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.

We established an enrichment culture of marine anaerobic ammonium oxidation (anammox) bacteria using an upflow column reactor fed with artificial sea water supplemented with nitrogen and minerals and inoculated with coastal surface sediment collected from Hiroshima Bay. After 2 months of reactor operation, simultaneous removal of NH4 + and NO2− was observed, suggesting that an anammox reaction was proceeding. A total nitrogen removal rate of 2.17 g-N L −1 day −1 was attained on day 594 while the nitrogen loading rate was 3.33 g-N L −1 day −1 . Phylogenetic analysis revealed that at least two dominant "Candidatus Scalindua" species were present in this reactor. Moreover, many uncultured bacteria and archaea, including candidate division or ammonia-oxidizing archaea, were present. Fluorescence in situ hybridization (FISH) revealed that anammox bacteria accounted for 85.5 ± 4.5% of the total bacteria at day 393. We also designed two oligonucleotide probes specific to each dominant "Candidatus Scalindua" species. A simultaneous FISH analysis using both probes showed that two different "Candidatus Scalindua" species were clearly recognizable and coexisted during reactor operation, although there was some variation in their abundance. The marine anammox bacteria enriched in this study have potential applications to the treatment of industrial wastewater containing high levels of ammonium and salt.

We investigated long-chain fatty acid (LCFA)-degrading anaerobic microbes by enrichment, isolation, and RNA-based stable isotope probing (SIP). Primary enrichment cultures were made with each of four LCFA substrates (palmitate, stearate, oleate, or linoleate, as the sole energy source) at 55°C or 37°C with two sources of anaerobic granular sludge as the inoculum. After several transfers, we obtained seven stable enrichment cultures in which LCFAs were converted to methane. The bacterial populations in these cultures were then subjected to 16S rRNA gene-based cloning, in situ hybridization, and RNA-SIP. In five of seven enrichment cultures, the predominant bacteria were affiliated with the family Syntrophomonadaceae. The other two enrichment cultures contained different bacterial populations in which the majority of members belonged to the phylum Firmicutes and the class Deltaproteobacteria. After several attempts to isolate these dominant bacteria, strain MPA, belonging to the family Syntrophomonadaceae, and strain TOL, affiliated with the phylum Firmicutes, were successfully isolated. Strain MPA converts palmitate to acetate and methane in syntrophic association with Methanospirillum hungatei. Even though strain TOL assimilated [13 C]palmitate in the original enrichment culture, strain TOL has not shown the ability to degrade LCFAs after isolation. These results suggest that microbes involved in the degradation of LCFAs under methanogenic conditions might not belong only to the family Syntrophomonadaceae, as most anaerobic LCFA-degrading microbes do, but may also be found in phylogenetically diverse bacterial groups.To date, anaerobic (methanogenic) treatment processes have been widely applied to the treatment of municipal and industrial waste and wastewater because of demonstrable performance and cost-saving advantages (30,34,45). To expand the applications of these processes, many engineers and researchers are now being challenged to treat more-complex waste and wastewater containing anthropogenic compounds and/or compounds that are recalcitrant to biodegradation (17). This type of treatment has been applied to lipid-rich wastes and wastewater since the early stages of development of anaerobic treatment technologies for the following reasons: (i) lipid-rich waste and wastewater are widely found in certain food processing industries such as dairy, edible oil, and slaughterhouses (22, 33); and (ii) lipids have a high theoretical methane yield in comparison with that of other organic substrates (22). However, most of the previous studies of methanogenic processes with lipid-rich wastewater found them to be less stable and able to accommodate lower organic-loading rates (see, for example, references 37 and 47) than other types of waste and wastewater. This may be due in part to the acute toxicity of long-chain fatty acids (LCFA), which are the main constituent and hydrolysate of lipids in the anaerobic consortium. LCFA give rise to substrate toxicity in anaerobic microbes (10,18,20) and tend to adsorb onto the bi...

Long-chain fatty acid (LCFA) degradation is a key step in methanogenic treatment of wastes/wastewaters containing high concentrations of lipids. However, despite the importance of LCFA-degrading bacteria, their natural diversity is little explored due to the limited availability of isolate information and the lack of appropriate molecular markers. We therefore investigated these microbes by using RNA-based stable isotope probing. We incubated four methanogenic sludges (mesophilic sludges MP and MBF and thermophilic sludges TP and JET) with 13 C-labeled palmitate (1 mM) as a substrate. After 8 to 19 days of incubation, we could detect 13 C-labeled bacterial rRNA. A density-resolved terminal restriction fragment length polymorphism fingerprinting analysis showed distinct bacterial populations in 13 C-labeled and unlabeled rRNA fractions. The bacterial populations in the 13 C-labeled rRNA fractions were identified by cloning and sequencing of reversetranscribed 16S rRNA. Diverse phylogenetic bacterial sequences were retrieved, including those of members of the family Syntrophaceae, clone cluster MST belonging to the class Deltaproteobacteria, Clostridium clusters III and IV, phylum Bacteroidetes, phylum Spirochaetes, and family Syntrophomonadaceae. Although Syntrophomonadaceae species are considered to be the major fatty acid-degrading syntrophic microorganisms under methanogenic conditions, they were detected in only two of the clone libraries. These results suggest that phylogenetically diverse bacterial groups were active in situ in the degradation of LCFA under methanogenic conditions.Lipid is a one of the major organic fractions of wastes/ wastewaters, and lipid-rich wastes/wastewaters are widely found in certain food processing industries, such as dairy, edible oil, and slaughterhouses (20). Because lipids have a high theoretical methane yield in comparison with other organic substances, methanogenic treatment has been applied to lipidrich wastes/wastewaters but resulted in low organic loading rates (see, for example, references 16 and 50) compared to that seen for other types of wastes/wastewaters. This is at least partly due to the acute toxicity of long-chain fatty acids (LCFA), which are the main constituent and hydrolysate of lipids in the anaerobic consortium. LCFA can cause substrate toxicity in anaerobic microorganisms (see, for example, references 18 and 44) and tend to adsorb onto the biomass and flow out of the reactor.Under methanogenic conditions, LCFA degradation requires a syntrophic association of LCFA-degrading anaerobes and hydrogenotrophic methanogens, because the oxidation of LCFA is thermodynamically unfavorable in such environments unless the consumption of reducing equivalents (hydrogen and/or formate) is coupled with oxidation (37). Due to the syntrophic metabolism and toxicity of LCFA, isolation of LCFA-degrading syntrophs is difficult. Thus, information on LCFA-degrading bacteria in pure culture is based on Syntrophomonas species (10, 21, 36, 47, 54) and on Thermosyntropha lipolytica...

Peptide nucleic acids (PNAs) are nucleic acid analogs having attractive properties such as quiet stability against nucleases and proteases, and they form strong complexes with complementary strands of DNA or RNA. Because of this attractive nature, PNA is often used in antisense technology to inhibit gene expression and microbial cell growth with high specificity. Many bacterial antisense or antiribosomal studies using PNA oligomers have been reported so far, and parameters to design effective antisense PNAs and to improve PNA cell entry for efficient inhibition of bacterial growth have been presented. However, there are still several obstacles such as low cellular uptake of PNA while applying antisense PNAs to a complex microbial community. On overcoming these problems, the PNA antisense technique might become a very attractive tool not only for controlling the microbial growth but also for further elucidating microbial ecology in complex microbial consortia. Here, we summarize and present recent studies on the development of antimicrobial PNAs targeting mRNAs and rRNAs. In addition, the application potentiality of antisense techniques in nonclinical biotechnology fields is discussed.

Syntrophomonas palmitatica sp. nov., an anaerobic, syntrophic, long-chain fatty-acid-oxidizing bacterium isolated from methanogenic sludge Natural lipids such as fats and oils are hydrolysed to longchain fatty acids (LCFAs) and glycerol. Under methanogenic conditions, LCFAs are further degraded by the syntrophic association of LCFA-oxidizing, hydrogen (and/ or formate)-producing fermentative bacteria and hydrogenotrophic methanogens, because the oxidation of LCFAs is thermodynamically unfavourable in such environments unless the consumption of hydrogen and/or formate is coupled with oxidation (Schink, 1997). Therefore, LCFAdegrading anaerobes can gain only a small amount of energy through these syntrophic reactions and thus their growth is generally slow. In addition, LCFAs can cause substrate toxicity in microbes. Consequently, isolation of LCFA-degrading bacteria has been difficult and, at the time of writing, only six species/subspecies have been described. Five of these belong to the family Syntrophomonadaceae within the phylum , 2007). There is only one species described to date belonging to the class Deltaproteobacteria, i.e. Syntrophus aciditrophicus (Jackson et al., 1999), which also degrades LCFAs syntrophically.Recently, we successfully isolated strain MPA T from methanogenic granular sludge that was taken from palm oil mill effluent treated in a mesophilic upflow anaerobic sludge blanket reactor (Hatamoto et al., 2007). In a previous study, strain MPA T was found to be able to grow on palmitate in co-culture with the hydrogenotrophic methanogen Methanospirillum hungatei JF-1 T (DSM 864 T ).Abbreviations: FAME, fatty acid methyl ester; LCFA, long-chain fatty acid.The GenBank/EMBL/DDBJ accession numbers for the rrnA and rrnB operons of the 16S rRNA gene sequence of strain MPA T are AB274039 and AB274040, respectively.A phase-contrast micrograph showing the cell morphology of strain MPA T is available as supplementary material with the online version of this paper.

Butyrate-degrading bacteria in four methanogenic sludges were studied by RNA-based stable isotope probing. Bacterial populations in the 13 C-labeled rRNA fractions were distinct from unlabeled fractions, and Syntrophaceae species, Tepidanaerobacter sp., and Clostridium spp. dominated. These results suggest that diverse microbes were active in butyrate degradation under methanogenic conditions.Butyrate is one of the important intermediates in the degradation of organic matter under methanogenic conditions (14,16). Under these conditions, butyrate degradation is carried out by a syntrophic association of butyrate-oxidizing bacteria and hydrogenotrophic methanogens, because of thermodynamic constraints (17). Due to the fastidious nature of this syntrophic metabolism, isolation of butyrate-degrading syntrophs has been difficult and thus information on butyratedegrading bacteria is based on some isolates belonging to the family Syntrophomonadaceae. Due to this lack of knowledge and the lack of appropriate molecular markers, culture-independent studies have focused only on species of the family Syntrophomonadaceae (5, 15, 25). Consequently, the natural diversity of syntrophic butyrate-degrading bacteria has not been studied in any detail.The recent development of stable isotope probing (SIP) enables metabolic function and taxonomic identity to be examined concurrently (3). Only one study on methanogenic butyrate degradation has been reported with this technique (1), but not within waste/wastewater-treated methanogenic sludges. SIP provides a potentially fruitful tool for identifying potential butyrate-degraders in a methanogenic environment. In this study, therefore we used RNA-based SIP (RNA-SIP) with [ 13 C 4 ]butyrate as a substrate to explore the microorganisms involved in butyrate degradation in four methanogenic sludges.Four methanogenic sludges were used in this study. Mesophilic granular sludge MP and thermophilic granular sludge TP were taken from two lab-scale multistage upflow anaerobic sludge blanket reactors treating palm oil mill effluent. Mesophilic anaerobic digester sludge treating palm oil mill effluent (sludge MBF) and thermophilic digester sludge treating municipal solid waste (sludge JET) were taken from commercial plants. Detailed properties of these sludges were described in our previous report (7). Incubation was carried out anaerobically at 37°C (for mesophilic sludges) or 55°C (for thermophilic sludges). The granular sludges TP and MP were preincubated with 5 mM butyrate because of prolonged storage at 4°C for over 2 years. Degradation of butyrate was monitored by measuring methane production using gas chromatography as described previously (6). Preincubation was conducted for 14 days, added butyrate was completely converted to methane, and then the sludge was sampled as an unlabeled control microbial consortium. Digester sludges MBF and JET were used immediately after sampling, and RNA extracted from unincubated sludges was used as an unlabeled control RNA. Incubation of stable isotope-label...

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