Community and Proteomic Analysis of Methanogenic Consortia Degrading Terephthalate

Degradation of terephthalate (TA) through microbial syntrophy under moderately thermophilic (46 to 50°C) methanogenic conditions was characterized by using a metagenomic approach (A. Lykidis et al., ISME J. 5:122–130, 2011). To further study the activities of key microorganisms responsible for the TA degradation, community analysis and shotgun proteomics were used. The results of hierarchical oligonucleotide primer extension analysis of PCR-amplified 16S rRNA genes indicated that Pelotomaculum, Methanosaeta, and Methanolinea were predominant in the TA-degrading biofilms. Metaproteomic analysis identified a total of 482 proteins and revealed a distinctive distribution pattern of microbial functions expressed in situ. The results confirmed that TA was degraded by Pelotomaculum spp. via the proposed decarboxylation and benzoyl-coenzyme A-dependent pathway. The intermediate by-products, including acetate, H₂/CO₂, and butyrate, were produced to support the growth of methanogens, as well as other microbial populations that could further degrade butyrate. Proteins related to energy production and conservation, and signal transduction mechanisms (that is, chemotaxis, PAS/GGDEF regulators, and stress proteins) were highly expressed, and these mechanisms were important for growth in energy-limited syntrophic ecosystems.     

Emission characteristics of fluorescent labels with respect to temperature changes and subsequent effects on DNA microchip studies

The effects of temperature, salt concentration, and formamide concentration on the emission characteristics of commonly used fluorescent labels were evaluated on DNA microchips. The emission intensities of different fluorophores without hybridization were observed to vary, each to a different extent, to mainly temperature changes. Rhodamine red, TAMRA (tetramethylrhodamine), and dyes from the carbocyanide group exhibited the largest variations, and Texas Red and Oregon Green exhibited the smallest variations. This temperature dependency was shown to affect results obtained during melting curve analysis in DNA microarray studies. To minimize the bias associated with the temperature-dependent emission of different fluorescent labels, a normalization step was proposed.     

Evaluating single-base-pair discriminating capability of planar oligonucleotide microchips using a non-equilibrium dissociation approach

The capability of planar rRNA-based oligonucleotide microarrays for single-base-pair discrimination was evaluated using an approach that compares the non-equilibrium dissociation profiles and dissociation temperatures (Tds) of all probe-target duplexes simultaneously. Three sets of 16S rRNA gene specific probes at different levels of specificity were used along with their counter probes for individual sets having either one or two mismatches (MM) to their targets at specific external (next to terminus) and various internal positions. Criteria based on the Td approach and a discrimination index (DI) were proven to be competent in discriminating PM from internal MM duplexes, but not always for external MM duplexes. Maximal DI for separating PM duplexes from ones with two and one internal MM usually occurred at temperatures approximately 5-10 degrees C and 10-15 degrees C, respectively, higher than the Tds of the PM duplexes. Washing buffer type and salt concentration, and MM number and position were shown statistically to affect dissociation profiles, Td, and single-base-pair discriminating capability. The reusability potential of the planar microchip was further demonstrated.     

Biogas production: evaluation of the influence of K2FeO4 pretreatment of maple leaves (Acer platanoides) on microbial consortia composition

Bioprocess and Biosystems Engineering - The potential of K2FeO4 as a pretreatment agent of a lignocellulosic material was examined on leaves of Acer platanodides as the sole substrate for biogas...     

Effects of target length on the hybridization efficiency and specificity of rRNA-based oligonucleotide microarrays

The effect of target size on microarray hybridization efficiencies and specificity was investigated using a set of 166 oligonucleotide probes targeting the 16S rRNA gene of Escherichia coli. The targets included unfragmented native rRNA, fragmented rRNA ( approximately 20 to 100 bp), PCR amplicons (93 to 1,480 bp), and three synthetic single-stranded DNA oligonucleotides (45 to 56 bp). Fluorescence intensities of probes hybridized with targets were categorized into classes I (81 to 100% relative to the control probe), II (61 to 80%), III (41 to 60%), IV (21 to 40%), V (6 to 20%), and VI (0 to 5%). Good hybridization efficiency was defined for those probes conferring intensities in classes I to IV; those in classes V and VI were regarded as weak and false-negative signals, respectively. Using unfragmented native rRNA, 13.9% of the probes had fluorescence intensities in classes I to IV, whereas the majority (57.8%) exhibited false-negative signals. Similar trends were observed for the 1,480-bp PCR amplicon (6.6% of the probes were in classes I to IV). In contrast, after hybridization of fragmented rRNA, the percentage of probes in classes I to IV rose to 83.1%. Likewise, when DNA target sizes were reduced from 1,480 bp to 45 bp, this percentage increased approximately 14-fold. Overall, microarray hybridization efficiencies and specificity were improved with fragmented rRNA (20 to 100 bp), short PCR amplicons (<150 bp), and synthetic targets (45 to 56 bp). Such an understanding is important to the application of DNA microarray technology in microbial community studies.     

Use of a Hierarchical Oligonucleotide Primer Extension Approach for Multiplexed Relative Abundance Analysis of Methanogens in Anaerobic Digestion Systems

In this study, we established a rapid multiplex method to detect the relative abundances of amplified 16S rRNA genes from known cultivatable methanogens at hierarchical specificities in anaerobic digestion systems treating industrial wastewater and sewage sludge. The method was based on the hierarchical oligonucleotide primer extension (HOPE) technique and combined with a set of 27 primers designed to target the total archaeal populations and methanogens from 22 genera within 4 taxonomic orders. After optimization for their specificities and detection sensitivity under the conditions of multiple single-nucleotide primer extension reactions, the HOPE approach was applied to analyze the methanogens in 19 consortium samples from 7 anaerobic treatment systems (i.e., 513 reactions). Among the samples, the methanogen populations detected with order-level primers accounted for >77.2% of the PCR-amplified 16S rRNA genes detected using an Archaea-specific primer. The archaeal communities typically consisted of 2 to 7 known methanogen genera within the Methanobacteriales, Methanomicrobiales, and Methanosarcinales and displayed population dynamic and spatial distributions in anaerobic reactor operations. Principal component analysis of the HOPE data further showed that the methanogen communities could be clustered into 3 distinctive groups, in accordance with the distribution of the Methanosaeta, Methanolinea, and Methanomethylovorans, respectively. This finding suggested that in addition to acetotrophic and hydrogenotrophic methanogens, the methylotrophic methanogens might play a key role in the anaerobic treatment of industrial wastewater. Overall, the results demonstrated that the HOPE approach is a specific, rapid, and multiplexing platform to determine the relative abundances of targeted methanogens in PCR-amplified 16S rRNA gene products.     

Quantitative multiplexing analysis of PCR-amplified ribosomal RNA genes by hierarchical oligonucleotide primer extension reaction

A method, termed hierarchical oligonucleotide primer extension (HOPE), is developed for quantitative, multiplexing detection of DNA targets present in PCR-amplified community 16S rRNA genes. It involves strand extension reaction and multiple oligonucleotide primers modified with different lengths of polyA at the 5′ end and targeting 16S rRNA genes at different phylogenetic specificities. On annealing to the targets, these primers are extended with a single fluorescently labeled dideoxynucleoside triphosphate or a dye-terminator. Using a DNA autosequencer, these extended primers are separated and identified by size and dye color, and quantified and normalized based on the fluorescence intensities and internal size standards. Using a primer-to-target ratio >1000, constant primer extension efficiencies can be obtained with individual primers to establish a ‘calibration factor’ between individual primers and a universal or domain-specific primer, providing the relative abundance of targeted rRNA genes with respect to total rRNA genes. HOPE up to 10-plexing is demonstrated to correctly identify 20 different bacterial strains, and quantify different Bacteroides spp. in 16S rRNA gene amplicons from different model bacteria mixtures and the influent and effluent of a wastewater treatment plant. Single mismatch discrimination with detection sensitivity of a target down to 0.01–0.05% of total DNA template is achieved.     

Emission Characteristics of Fluorescent Labels with Respect to Temperature Changes and Subsequent Effects on DNA Microchip Studies

The effects of temperature, salt concentration, and formamide concentration on the emission characteristics of commonly used fluorescent labels were evaluated on DNA microchips. The emission intensities of different fluorophores without hybridization were observed to vary, each to a different extent, to mainly temperature changes. Rhodamine red, TAMRA (tetramethylrhodamine), and dyes from the carbocyanide group exhibited the largest variations, and Texas Red and Oregon Green exhibited the smallest variations. This temperature dependency was shown to affect results obtained during melting curve analysis in DNA microarray studies. To minimize the bias associated with the temperature-dependent emission of different fluorescent labels, a normalization step was proposed.     

Relative abundance of Bacteroides spp. in stools and wastewaters as determined by hierarchical oligonucleotide primer extension

A molecular method, termed hierarchical oligonucleotide primer extension (HOPE), was used to determine the relative abundances of predominant Bacteroides spp. present in fecal microbiota and wastewaters. For this analysis, genomic DNA in feces of healthy human adults, bovines, and swine and in wastewaters was extracted and total bacterial 16S rRNA genes were PCR amplified and used as the DNA templates for HOPE. Nineteen oligonucleotide primers were designed to detect 14 Bacteroides spp. at different hierarchical levels (domain, order, cluster, and species) and were arranged into and used in six multiplex HOPE reaction mixtures. Results showed that species like B. vulgatus, B. thetaiotaomicron, B. caccae, B. uniformis, B. fragilis, B. eggerthii, and B. massiliensis could be individually detected in human feces at abundances corresponding to as little as 0.1% of PCR-amplified 16S rRNA genes. Minor species like B. pyogenes, B. salyersiae, and B. nordii were detected only collectively using a primer that targeted the B. fragilis subgroup (corresponding to approximately 0.2% of PCR-amplified 16S rRNA genes). Furthermore, Bac303-related targets (i.e., most Bacteroidales) were observed to account for 28 to 44% of PCR-amplified 16S rRNA genes from human fecal microbiota, and their abundances were higher than those detected in the bovine and swine fecal microbiota and in wastewaters by factors of five and two, respectively. These results were comparable to those obtained by quantitative PCR and to those reported previously from studies using whole-cell fluorescence hybridization and 16S rRNA clone library methods, supporting the conclusion that HOPE can be a sensitive, specific, and rapid method to determine the relative abundances of Bacteroides spp. predominant in fecal samples.