In situ detection of microorganisms by fluorescence in situ hybridization (FISH) is a powerful tool for environmental microbiology, but analyses can be hampered by low rRNA content in target organisms, especially in oligotrophic environments. Here, we present a non-enzymatic, hybridization chain reaction (HCR)-based signal amplified in situ whole-cell detection technique (in situ DNA-HCR). The components of the amplification buffer were optimized to polymerize DNA amplifier probes for in situ DNA-HCR. In situ hybridization of initiator probes followed by signal amplification via HCR produced bright signals with high specificity and probe permeation into cells. The detection rates for Bacteria in a seawater sample and Archaea in anaerobic sludge samples were comparable with or greater than those obtained by catalyzed reporter deposition (CARD)-FISH or standard FISH. Detection of multiple organisms (Bacteria, Archaea and Methanosaetaceae) in an anaerobic sludge sample was achieved by simultaneous in situ DNA-HCR. In summary, in situ DNA-HCR is a simple and easy technique for detecting single microbial cells and enhancing understanding of the ecology and behaviour of environmental microorganisms in situ.

The production of soluble microbial products (SMP) in anaerobic systems was evaluated using chemostat reactors. Results from steady-state and tracer experiments with (14)C-glucose and (14)C-acetate showed that significant amounts of SMP were produced during the acidogenesis of glucose, but that SMP did not accumulate during methanogenesis from acetate. In addition, at a retention time of 40 days, SMP comprised almost all of the effluent COD from the glucose-fed chemostat. For shorter retention times, as low as 10 days, the SMP concentration remained almost constant, but its significance in the effluent COD was reduced due to the accumulation of intermediate volatile fatty acids. The results from a (14)C-tracer experiment in the glucose-fed chemostat were used to evaluate the importance of including SMP formation and degradation in kinetic modeling of the methanogenic chemostats. Three models were evaluated: a model without SMP production, a model with SMP production but no degradation, and a model with SMP production and degradation, The results of this kinetic analysis indicate that the model that includes SMP production and degradation was the only one able to adequately represent the fate of (14)C in the tracer experiment. The kinetic parameters were successfully used to predict steady-state concentrations of SMP and to characterize the formation and degradation characteristics of the SMP.

Aerobic ammonia-oxidizing archaea (AOA) play a crucial role in the global nitrogen cycle by oxidizing ammonia to nitrite, and nitric oxide (NO) is a key intermediate in AOA for sustaining aerobic ammonia oxidation activity. We herein heterologously expressed the NO-forming, copper-containing, dissimilatory nitrite reductase (NirK) from Nitrososphaera viennensis and investigated its enzymatic properties. The recombinant protein catalyzed the reduction of 15NO2− to 15NO, the oxidation of hydroxylamine (15NH2OH) to 15NO, and the production of 14–15N2O from 15NH2OH and 14NO2−. To the best of our knowledge, the present study is the first to document the enzymatic properties of AOA NirK.

Fluorescence in-situ hybridization (FISH) and DO microelectrodes were applied to biofilms developed in a novel reactor named DHS (downflow hanging sponge-cubes), to investigate the mechanisms of simultaneous carbon removal and nitrification. The DHS reactor was employed as an aerobic post-treatment process after an UASB anaerobic pre-treatment process receiving a municipal sewage. The presence ratio of Nitrosomonas and Nitrobacter cells to total cells of the DHS biomass was estimated by FISH technique to be 1.4% and 0.18%, respectively. Cell concentrations of both nitrifying bacteria were in good agreement with the magnitudes of ammonia-oxidizing and nitrite-oxidizing activities evaluated from batch tests. The habitats of both nitrifiers were the interior space of sponge-cubes, rather than within the biofilms attached onto sponge-cube surfaces. DO microelectrodes verify that the sponge-cubes insides were kept aerobically throughout the whole reactor height excluding the inlet vicinity portion.

A method based on sequence-specific cleavage of rRNA with ribonuclease H was used to detect almost all known cultivable methanogens in anaerobic biological treatment systems. To do so, a total of 40 scissor probes in different phylogeny specificities were designed or modified from previous studies, optimized for their specificities under digestion conditions with 32 methanogenic reference strains, and then applied to detect methanogens in sludge samples taken from 6 different anaerobic treatment processes. Among these processes, known aceticlastic and hydrogenotrophic groups of methanogens from the families Methanosarcinaceae, Methanosaetaceae, Methanobacteriaceae, Methanothermaceae and Methanocaldococcaceae could be successfully detected and identified down to the genus level. Within the aceticlastic methanogens, the abundances of mesophilic Methanosaeta accounted for 5.7-48.5% of the total archaeal populations in mesophilic anaerobic processes, and those of Methanosarcina represented 41.7% of the total archaeal populations in thermophilic processes. For hydrogenotrophic methanogens, members of the Methanomicrobiales, Methanobrevibacter and Methanobacterium were detected in mesophilic processes (1.2-17.2%), whereas those of Methanothermobacter, Methanothermaceae and Methanocaldococcaceae were detected in thermophilic process (2.0-4.8%). Overall results suggested that those hierarchical scissor probes developed could be effective for rapid and possibly on-site monitoring of targeted methanogens in different microbial environments.