Distribution, Localization, and Phylogeny of Abundant Populations of Crenarchaeota in Anaerobic Granular Sludge

Abstract

Eight anaerobic granular sludges were surveyed for Crenarchaeota using rRNA gene cloning. Microbial arrangement and substrate uptake patterns were elucidated by fluorescent in situ hybridization and beta imaging. Group 1.3 Crenarchaeota represented up to 50% of Archaea and 25% of the total microbiota in five sludges. Crenarchaeota were localized in close association with methanogenic Archaea.Crenarchaeal phylogeny has been fundamentally changed by the isolation of novel small-subunit rRNA gene clones from mesophilic and psychrophilic habitats. Indeed, rRNA gene sequences representing nonthermophilic clades of Crenarchaeota have been reported from, for example, mesophilic soils in disparate locations (e.g., the United States [3], Japan [14], and Finland [12]) and granular biofilms from psychrophilic anaerobic bioreactors treating various wastewaters (7,8,9,16).Notwithstanding the many reports documenting their presence in submesophilic environmental samples, the absence of nonthermophilic isolates has hindered a biochemical and physiological characterization of these organisms. Thus, the ecological roles and physiological functions of these abundant and cosmopolitan Crenarchaeota remain a mystery. While the unusual properties of thermophilic Crenarchaeota have attracted the attention of both exobiologists wishing to study microbial evolution and biotechnology companies wishing to exploit the hyperthermotolerance of crenarchaeal cellular enzymes, the widespread occurrence of nonthermophilic Crenarchaeota, particularly in engineered environments such as anaerobic digesters, means that the biotechnological significance and potential of these organisms should now be explored. This paper describes observations regarding the prevalence and spatial distribution of Crenarchaeota in anaerobic granules from wastewater treatment reactors. The microbial community structure was determined by using 16S rRNA clone library analysis. Fluorescent in situ hybridization (FISH) was applied in conjunction with radioactive tracer techniques and microbeta imaging to investigate substrate uptake patterns. This is the first application of this technology to anaerobic granular biofilms and represents an advancement in terms of both microbial ecology and efforts to describe the role of microorganisms involved in wastewater treatment.Eight anaerobic granular sludges, S1 to S8, were obtained from various full (37°C)-and lab (15°C)-scale anaerobic biological wastewater treatment plants. S1 was from a full-scale upflow anaerobic sludge bed treatment plant treating citric acid production wastewater at Archer Daniels Midland, Ringaskiddy, County Cork, Ireland; S2 was from a lab-scale expanded granular sludge bed-anaerobic filter (EGSB-AF) reactor treating volatile fatty acid-based wastewater; S3 and S4 were from lab-scale upflow anaerobic sludge bed reactors used to treat acetate-based and propionate/butyrate/ethanol-based wastewaters, respectively; S5 and S6 were from full-scale internal circulation (IC) reactors at Archer Daniels Midland, Coun...

Quantitative detection of culturable methanogenic archaea abundance in anaerobic treatment systems using the sequence-specific rRNA cleavage method

“…Approximately 51% and 21% of the clones analyzed for reactors 1 and 2, respectively, were closely related to uncultured archaeal 16S rRNA gene clones that were not targeted by our probe set (Figure 3). In addition to these uncultured methanogen-like groups, it is known that clones assigned to the candidate taxon WSA2 of the Euryarchaeota and the subphylum C2 of the Crenarchaeota have been retrieved from some methanogenic sludges in abundance (Chouari et al, 2005;Collins et al, 2005), although the archaeal rRNA gene cloning analysis for the sludges in this study showed no detection of such phylotypes. Thus there is a further need to develop additional scissor probes to cover these uncultured Archaea to better determine the abundance of the archaeal populations in natural and engineered environments in the future.…”

Archaea of the Miscellaneous Crenarchaeotal Group are abundant, diverse and widespread in marine sediments

“…MCG are present in terrestrial hot springs , deep oceanic subsurface sediments (Parkes et al, 2005), deep terrestrial subsurface (Inagaki et al, 2003), continental shelf sediments (Vetriani et al, 1998), ancient marine sapropels (Coolen et al, 2002), petroleum-contaminated soil (Kasai et al, 2005), termite guts (Friedrich et al, 2001), mud volcanoes (Heijs et al, 2007), methane hydrate-containing marine sediments (Inagaki et al, 2006), landfill leachate (Huang et al, 2003), anaerobic wastewater reactors (Collins et al, 2005), sulfidic springs (Elshahed et al, 2003), brackish lakes (Hershberger et al, 1996) and coastal salt marshes (Castro et al, 2004). Only 28 out of 4720 MCG sequences were retrieved from potentially oxic habitats (for example, database releases EU370096 and FJ560325).…”

Comparative Analysis of Bacterial and Archaeal Communities in Methanogenic Sludge Granules from Upflow Anaerobic Sludge Blanket Reactors Treating Various Food-Processing, High-Strength Organic Wastewaters

“…According to these studies, the 16S rRNA gene clones retrieved from UASB sludges indicated the major microbial constituents to be those of the phyla Proteobacteria, Chloroflexi, Firmicutes, Spirochaetes, and Bacteroidetes in the domain Bacteria, and those of the classes Methanomicrobia, Methanobacteria, and Thermoplasmata in the domain Archaea (47). In addition, such studies have also shown that a large number of the clones assigned to candidate phyla (known as 'clone clusters') were frequently found in such ecosystems (7,8,10,20,31,48,55). However, there are still obstacles to precisely determining and monitoring the entire microbial community of sludge because of the limited number of datasets reported so far.…”

“…So far, molecular-based community analyses have been performed for UASB granular sludges treating wastewater from a paper factory (41), a terephthalatemanufacturing plant (53), a beer brewery (13), and sucrose/ propionate/acetate-based artificial wastewater (44). In addition, the molecular characterization of UASB granules targeting specific microbial groups has also been reported (10,20,31). According to these studies, the 16S rRNA gene clones retrieved from UASB sludges indicated the major microbial constituents to be those of the phyla Proteobacteria, Chloroflexi, Firmicutes, Spirochaetes, and Bacteroidetes in the domain Bacteria, and those of the classes Methanomicrobia, Methanobacteria, and Thermoplasmata in the domain Archaea (47).…”