Identity, abundance and physiology of Aquaspirillum-related filamentous bacteria in activated sludge.

Microcolony-forming bacteria closely related to the genus Aquaspirillum in the Betaproteobacteria were recently observed to be abundant in many nutrient removal wastewater treatment plants. The developed oligonucleotide probe, Aqs997, however, occasionally also targeted some filamentous bacteria in activated sludge samples when fluorescence in situ hybridization was performed. In this study, the identity, abundance, and ecophysiology of these Aqs997-positive filamentous bacteria were studied in detail. Most of the Aqs997-positive filamentous bacteria could morphologically be identified as either Eikelboom Type 1701, Type 0041/0675 or possibly Type 1851, all characterized by epiphytic growth. They were found in almost all 21 wastewater treatment plants investigated. Two morphotypes were found. Type A filaments, which seemed to be the same genotype as the microcolony-forming bacteria targeted by probe Aqs997.Type B filaments also hybridized with probe GNS941, specific for the Chloroflexi phylum, so the true identity remains unclear. Aqs997-positive filaments usually stained Gram-negative, but Gram-positive filaments were also found, stressing the difficulties in identifying bacteria from morphology and simple staining results. Studies on the ecophysiology by microautoradiography showed that Aqs997-positive filamentous bacteria did not consume acetate and glucose, while some took up butyrate, mannose, and certain amino acids. Most likely, some Aqs997-positive filamentous bacteria were able to perform full denitrification such as the Aqs997-positive microcolony-forming bacteria, and some were able to store polyhydroxyalkanoates under anaerobic conditions, potentially being glycogen accumulating organisms.

Floc-forming properties of polyphosphate accumulating organisms in activated sludge.

The physico-chemical characteristics of polyphosphate-accumulating organisms (PAO) involved in enhanced biological phosphorus removal (EBPR) was investigated in order to find a novel method for phosphorus recovery. If the physico-chemical characteristics of PAO are different from those of other main floc components, it may be possible to enrich PAO in bulk water or in the floc material for improved recovery of phosphorus. A combination of shear tests, chemical manipulation, and quantification of PAO by fluorescence in situ hybridization was applied. The microcolony strength of both Rhodocyclus-related PAO and Actinobacteria-related PAO was generally high as no treatment could break up more than 20% of all PAO in microcolonies. In contrast, it was possible to remove 20-40% of the organic matter and other bacterial cells by applying a high pH value or adding EDTA. With that a selective enrichment of PAO in the remaining floc material was possible. The feasibility of applying this selective PAO enrichment in flocs remains to be evaluated in full-scale plants for P-recovery.

Microbial diversity in biofilms from corroding heating systems.

Culture-independent investigations of the bacterial diversity and activity in district heating systems with and without corrosion did not make it possible to relate one group of microorganisms with the observed corrosion. Fluorescence in situ hybridization by oligonucleotide probes revealed the dominance of beta-proteobacteria, sulphate reducing prokaryotes and alpha-proteobacteria. Analysis of a clone library from one Danish heating (DH) system showed that the most sequences formed two clusters within the alpha-proteobacteria affiliated to the families Rhizobiaceae and Acetobacteraceae and two clusters within the beta-proteobacteria belonging to the family Comamonadaceae. Functional groups were determined by microautoradiography showing aerobic and anaerobic bacteria (sulphate reducing and methanogenic bacteria). The corrosion study showed that pitting corrosion rates were five to ten times higher than the general corrosion rates, suggesting the presence of biocorrosion. The results indicate that several bacterial groups could be involved in corrosion of DH system piping including sulphate reducing prokaryotes, Acidovorax (within the beta-proteobacteria), methanogenic bacteria and others.

Monitoring of microbial souring in chemically treated, produced-water biofilm systems using molecular techniques.

The identification of bacteria in oil production facilities has previously been based on culture techniques. However, cultivation of bacteria from these often-extreme environments can lead to errors in identifying the microbial community members. In this study, molecular techniques including fluorescence in situ hybridization, PCR, denaturing gradient gel electrophoresis, and sequencing were used to track changes in bacterial biofilm populations treated with nitrate, nitrite, or nitrate+molybdate as agents for the control of sulfide production. Results indicated that nitrite and nitrate+molybdate reduced sulfide production, while nitrate alone had no effect on sulfide generation. No long-term effect on sulfide production was observed. Initial sulfate-reducing bacterial numbers were not influenced by the chemical treatments, although a significant increase in sulfate-reducing bacteria was observed after termination of the treatments. Molecular analysis showed a diverse bacterial population, but no major shifts in the population due to treatment effects were observed.

Bacterial composition of activated sludge--importance for floc and sludge properties.

Activated sludge flocs consist of numerous constituents which, together with other factors, are responsible for floc structure and floc properties. These properties largely determine the sludge properties such as flocculation, settling and dewaterability. In this paper we briefly review the present knowledge about the role of bacteria in relation to floc and sludge properties, and we present a new approach to investigate the identity and function of the bacteria in the activated sludge flocs. The approach includes identification of the important bacteria and a characterization of their physiological and functional properties. It is carried out by use of culture-independent molecular biological methods linked with other methods to study the physiology and function, maintaining a single cell resolution. Using this approach it was found that floc-forming properties differed among the various bacterial groups, e.g. that different microcolony-forming bacteria had very different sensitivities to shear and that some of them deflocculated under anaerobic conditions. In our opinion, the approach to combine identity with functional analysis of the dominant bacteria in activated sludge by in situ methods is a very promising way to investigate correlations between presence of specific bacteria, and floc and sludge properties that are of interest.

Micromanipulation and further identification of FISH-labelled microcolonies of a dominant denitrifying bacterium in activated sludge.

The activated sludge process relies on the formation of strong microbial flocs. The knowledge about dominant floc-forming bacteria is at present very limited, especially from a phylogenetic perspective. In this study, numerous microcolonies in the activated sludge flocs were found to be targeted by a Betaproteobacteria-group-specific oligonucleotide probe using fluorescence in situ hybridization (FISH). Some of these were micromanipulated and further identified by reverse transcriptase polymerase chain reaction (RT-PCR) and sequencing to belong to the Aquaspirillum genus in the Neisseriaceae family. A specific oligonucleotide probe, Aqs997, was designed to target the identified bacteria. A survey in nine different wastewater treatment plants with nutrient removal (WWTP) showed a high abundance of bacteria hybridizing to the oligonucleotide probe developed. Microautoradiography (MAR) combined with FISH on activated sludge incubated with radiolabelled substrate showed uptake of substrate with oxygen, nitrate and nitrite as electron acceptor demonstrating a denitrifying potential of the bacteria investigated. The Aquaspirillum-related bacteria seemed to be abundant denitrifiers in WWTPs with nitrogen removal and they were particularly numerous in plants mainly receiving domestic wastewater, where they constituted up to 30% of all bacteria.

Biogeochemical and molecular signatures of anaerobic methane oxidation in a marine sediment.

Anaerobic methane oxidation was investigated in 6-m-long cores of marine sediment from Aarhus Bay, Denmark. Measured concentration profiles for methane and sulfate, as well as in situ rates determined with isotope tracers, indicated that there was a narrow zone of anaerobic methane oxidation about 150 cm below the sediment surface. Methane could account for 52% of the electron donor requirement for the peak sulfate reduction rate detected in the sulfate-methane transition zone. Molecular signatures of organisms present in the transition zone were detected by using selective PCR primers for sulfate-reducing bacteria and for Archaea. One primer pair amplified the dissimilatory sulfite reductase (DSR) gene of sulfate-reducing bacteria, whereas another primer (ANME) was designed to amplify archaeal sequences found in a recent study of sediments from the Eel River Basin, as these bacteria have been suggested to be anaerobic methane oxidizers (K. U. Hinrichs, J. M. Hayes, S. P. Sylva, P. G. Brewer, and E. F. DeLong, Nature 398:802-805, 1999). Amplification with the primer pairs produced more amplificate of both target genes with samples from the sulfate-methane transition zone than with samples from the surrounding sediment. Phylogenetic analysis of the DSR gene sequences retrieved from the transition zone revealed that they all belonged to a novel deeply branching lineage of diverse DSR gene sequences not related to any previously described DSR gene sequence. In contrast, DSR gene sequences found in the top sediment were related to environmental sequences from other estuarine sediments and to sequences of members of the genera Desulfonema, Desulfococcus, and Desulfosarcina. Phylogenetic analysis of 16S rRNA sequences obtained with the primers targeting the archaeal group of possible anaerobic methane oxidizers revealed two clusters of ANME sequences, both of which were affiliated with sequences from the Eel River Basin.

Desulfomusa hansenii gen. nov., sp. nov., a novel marine propionate-degrading, sulfate-reducing bacterium isolated from Zostera marina roots.

The physiology and phylogeny of a novel sulfate-reducing bacterium, isolated from surface-sterilized roots of the marine macrophyte Zostera marina, are presented. The strain, designated P1T, was enriched and isolated in defined oxygen-free, bicarbonate-buffered, iron-reduced seawater medium with propionate as sole carbon source and electron donor and sulfate as electron acceptor. Strain P1T had a rod-shaped, slightly curved cell morphology and was motile by means of a single polar flagellum. Cells generally aggregated in clumps throughout the growth phase. High CaCl2 (10 mM) and MgCl2 (50 mM) concentrations were required for optimum growth. In addition to propionate, strain P1T utilized fumarate, succinate, pyruvate, ethanol, butanol and alanine. Oxidation of propionate was incomplete and acetate was formed in stoichiometric amounts. Strain P1T thus resembles members of the sulfate-reducing genera Desulfobulbus and Desulforhopalus, which both oxidize propionate incompletely and form acetate in addition to CO2. However, sequence analysis of the small-subunit rDNA and the dissimilatory sulfite reductase gene revealed that strain P1T was unrelated to the incomplete oxidizers Desulfobulbus and Desulforhopalus and that it constitutes a novel lineage affiliated with the genera Desulfococcus, Desulfosarcina, Desulfonema and 'Desulfobotulus'. Members of this branch, with the exception of 'Desulfobotulus sapovorans', oxidize a variety of substrates completely to CO2. Strain P1T (= DSM 12642T = ATCC 700811T) is therefore proposed as Desulfomusa hansenii gen. nov., sp. nov. Strain p1T thus illustrates the difficulty of extrapolating rRNA similarities to physiology and/or ecological function.