregate. Hence fluorescence lectin bar-coding (FLBC) serves as a basis for a subsequent tailor-made fluorescence lectin-binding analysis (FLBA) of a particular biofilm. So far, the lectin approach represents the only tool for in situ characterization of the glycoconjugate makeup in biofilm systems. Furthermore, lectin staining lends itself to other fluorescence techniques in order to correlate it with cellular biofilm constituents in general and glycoconjugate producers in particular.

Aerobic granular sludge is based on self-granulated flocs forming mobile biofilms with a gel-like consistence. Bacterial and structural dynamics from flocs to granules were followed in anaerobic-aerobic sequencing batch reactors fed with synthetic wastewater, namely a bubble column (BC-SBR) operated under wash-out conditions for fast granulation, and two stirred-tank enrichments of Accumulibacter (PAO-SBR) and Competibacter (GAO-SBR) operated at steady-state. In the BC-SBR, granules formed within two weeks by swelling of Zoogloea colonies around flocs, developing subsequently smooth zoogloeal biofilms. However, Zoogloea predominance (37-79%) led to deteriorated nutrient removal during the first months of reactor operation. Upon maturation, improved nitrification (80-100%), nitrogen removal (43-83%), and high but unstable dephosphatation (75-100%) were obtained. Proliferation of dense clusters of nitrifiers, Accumulibacter, and Competibacter from granule cores outwards resulted in heterogeneous bioaggregates, inside which only low abundance Zoogloea (

Despite the ubiquity of Mn oxides in natural environments, there are only a few observations of biological Mn(II) oxidation at pH < 6. The lack of low pH Mn-oxidizing bacteria (MOB) isolates limits our understanding of how pH influences biological Mn(II) oxidation in extreme environments. Here, we report that a novel MOB isolate, Mesorhizobium australicum strain T-G1, isolated from an acidic and metalliferous uranium mining area, can oxidize Mn(II) at both acidic and neutral pH using different enzymatic pathways. X-ray diffraction (XRD), Raman spectroscopy, and scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDS) revealed that T-G1 initiated bixbyite-like Mn oxide formation at pH 5.5 which coincided with multi-copper oxidase (MCO) expression from early exponential phase to late stationary phase. In contrast, reactive oxygen species (ROS), particularly superoxide, appeared to be more important for T-G1 mediated Mn(II) oxidation at neutral pH. ROS was produced in parallel with the occurrence of Mn(II) oxidation at pH 7.2 from early stationary phase. Solid phase Mn oxides did not precipitate, which is consistent with the presence of a high amount of H2O2 and lower activity of catalase in the liquid culture at pH 7.2. Our results show that M. australicum T-G1, an acid tolerant MOB, can initiate Mn(II) oxidation by varying its oxidation mechanisms depending on the pH and may play an important role in low pH manganese biogeochemical cycling.

We investigated the ability of bacterial communities to colonize and dissolve two biogenic carbonates (Foraminifera and oyster shells). Bacterial carbonate dissolution in the upper water column is postulated to be driven by metabolic activity of bacteria directly colonising carbonate surfaces and the subsequent development of acidic microenvironments. We employed a combination of microsensor measurements, scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) and image analysis and molecular documentation of colonising bacteria to monitor microbial processes and document changes in shell surface topography. Bacterial communities rapidly colonised shell surfaces, forming dense biofilms with extracellular polymeric substance (EPS) deposits. Despite this, we found no evidence of bacterially mediated carbonate dissolution. Dissolution was not indicated by Ca²⁺ microprofiles, nor was changes in shell surface structure related to the presence of colonizing bacteria. Given the short time (days) settling carbonate material is actually in the twilight zone (500-1000 m), it is highly unlikely that microbial metabolic activity on directly colonised shells plays a significant role in dissolving settling carbonates in the shallow ocean.