Biofilms contain bacterial cells that are in a wide range of physiological states. Within a biofilm population, cells with diverse genotypes and phenotypes that express distinct metabolic pathways, stress responses and other specific biological activities are juxtaposed. The mechanisms that contribute to this genetic and physiological heterogeneity include microscale chemical gradients, adaptation to local environmental conditions, stochastic gene expression and the genotypic variation that occurs through mutation and selection. Here, we discuss the processes that generate chemical gradients in biofilms, the genetic and physiological responses of the bacteria as they adapt to these gradients and the techniques that can be used to visualize and measure the microscale physiological heterogeneities of bacteria in biofilms.

Physiological heterogeneity in biofilms.

Nitrogen is an essential component of all living organisms and the main nutrient limiting life on our planet By far, the largest inventory of freely accessible nitrogen is atmospheric dinitrogen, but most organisms rely on more bioavailable forms of nitrogen, such as ammonium and nitrate, for growth The availability of these substrates depends on diverse nitrogen-transforming reactions that are carried out by complex networks of metabolically versatile microorganisms In this Review, we summarize our current understanding of the microbial nitrogen-cycling network, including novel processes, their underlying biochemical pathways, the involved microorganisms, their environmental importance and industrial applications

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The microbial nitrogen-cycling network

In most natural environments, association with a surface in a structure known as a biofilm is the prevailing microbial lifestyle. Surface association is an efficient means of lingering in a favorable microenvironment rather than being swept away by the current. Taken to the extreme, we may view the

Biofilm, City of Microbes

▪ Abstract The eutrophication of many ecosystems in recent decades has led to an increased interest in the ecology of nitrogen transformation. Chemolitho-autotrophic ammonia-oxidizing bacteria are responsible for the rate-limiting step of nitrification in a wide variety of environments, making them important in the global cycling of nitrogen. These organisms are unique in their ability to use the conversion of ammonia to nitrite as their sole energy source. Because of the importance of this functional group of bacteria, understanding of their ecology and physiology has become a subject of intense research over recent years. The monophyletic nature of these bacteria in terrestrial environments has facilitated molecular biological approaches in studying their ecology, and progress in this field has been rapid. The ammonia-oxidizing bacteria of the β-subclass Proteobacteria have become somewhat of a model system within molecular microbial ecology, and this chapter reviews recent progress in our knowledge of ...

Ammonia-Oxidizing Bacteria: A Model for Molecular Microbial Ecology

Much of what makes life in a microbial biofilm different from life in a free aqueous suspension can be explained by invoking the phenomenon of diffusion. This article discusses the profound influence of the physics of the diffusion process on the chemistry and biology of the biofilm mode of growth.

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Diffusion in biofilms

We investigated the in situ spatial organization of ammonia-oxidizing and nitrite-oxidizing bacteria in domestic wastewater biofilms and autotrophic nitrifying biofilms by using microsensors and fluorescent in situ hybridization (FISH) performed with 16S rRNA-targeted oligonucleotide probes. The combination of these techniques made it possible to relate in situ microbial activity directly to the occurrence of nitrifying bacterial populations. In situ hybridization revealed that bacteria belonging to the genus Nitrosomonas were the numerically dominant ammonia-oxidizing bacteria in both types of biofilms. Bacteria belonging to the genus Nitrobacter were not detected; instead, Nitrospira-like bacteria were the main nitrite-oxidizing bacteria in both types of biofilms. Nitrospira-like cells formed irregularly shaped aggregates consisting of small microcolonies, which clustered around the clusters of ammonia oxidizers. Whereas most of the ammonia-oxidizing bacteria were present throughout the biofilms, the nitrite-oxidizing bacteria were restricted to the active nitrite-oxidizing zones, which were in the inner parts of the biofilms. Microelectrode measurements showed that the active ammonia-oxidizing zone was located in the outer part of a biofilm, whereas the active nitrite-oxidizing zone was located just below the ammonia-oxidizing zone and overlapped the location of nitrite-oxidizing bacteria, as determined by FISH.

In Situ Analysis of Nitrifying Biofilms as Determined by In Situ Hybridization and the Use of Microelectrodes

https://ir.lib.hiroshima-u.ac.jp/files/public/2/20587/20141016135304481485/WR_41-8_1623.pdf

Development of high-rate anaerobic ammonium-oxidizing (ANAMMOX) biofilm reactors

Microbially induced concrete corrosion (MICC) in sewer systems has been a serious problem for a long time. A better understanding of the succession of microbial community members responsible for the production of sulfuric acid is essential for the efficient control of MICC. In this study, the succession of sulfur-oxidizing bacteria (SOB) in the bacterial community on corroding concrete in a sewer system in situ was investigated over 1 year by culture-independent 16S rRNA gene-based molecular techniques. Results revealed that at least six phylotypes of SOB species were involved in the MICC process, and the predominant SOB species shifted in the following order: Thiothrix sp., Thiobacillus plumbophilus, Thiomonas intermedia, Halothiobacillus neapolitanus, Acidiphilium acidophilum, and Acidithiobacillus thiooxidans. A. thiooxidans, a hyperacidophilic SOB, was the most dominant (accounting for 70% of EUB338-mixed probe-hybridized cells) in the heavily corroded concrete after 1 year. This succession of SOB species could be dependent on the pH of the concrete surface as well as on trophic properties (e.g., autotrophic or mixotrophic) and on the ability of the SOB to utilize different sulfur compounds (e.g., H2S, S0, and S2O32−). In addition, diverse heterotrophic bacterial species (e.g., halo-tolerant, neutrophilic, and acidophilic bacteria) were associated with these SOB. The microbial succession of these microorganisms was involved in the colonization of the concrete and the production of sulfuric acid. Furthermore, the vertical distribution of microbial community members revealed that A. thiooxidans was the most dominant throughout the heavily corroded concrete (gypsum) layer and that A. thiooxidans was most abundant at the highest surface (1.5-mm) layer and decreased logarithmically with depth because of oxygen and H2S transport limitations. This suggested that the production of sulfuric acid by A. thiooxidans occurred mainly on the concrete surface and the sulfuric acid produced penetrated through the corroded concrete layer and reacted with the sound concrete below.

Succession of Sulfur-Oxidizing Bacteria in the Microbial Community on Corroding Concrete in Sewer Systems

The present study investigated the phylogenetic affiliation and physiological characteristics of bacteria responsible for anaerobic ammonium oxidization (anammox); these bacteria were enriched in an anammox reactor with a nitrogen removal rate of 26.0 kg N m−3 day−1. The anammox bacteria were identified as representing ‘Candidatus Brocadia sinica’ on the basis of phylogenetic analysis of rRNA operon sequences. Physiological characteristics examined were growth rate, kinetics of ammonium oxidation and nitrite reduction, temperature, pH and inhibition of anammox. The maximum specific growth rate (μmax) was 0.0041 h−1, corresponding to a doubling time of 7 days. The half-saturation constants (K
s) for ammonium and nitrite of ‘Ca. B. sinica’ were 28±4 and 86±4 µM, respectively, higher than those of ‘Candidatus Brocadia anammoxidans’ and ‘Candidatus Kuenenia stuttgartiensis’. The temperature and pH ranges of anammox activity were 25–45 °C and pH 6.5–8.8, respectively. Anammox activity was inhibited in the presence of nitrite (50 % inhibition at 16 mM), ethanol (91 % at 1 mM) and methanol (86 % at 1 mM). Anammox activities were 80 and 70 % of baseline in the presence of 20 mM phosphorus and 3 % salinity, respectively. The yield of biomass and dissolved organic carbon production in the culture supernatant were 0.062 and 0.005 mol C (mol 
NH
4
+
)−1, respectively. This study compared physiological differences between three anammox bacterial enrichment cultures to provide a better understanding of anammox niche specificity in natural and man-made ecosystems.

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Physiological characteristics of the anaerobic ammonium-oxidizing bacterium 'Candidatus Brocadia sinica'.

Summary
Anaerobic ammonium oxidation (anammox) is a microbial process in which NH4+ is oxidized to N2 gas with NO2− as an electron acceptor. The anammox process is mediated by bacterial members affiliated with the phylum Planctomycetes, which are ubiquitously detected from anoxic natural and man-made ecosystems and a key player in the global nitrogen cycle. In the past two decades, phylogenetically different anammox bacteria have been recognized in natural and synthetic ecosystems (i.e. ‘Candidatus Kuenenia’, ‘Candidatus Brocadia’, ‘Candidatus Jettenia’, ‘Candidatus Anammoxoglobus’ and ‘Candidatus Scalindua’ genera), and the geographic distributions of these anammox bacteria indicate that they have genus-specific or species-specific habitats. Recently, we revealed the physiological characteristics of ‘Ca. Jettenia’ in addition to ‘Ca. Kuenenia’, ‘Ca. Brocadia’ and ‘Ca. Scalindua’, and, as a result, it is possible to compare the physiological characteristics of the anammox bacteria and discuss their niche partitioning. Therefore, we summarize the current knowledge of anammox bacterial ecology and physiology in this review to assess the potential ecological niche partitioning of anammox bacteria in natural and synthetic ecosystems.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/1462-2920.13134

Hisashi Satoh

Papers

In Situ Analysis of Nitrifying Biofilms as Determined by In Situ Hybridization and the Use of Microelectrodes

Development of high-rate anaerobic ammonium-oxidizing (ANAMMOX) biofilm reactors

Succession of Sulfur-Oxidizing Bacteria in the Microbial Community on Corroding Concrete in Sewer Systems

Physiological characteristics of the anaerobic ammonium-oxidizing bacterium 'Candidatus Brocadia sinica'.

Ecology and physiology of anaerobic ammonium oxidizing bacteria