Characterization of wheat straw-degrading anaerobic alkali-tolerant mixed cultures from soda lake sediments by molecular and cultivation techniques

Alkaline pretreatment has the potential to enhance the anaerobic digestion of lignocellulosic biomass to biogas. However, the elevated pH of the substrate may require alkalitolerant microbial communities for an effective digestion. Three mixed anaerobic lignocellulolytic cultures were enriched from sediments from two soda lakes with wheat straw as substrate under alkaline (pH 9) mesophilic (37°C) and thermophilic (55°C) conditions. The gas production of the three cultures ceased after 4 to 5 weeks, and the produced gas was composed of carbon dioxide and methane. The main liquid intermediates were acetate and propionate. The physiological behavior of the cultures was stable even after several transfers. The enrichment process was also followed by molecular fingerprinting (terminal restriction fragment length polymorphism) of the bacterial 16S rRNA gene and of the mcrA/mrtA functional gene for methanogens. The main shift in the microbial community composition occurred between the sediment samples and the first enrichment, whereas the structure was stable in the following transfers. The bacterial communities mainly consisted of Sphingobacteriales, Clostridiales and Spirochaeta, but differed at genus level. Methanothermobacter and Methanosarcina genera and the order Methanomicrobiales were predominant methanogenes in the obtained cultures. Additionally, single cellulolytic microorganisms were isolated from enrichment cultures and identified as members of the alkaliphilic or alkalitolerant genera. The results show that anaerobic alkaline habitats harbor diverse microbial communities, which can degrade lignocellulose effectively and are therefore a potential resource for improving anaerobic digestion.     

Molecular characterization of dichloromethane-degrading Hyphomicrobium strains using 16S rDNA and DCM dehalogenase gene sequences

A phylogenetic analysis of 6 strains of dichloromethane (DCM) utilizing bacteria was performed. Based on the almost complete 16S rDNA sequence determination, all strains clustered together and showed high sequence similarity to Hyphomicrobium denitrificans, except for the strain MC8b, which is only moderately related to them and probably represents a distinct species. The 16S rDNA-based phylogenetic tree was compared to the one obtained from the DNA sequence data of the dcmA gene coding DCM dehalogenase, the key enzyme of DCM utilization. The topology of the two trees is in good agreement and may suggest an ancient origin of DCM dehalogenase, but also raises questions about the original role of the enzyme.     

Stable carbon isotope fractionation during degradation of dichloromethane by methylotrophic bacteria

Stable carbon isotope fractionation during dichloromethane (DCM) degradation by methylotrophic bacteria was investigated under aerobic and nitrate-reducing conditions. The strains studied comprise several Hyphomicrobium strains, Methylobacterium, Methylopila, Methylophilus and Methylorhabdus spp. that are considered to degrade DCM by a glutathione (GSH)-dependent dehalogenase enzyme system in the initial step. The stable carbon isotope fractionation factors (alphaC) of the strains varied under aerobic conditions between 1.043 and 1.071 and under nitrate-reducing conditions between 1.048 and 1.065. Comparison of isotope fractionation under aerobic and nitrate-reducing conditions by individual strains revealed only minor to no differences. The variability in isotope fractionation among strains was found to be related to the polymorphism of the functional genes encoding the DCM dehalogenase.     

Rhizome-associated bacterial communities of healthy and declining reed stands in Lake Velencei,Hungary

Lake Velencei is a shallow soda lake with extensive reed coverage. In this study, the bacterial communities of reed (Phragmites australis (Cav.) Trin. ex Steudel) rhizomes from healthy and declining stands were compared. Inner and outer rhizome surfaces were sampled. Samples were plated and isolated in September 1998 and June 1999. Phenotypic data of 371 bacterial strains were used for cluster analysis. Identification of phena was based on partial 16S rDNA sequence analysis of representative strains. Healthy reed stand rhizomes in fall 1998 were dominantly colonised by facultatively fermentative organisms, like Erwinia billingiae, Aeromonas sobria, Pantoea agglomerans, and Pseudomonas azotoformans. In the June 1999 sample, mainly Kocuria rosea and various Bacillus spp. dominated. In declining stands of September 1998, a saprotrophic community was found: Acinetobacter spp., Aeromonas hydrophila, Curtobacterium luteum, Agrobacterium vitis, and two further groups representing presumably new taxa. In June 1999, reed rhizomes were colonised by Kocuria rosea, but Dietzia maris and Bacillus cohnii could be isolated as well. Healthy and declining reed stand rhizomes can be distinguished based on the culturable bacterial community. No obligately plant pathogenic bacteria were found, however the possibility of a local, opportunistic bacterial invasion can not be ruled out (e.g. Curtobacterium). The presence of potentially beneficial bacterial species was demonstrated in the healthy reed rhizome rhizosphere (e.g. Pseudomonas azotoformans, Pantoea agglomerans).

     

Single-nucleotide primer extension assay for detection and sequence typing of "Dehalococcoides" spp

A single-nucleotide primer extension (SNuPE) assay in combination with taxon-specific 16S rRNA gene PCR analysis was developed for the detection and typing of populations of the genus "Dehalococcoides". The specificity of the assay was evaluated with 16S rRNA gene sequences obtained from an isolate and an environmental sample representing two Dehalococcoides subgroups, i.e., the Cornell and the Pinellas subgroups. Only one sequence type, belonging to the Pinellas subgroup, was detected in a Bitterfeld-Wolfen region aquifer containing chlorinated ethenes as the main contaminants. The three-primer hybridization assay thus provided a fast and easy-to-implement method for confirming the specificity of taxon-specific PCR and allowed rapid additional taxonomic classification into subgroups. This study demonstrates the great potential of SNuPE as a novel approach for rapid parallel detection of microorganisms and typing of different nucleic acid signature sequences from environmental samples.     

Evaluation of Single-Nucleotide Primer Extension for Detection and Typing of Phylogenetic Markers Used for Investigation of Microbial Communities

Single-nucleotide primer extension (SNuPE) is an emerging tool for parallel detection of DNA sequences of different target microorganisms. The specificity and sensitivity of the SNuPE method were assessed by performing single and multiplex reactions using defined template mixtures of 16S rRNA gene PCR products obtained from pure bacterial cultures. The mismatch discrimination potential of primer extension was investigated by introducing different single and multiple primer-target mismatches. The type and position of the mismatch had significant effects on the specificity of the assay. While a 3′-terminal mismatch has a considerable effect on the fidelity of the extension reaction, the internal mismatches influenced hybridization mostly by destabilizing the hybrid duplex. Thus, carefully choosing primer-mismatch positions should result in a high signal-to-noise ratio and prevent any nonspecific extension. Cyclic fluorescent labeling of the hybridized primers via extension also resulted in a significant increase in the detection sensitivity of the PCR. In multiplex reactions, the signal ratios detected after specific primer extension correlated with the original template ratios. In addition, reverse-transcribed 16S rRNA was successfully used as a nonamplified template to prove the applicability of SNuPE in a PCR-independent manner. In conclusion, this study demonstrates the great potential of SNuPE for simultaneous detection and typing of various nucleic acid sequences from both environmental and engineered samples.Fast detection, differentiation, and identification of bacteria are crucial tasks in clinical, food, and environmental microbiology. Cultivation-independent tools not only save time compared to cultivation-based techniques but also allow access to the difficult-to-cultivate part of a microbial community. Molecular detection methods are usually based on hybridization of oligonucleotide probes to signature sequences (phylogenetically informative regions) in the nucleic acids (RNA or DNA) of the target microorganisms. Verification of the hybridization event can be accomplished by detection of hybridized labeled probes in situ (e.g., fluorescence in situ hybridization [FISH]) or ex situ (dot blot hybridization). Combining two specific oligonucleotides in a PCR increases the sensitivity of specific detection, while real-time monitoring of the amplification product formed allows quantification of the original template (for a review, see reference 17). Multiple detection can be achieved by using more than one primer pair targeting several loci in multiplex PCR assays (for a review, see reference 32). However, the main disadvantages of FISH are its restricted capability for parallel analysis of several target groups in the same sample and limitations in probe design due to differences in accessibility of the probes to their target sites (3, 7). Moreover, detection of slowly growing bacteria with low ribosome contents requires labor-intensive techniques (30, 36). Multiplex PCR also has limitations for multiplexing and challenges for primer design (32).Recently, single-nucleotide primer extension (SNuPE) was proposed as a fast, semiquantitative multiplex detection tool for analyzing sequence variants. This method is frequently used for determination of single-nucleotide polymorphisms and benefits from the fidelity of dideoxynucleoside triphosphate (ddNTP) incorporation catalyzed by a DNA polymerase. When primer extension takes place on a solid support, the method is called minisequencing (35, 37), while a reaction in solution is referred to as SNuPE (34) or single-base extension (15). These methods were originally developed for routine medical diagnosis of genetic disorders (23, 35) or for use in forensic research (38). Different versions of the primer extension technique have also been used recently for fast identification and genotyping of microbial strains (9, 31). Recent studies showed that detection of a hybridization event via labeling of the hybridized primer in the extension reaction is possible. However, the use of this method as a detection tool in applied and environmental microbiology has not been fully exploited so far. Rudi and coworkers were the first workers who used a minisequencing approach with PCR products from environmental DNA to detect toxic cyanobacteria by labeling only one of the four ddNTPs used in the reaction (27). Multiplexing was accomplished by hybridizing the labeled products to complementary oligonucleotides in an array format. In combination with antibody-based chromogenic visualization, genetic profiles of cyanobacterial diversity (28), microbial communities in vegetable salads (25), and Listeria strains (26) were obtained. However, this approach is labor-intensive and time-consuming and requires specific equipment; furthermore, the primer is restricted to certain positions since only one terminator nucleotide is labeled.An alternative strategy for multiplexing in solution benefits from incorporation of four differently labeled ddNTPs and attachment of mobility modifiers to the different primers. Subsequent separation using capillary electrophoresis and laser-induced fluorescence detection results in a very fast assay that is easy to interpret. Determination of the incorporated nucleotide provides additional proof of the assay specificity or may even provide extra phylogenetic information. The first application of primer extension with four differently labeled ddNTPs in environmental microbiology was the use of this method by Wu and Liu (41) for multiplex detection of different Bacteroides spp. This study also addressed different methodological issues and aspects, such as the effects of noncomplementary tail length, annealing temperature, cycle number, and primer-to-template ratio on extension efficiency. In a previous study, Nikolausz et al. (19) reported development and application of a multiplex SNuPE assay for detection and typing of “Dehalococcoides” sp. sequences obtained from chloroethene-contaminated groundwater samples. However, there still has not been a systematic evaluation of factors that affect primer design and the discriminatory power of primer extension. Moreover, quantitative aspects of the method have not been thoroughly addressed so far.The present study focused on these crucial issues by investigating the effects of the type, number, and position of primer mismatches on the extension efficiency and hence the specificity. Furthermore, quantitative aspects of SNuPE were investigated in a model community experiment by using defined template mixtures of 16S rRNA gene PCR products or reverse-transcribed RNA.     

Observation of bias associated with re-amplification of DNA isolated from denaturing gradient gels

DNA from environmental PCR products separated by denaturing gradient gel electrophoresis (DGGE) was isolated from the background smear rather than from discrete bands of the DGGE gel. The "interband" region was considered as a potential source of less dominant members of natural microbial communities. Surprisingly, instead of detecting new bands from the re-amplified PCR products, patterns very similar to the original ones were obtained regardless of the position of the "interband" region. The results suggest that the separation of amplicons by DGGE may not be perfect and band re-amplification based sequence analyses need careful interpretation.     

Enrichment of lignocellulose-degrading microbial communities from natural and engineered methanogenic environments

The aim of this study was to develop an effective bioaugmentation concept for anaerobic digesters treating lignocellulosic biomass such as straw. For that purpose, lignocellulose-degrading methanogenic communities were enriched on wheat straw from cow and goat rumen fluid as well as from a biogas reactor acclimated to lignocellulosic biomass (sorghum as mono-substrate). The bacterial communities of the enriched cultures and the different inocula were examined by 454 amplicon sequencing of 16S rRNA genes while the methanogenic archaeal communities were analyzed by terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of the mcrA gene. Bacteroidetes was the most abundant phylum in all samples. Within the Bacteroidetes phylum, Bacteroidaceae was the most abundant family in the rumen-derived enrichment cultures, whereas Porphyromonadaceae was the predominant one in the reactor-derived culture. Additionally, the enrichment procedure increased the relative abundance of Ruminococcaceae (phylum: Firmicutes) in all cultures. T-RFLP profiles of the mcrA gene amplicons highlighted that the ruminal methanogenic communities were composed of hydrogenotrophic methanogens dominated by the order Methanobacteriales regardless of the host species. The methanogenic communities changed significantly during the enrichment procedure, but still the strict hydrogenotrophic Methanobacteriales and Methanomicrobiales were the predominant orders in the enrichment cultures. The bioaugmentation potential of the enriched methanogenic cultures will be evaluated in further studies.