Detection and Quantification of Functional Genes of Cellulose- Degrading,Fermentative, and Sulfate-Reducing Bacteria and Methanogenic Archaea

Cellulose degradation, fermentation, sulfate reduction, and methanogenesis are microbial processes that coexist in a variety of natural and engineered anaerobic environments. Compared to the study of 16S rRNA genes, the study of the genes encoding the enzymes responsible for these phylogenetically diverse functions is advantageous because it provides direct functional information. However, no methods are available for the broad quantification of these genes from uncultured microbes characteristic of complex environments. In this study, consensus degenerate hybrid oligonucleotide primers were designed and validated to amplify both sequenced and unsequenced glycoside hydrolase genes of cellulose-degrading bacteria, hydA genes of fermentative bacteria, dsrA genes of sulfate-reducing bacteria, and mcrA genes of methanogenic archaea. Specificity was verified in silico and by cloning and sequencing of PCR products obtained from an environmental sample characterized by the target functions. The primer pairs were further adapted to quantitative PCR (Q-PCR), and the method was demonstrated on samples obtained from two sulfate-reducing bioreactors treating mine drainage, one lignocellulose based and the other ethanol fed. As expected, the Q-PCR analysis revealed that the lignocellulose-based bioreactor contained higher numbers of cellulose degraders, fermenters, and methanogens, while the ethanol-fed bioreactor was enriched in sulfate reducers. The suite of primers developed represents a significant advance over prior work, which, for the most part, has targeted only pure cultures or has suffered from low specificity. Furthermore, ensuring the suitability of the primers for Q-PCR provided broad quantitative access to genes that drive critical anaerobic catalytic processes.The gene encoding the 16S small ribosomal subunit has served as a highly suitable target for studying bacterial species. When one obtains 16S rRNA gene sequence information, it is sometimes possible to infer function from an identical match to a well-characterized pure culture. More commonly, however, the similarity to pure cultures is low, and/or the highest similarities correspond to 16S rRNA gene sequences identified without isolation or phenotypic characterization. In either case, care must be taken, because distinct phenotypes [e.g., dissimilatory Fe(III) reduction, chlorate reduction] are found in microorganisms with highly similar (e.g., 99.5%) 16S rRNA gene sequences (1). In addition, 16S rRNA gene surveys of broad phylogenetic groups can be time-, labor-, and cost-intensive. For example, it is estimated that the 16S rRNA gene-based detection of all recognized lineages of sulfate-reducing bacteria (SRB) would require approximately 132 16S rRNA gene-targeted microarray probes (32).A more-direct approach for the study of microbes that span phylogenetic groups is to target them as a physiologically coherent guild by using specific genetic markers (functional genes) for the functions of interest. Functional genes have been successfully targeted in bioremediation studies to investigate microbial populations responsible for the degradation of various contaminants. Some examples include the use of the large alpha subunit of benzylsuccinate synthase to monitor anaerobic hydrocarbon-degrading bacteria (5), the monitoring of ars genes for the identification and quantification of arsenic-metabolizing bacteria (45), and the detection of catechol 1,2-dioxygenase in aromatic-hydrocarbon-degrading Rhodococcus spp. (48). In the field of mine drainage/metal remediation, functional genes have been used to target SRB (17, 26), but the methods have suffered both from a lack of broad specificity for SRB and from the inability to distinguish SRB from sulfur-oxidizing bacteria (SOB). A general challenge to the functional-gene approach has been the relative lack of characterization and unavailability of target sequences. As a consequence, the primer sets that are available tend to be more relevant to pure cultures than to complex environmental samples.Microbial communities in natural and engineered anaerobic environments that utilize cellulose as the primary carbon source, such as those in rumina (56), termite guts (54), decomposing wood (7), sulfate-reducing and methanogenic sediments (9, 22), wetlands (28), and sulfate-reducing bioreactors (26), are particularly challenging to characterize. 16S rRNA gene-based studies have revealed the complexity of these microbial communities and their high levels of phylogenetic and functional diversity. In such anaerobic environments, mineralization of complex organic matter occurs through the concerted action of a variety of microorganisms. Primary fermenters, such as cellulose degraders, break down the complex molecules and ferment the hydrolysis products. Secondary fermenters also ferment the hydrolysis products. When sulfate is available, SRB utilize the fermentation products as carbon and energy sources. In addition, methanogens can also utilize some of the fermentation products. In many cases, functionally important members, such as SRB, are present only as a small fraction of the community (36, 38), making them difficult to detect by use of 16S rRNA gene-targeted fingerprinting methods. Furthermore, the phylogenetic diversity of cellulose degraders, fermenters, and SRB prevents their quantification using a small number of 16S rRNA gene-targeted probes.In this study, degenerate PCR primers were developed, validated, and demonstrated for the amplification of key functional groups in anaerobic environments possessing genes encoding glycoside hydrolases of families 5 (collectively designated cel5 in this study) and 48 (collectively designated cel48 in this study) (cellulose degradation), the alpha subunit of iron hydrogenase (hydA) (fermentation), dissimilatory sulfite reductase (dsrA) (sulfate reduction), and methyl coenzyme M reductase (mcrA) (methanogenesis). This work is particularly novel considering that the vast majority of existing methods are suitable only for pure cultures, especially in the cases of cel5, cel48, and hydA (21, 44, 47). Thus, the approach provides access to uncultured and unsequenced markers, a critical feature for the study of key anaerobic processes in complex environments. Specificity was also enhanced where possible, notably in the case of dsrA, for which existing primers either do not distinguish SRB from SOB (14, 17) or have good alignment with only a narrow range of SRB (31, 52). Finally, all primers in this study were designed and validated for quantitative PCR (Q-PCR), in order to provide valuable quantitative functional information about complex anaerobic communities. The approach is demonstrated on mine drainage remediation systems and is expected to be of broad value to a variety of fields, including advancing the understanding of biohydrogen production, global carbon cycling, and other important biogeochemical processes.