Identification of a Chlorodibenzo-p-dioxin Dechlorinating Dehalococcoides mccartyi by Stable Isotope Probing.

Polychlorinated dibenzo-p-dioxins (PCDDs) are released into the environment from a variety of both anthropogenic and natural sources. While highly chlorinated dibenzo-p-dioxins are persistent under oxic conditions, in anoxic environments, these organohalogens can be reductively dechlorinated to less chlorinated compounds that are then more amenable to subsequent aerobic degradation. Identifying the microorganisms responsible for dechlorination is an important step in developing bioremediation approaches. In this study, we demonstrated the use of a DNA-stable isotope probing (SIP) approach to identify the bacteria active in dechlorination of PCDDs in river sediments, with 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TeCDD) as a model. In addition, pyrosequencing of reverse transcribed 16S rRNA of TeCDD dechlorinating enrichment cultures was used to reveal active members of the bacterial community. A set of operational taxonomic units (OTUs) responded positively to the addition of 1,2,3,4-TeCDD in SIP microcosms assimilating 13C-acetate as the carbon source. Analysis of bacterial community profiles of the 13C labeled heavy DNA fraction revealed that an OTU corresponding to Dehalococcoides mccartyi accounted for a significantly greater abundance in cultures amended with 1,2,3,4-TeCDD than in cultures without 1,2,3,4-TeCDD. This implies the involvement of this Dehalococcoides mccartyi strain in the reductive dechlorination of 1,2,3,4-TeCDD and suggests the applicability of SIP for a robust assessment of the bioremediation potential of organohalogen contaminated sites.

Identification of a Ruminococcaceae Species as the Methyl tert-Butyl Ether (MTBE) Degrading Bacterium in a Methanogenic Consortium.

The widespread use of methyl tert-butyl ether (MTBE) has caused major contamination of groundwater sources and is a concern due to its taste and odor problems, as well as its toxicity. MTBE can be degraded anaerobically which makes bioremediation of contaminated aquifers a potential solution. Nevertheless, the organisms and mechanisms that are responsible for anaerobic MTBE degradation are still unknown. The aim of our research was to identify the organisms actively degrading MTBE. For this purpose we characterized an anaerobic methanogenic culture enriched with MTBE as the sole carbon source from the New Jersey Arthur Kill intertidal strait sediment. The cultures were analyzed using stable isotope probing (SIP) combined with terminal restriction fragment length polymorphism (T-RFLP), high-throughput sequencing and clone library analysis of bacterial 16S rRNA genes. The sequence data indicated that phylotypes belonging to the Ruminococcaceae in the Firmicutes were predominant in the methanogenic cultures. SIP experiments also showed sequential incorporation of the (13)C labeled MTBE by the bacterial community with a bacterium most closely related to Saccharofermentans acetigenes identified as the bacterium active in O-demethylation of MTBE. Identification of the microorganisms responsible for the activity will help us better understand anaerobic MTBE degradation processes in the field and determine biomarkers for monitoring natural attenuation.

Identification of Anaerobic Aniline-Degrading Bacteria at a Contaminated Industrial Site.

Anaerobic aniline biodegradation was investigated under different electron-accepting conditions using contaminated canal and groundwater aquifer sediments from an industrial site. Aniline loss was observed in nitrate- and sulfate-amended microcosms and in microcosms established to promote methanogenic conditions. Lag times of 37 days (sulfate amended) to more than 100 days (methanogenic) were observed prior to activity. Time-series DNA-stable isotope probing (SIP) was used to identify bacteria that incorporated (13)C-labeled aniline in the microcosms established to promote methanogenic conditions. In microcosms from heavily contaminated aquifer sediments, a phylotype with 92.7% sequence similarity to Ignavibacterium album was identified as a dominant aniline degrader as indicated by incorporation of (13)C-aniline into its DNA. In microcosms from contaminated canal sediments, a bacterial phylotype within the family Anaerolineaceae, but without a match to any known genus, demonstrated the assimilation of (13)C-aniline. Acidovorax spp. were also identified as putative aniline degraders in both of these two treatments, indicating that these species were present and active in both the canal and aquifer sediments. There were multiple bacterial phylotypes associated with anaerobic degradation of aniline at this complex industrial site, which suggests that anaerobic transformation of aniline is an important process at the site. Furthermore, the aniline degrading phylotypes identified in the current study are not related to any known aniline-degrading bacteria. The identification of novel putative aniline degraders expands current knowledge regarding the potential fate of aniline under anaerobic conditions.

Phase preference by active, acetate-utilizing bacteria at the rifle, CO integrated field research challenge site.

Previous experiments at the Rifle, Colorado Integrated Field Research Challenge (IFRC) site demonstrated that field-scale addition of acetate to groundwater reduced the ambient soluble uranium concentration. In this report, sediment samples collected before and after acetate field addition were used to assess the active microbes via (13)C acetate stable isotope probing on 3 phases [coarse sand, fines (8-approximately 150 µm), groundwater (0.2-8 µm)] over a 24-day time frame. TRFLP results generally indicated a stronger signal in (13)C-DNA in the "fines" fraction compared to the sand and groundwater. Before the field-scale acetate addition, a Geobacter-like group primarily synthesized (13)C-DNA in the groundwater phase, an alpha Proteobacterium primarily grew on the fines/sands, and an Acinetobacter sp. and Decholoromonas-like OTU utilized much of the (13)C acetate in both groundwater and particle-associated phases. At the termination of the field-scale acetate addition, the Geobacter-like species was active on the solid phases rather than the groundwater, while the other bacterial groups had very reduced newly synthesized DNA signal. These findings will help to delineate the acetate utilization patterns of bacteria in the field and can lead to improved methods for stimulating distinct microbial populations in situ.

Detection of 2,4,6-trinitrotoluene-utilizing anaerobic bacteria by 15N and 13C incorporation.

2,4,6-Trinitrotoluene ((15)N or (13)C labeled) was added to Norfolk Harbor sediments to test whether anaerobic bacteria use TNT for growth. Stable-isotope probing (SIP)-terminal restriction fragment length polymorphism (TRFLP) detected peaks in the [(15)N]TNT cultures (60, 163, and 168 bp). The 60-bp peak was also present in the [(13)C]TNT cultures and was related to Lysobacter taiwanensis.

Species-level evaluation of the human respiratory microbiome.

BACKGROUND: Changes to human respiratory tract microbiome may contribute significantly to the progression of respiratory diseases. However, there are few studies examining the relative abundance of microbial communities at the species level along the human respiratory tract. FINDINGS: Bronchoalveolar lavage, throat swab, mouth rinse, and nasal swab samples were collected from 5 participants. Bacterial ribosomal operons were sequenced using the Oxford Nanopore MinION to determine the relative abundance of bacterial species in 4 compartments along the respiratory tract. More than 1.8 million raw operon reads were obtained from the participants with ∼600,000 rRNA reads passing quality assurance/quality control (70-95% identify; >1,200 bp alignment) by Discontiguous MegaBLAST against the EZ BioCloud 16S rRNA gene database. Nearly 3,600 bacterial species were detected overall (>750 bacterial species within the 5 dominant phyla: Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Fusobacteria. The relative abundance of bacterial species along the respiratory tract indicated that most microbes (95%) were being passively transported from outside into the lung. However, a small percentage (<5%) of bacterial species were at higher abundance within the lavage samples. The most abundant lung-enriched bacterial species were Veillonella dispar and Veillonella atypica while the most abundant mouth-associated bacterial species were Streptococcus infantis and Streptococcus mitis. CONCLUSIONS: Most bacteria detected in lower respiratory samples do not seem to colonize the lung. However, >100 bacterial species were found to be enriched in bronchoalveolar lavage samples (compared to mouth/nose) and may play a substantial role in lung health.

Arctic tundra soil bacterial communities active at subzero temperatures detected by stable isotope probing.

Arctic soils store vast amounts of carbon and are subject to intense climate change. While the effects of thaw on the composition and activities of Arctic tundra microorganisms has been examined extensively, little is known about the consequences of temperature fluctuations within the subzero range in seasonally frozen or permafrost soils. This study identified tundra soil bacteria active at subzero temperatures using stable isotope probing (SIP). Soils from Kilpisjärvi, Finland, were amended with 13C-cellobiose and incubated at 0, -4 and -16°C for up to 40 weeks. 16S rRNA gene sequence analysis of 13C-labelled DNA revealed distinct subzero-active bacterial taxa. The SIP experiments demonstrated that diverse bacteria, including members of Candidatus Saccharibacteria, Melioribacteraceae, Verrucomicrobiaceae, Burkholderiaceae, Acetobacteraceae, Armatimonadaceae and Planctomycetaceae, were capable of synthesising 13C-DNA at subzero temperatures. Differences in subzero temperature optima were observed, for example, with members of Oxalobacteraceae and Rhizobiaceae found to be more active at 0°C than at -4°C or -16°C, whereas Melioribacteriaceae were active at all subzero temperatures tested. Phylogeny of 13C-labelled 16S rRNA genes from the Melioribacteriaceae, Verrucomicrobiaceae and Candidatus Saccharibacteria suggested that these taxa formed subzero-active clusters closely related to members from other cryo-environments. This study demonstrates that subzero temperatures impact active bacterial community composition and activity, which may influence biogeochemical cycles.

Characterization of nitrifying, denitrifying, and overall bacterial communities in permeable marine sediments of the northeastern Gulf of Mexico.

Sandy or permeable sediment deposits cover the majority of the shallow ocean seafloor, and yet the associated bacterial communities remain poorly described. The objective of this study was to expand the characterization of bacterial community diversity in permeable sediment impacted by advective pore water exchange and to assess effects of spatial, temporal, hydrodynamic, and geochemical gradients. Terminal restriction fragment length polymorphism (TRFLP) was used to analyze nearly 100 sediment samples collected from two northeastern Gulf of Mexico subtidal sites that primarily differed in their hydrodynamic conditions. Communities were described across multiple taxonomic levels using universal bacterial small subunit (SSU) rRNA targets (RNA- and DNA-based) and functional markers for nitrification (amoA) and denitrification (nosZ). Clonal analysis of SSU rRNA targets identified several taxa not previously detected in sandy sediments (i.e., Acidobacteria, Actinobacteria, Chloroflexi, Cyanobacteria, and Firmicutes). Sequence diversity was high among the overall bacterial and denitrifying communities, with members of the Alphaproteobacteria predominant in both. Diversity of bacterial nitrifiers (amoA) remained comparatively low and did not covary with the other gene targets. TRFLP fingerprinting revealed changes in sequence diversity from the family to species level across sediment depth and study site. The high diversity of facultative denitrifiers was consistent with the high permeability, deeper oxygen penetration, and high rates of aerobic respiration determined in these sediments. The high relative abundance of Gammaproteobacteria in RNA clone libraries suggests that this group may be poised to respond to short-term periodic pulses of growth substrates, and this observation warrants further investigation.

Discerning autotrophy, mixotrophy and heterotrophy in marine TACK archaea from the North Atlantic.

DNA stable isotope probing (SIP) was used to track the uptake of organic and inorganic carbon sources for TACK archaea (Thaumarchaeota/Aigarchaeota/Crenarchaeota/Korarchaeota) on a cruise of opportunity in the North Atlantic. Due to water limitations, duplicate samples from the deep photic (60-115 m), the mesopelagic zones (local oxygen minimum; 215-835 m) and the bathypelagic zone (2085-2835 m) were amended with various combinations of 12C- or 13C-acetate/urea/bicarbonate to assess cellular carbon acquisition. The SIP results indicated the majority of TACK archaeal operational taxonomic units (OTUs) incorporated 13C from acetate and/or urea into newly synthesized DNA within 48 h. A small fraction (16%) of the OTUs, often representing the most dominant members of the archaeal community, were able to incorporate bicarbonate in addition to organic substrates. Only two TACK archaeal OTUs were found to incorporate bicarbonate but not urea or acetate. These results further demonstrate the utility of SIP to elucidate the metabolic capability of mesothermal archaea in distinct oceanic settings and suggest that TACK archaea play a role in organic carbon recycling in the mid-depth to deep ocean.

Sulfate-Reducing Naphthalene Degraders Are Picky Eaters.

Polycyclic aromatic hydrocarbons (PAHs) are common organic contaminants found in anoxic environments. The capacity for PAH biodegradation in unimpacted environments, however, has been understudied. Here we investigate the enrichment, selection, and sustainability of a microbial community from a pristine environment on naphthalene as the only amended carbon source. Pristine coastal sediments were obtained from the Jacques Cousteau National Estuarine Research Reserve in Tuckerton, New Jersey, an ecological reserve which has no direct input or source of hydrocarbons. After an initial exposure to naphthalene, primary anaerobic transfer cultures completely degraded 500 µM naphthalene within 139 days. Subsequent transfer cultures mineralized naphthalene within 21 days with stoichiometric sulfate loss. Enriched cultures efficiently utilized only naphthalene and 2-methylnaphthalene from the hydrocarbon mixtures in crude oil. To determine the microorganisms responsible for naphthalene degradation, stable isotope probing was utilized on cultures amended with fully labeled 13C-naphthalene as substrate. Three organisms were found to unambiguously synthesize 13C-DNA from 13C-naphthalene within 7 days. Phylogenetic analysis revealed that 16S rRNA genes from two of these organisms are closely related to the known naphthalene degrading isolates NaphS2 and NaphS3 from PAH-contaminated sites. A third 16S rRNA gene was only distantly related to its closest relative and may represent a novel naphthalene degrading microbe from this environment.

Profiling bacterial communities by MinION sequencing of ribosomal operons.

BACKGROUND: An approach utilizing the long-read capability of the Oxford Nanopore MinION to rapidly sequence bacterial ribosomal operons of complex natural communities was developed. Microbial fingerprinting employs domain-specific forward primers (16S rRNA subunit), reverse primers (23S rRNA subunit), and a high-fidelity Taq polymerase with proofreading capabilities. Amplicons contained both ribosomal subunits for broad-based phylogenetic assignment (~ 3900 bp of sequence), plus the intergenic spacer (ITS) region (~ 300 bp) for potential strain-specific identification. RESULTS: To test the approach, bacterial rRNA operons (~ 4200 bp) were amplified from six DNA samples employing a mixture of farm soil and bioreactor DNA in known concentrations. Each DNA sample mixture was barcoded, sequenced in quadruplicate (n = 24), on two separate 6-h runs using the MinION system (R7.3 flow cell; MAP005 and 006 chemistry). From nearly 90,000 MinION reads, roughly 33,000 forward and reverse sequences were obtained. This yielded over 10,000 2D sequences which were analyzed using a simplified data analysis pipeline based on NCBI Blast and assembly with Geneious software. The method could detect over 1000 operational taxonomic units in the sample sets in a quantitative manner. Global sequence coverage for the various rRNA operons ranged from 1 to 1951x. An iterative assembly scheme was developed to reconstruct those rRNA operons with > 35x coverage from a set of 30 operational taxonomic units (OTUs) among the Proteobacteria, Actinobacteria, Acidobacteria, Firmicutes, and Gemmatimonadetes. Phylogenetic analysis of the 16S rRNA and 23S rRNA genes from each operon demonstrated similar tree topologies with species/strain-level resolution. CONCLUSIONS: This sequencing method represents a cost-effective way to profile microbial communities. Because the MinION is small, portable, and runs on a laptop, the possibility of microbiota characterization in the field or on robotic platforms becomes realistic.

Comparison of a modified phenol/chloroform and commercial-kit methods for extracting DNA from horse fecal material.

There are many choices for methods of extracting bacterial DNA for Next Generation Sequencing (NGS) from fecal samples. Here, we compare our modifications of a phenol/chloroform extraction method plus an inhibitor removal solution (C3) (ph/Chl+C3) to the PowerFecal® DNA Isolation Kit (MoBio-K). DNA quality and quantity coupled to NGS results were used to assess differences in relative abundance, Shannon diversity index, unique species, and principle coordinate analysis (PCoA) between biological replicates. Six replicate samples, taken from a single ball of horse feces manually collected from the rectum, were subjected to each extraction method. The Ph/Chl+C3 method produced 100× higher DNA yields with less shearing than the MoBio-K method. To assess the methods, the two method samples were sent for sequencing of the bacterial V3-V4 region of 16S rRNA gene using the Illumina MiSeq platform. The relative abundance of Bacteroidetes was greater and there were more unique species assigned to this group in MoBio-K than in Ph/Chl+C3 (P<0.05). In contrast, Firmicutes had greater relative abundance and more unique species in Ph/Chl+C3 extracts than in MoBio-K (P<0.05). The other major bacterial phyla were equally abundant in samples using both extraction methods. Alpha diversity and Shannon Weaver indices showed greater evenness of bacterial distribution in Ph/Chl+C3 compared with MoBio-K (P<0.05), but there was no difference in the OTU richness. Principle coordinate analysis (PCoA) indicated a distinct separation between the two methods (P<0.05) and tighter clustering (less variability) in Ph/Chl+C3 than in MoBio-K. These results suggest that the Ph/Chl+C3 may be preferred for research to identify specific Firmicutes taxa such as Clostridium, and Bacillus. However; MoBio-K may be a better choice for projects focusing on Bacteroidetes abundance. The Ph/Chl+C3 method required less time, but has some safety concerns associated with exposure and disposal of phenol and chloroform. While the MoBio-K may be better choice for researchers with less access to safety equipment like a fume hood.

The Effect of Diet and Exercise on Intestinal Integrity and Microbial Diversity in Mice.

BACKGROUND: The gut microbiota is now known to play an important role contributing to inflammatory-based chronic diseases. This study examined intestinal integrity/inflammation and the gut microbial communities in sedentary and exercising mice presented with a normal or high-fat diet. METHODS: Thirty-six, 6-week old C57BL/6NTac male mice were fed a normal or high-fat diet for 12-weeks and randomly assigned to exercise or sedentary groups. After 12 weeks animals were sacrificed and duodenum/ileum tissues were fixed for immunohistochemistry for occludin, E-cadherin, and cyclooxygenase-2 (COX-2). The bacterial communities were assayed in fecal samples using terminal restriction fragment length polymorphism (TRFLP) analysis and pyrosequencing of 16S rRNA gene amplicons. RESULTS: Lean sedentary (LS) mice presented normal histologic villi while obese sedentary (OS) mice had similar villi height with more than twice the width of the LS animals. Both lean (LX) and obese exercise (OX) mice duodenum and ileum were histologically normal. COX-2 expression was the greatest in the OS group, followed by LS, LX and OX. The TRFLP and pyrosequencing indicated that members of the Clostridiales order were predominant in all diet groups. Specific phylotypes were observed with exercise, including Faecalibacterium prausnitzi, Clostridium spp., and Allobaculum spp. CONCLUSION: These data suggest that exercise has a strong influence on gut integrity and host microbiome which points to the necessity for more mechanistic studies of the interactions between specific bacteria in the gut and its host.

BioDry: An Inexpensive, Low-Power Method to Preserve Aquatic Microbial Biomass at Room Temperature.

This report describes BioDry (patent pending), a method for reliably preserving the biomolecules associated with aquatic microbial biomass samples, without the need of hazardous materials (e.g. liquid nitrogen, preservatives, etc.), freezing, or bulky storage/sampling equipment. Gel electrophoresis analysis of nucleic acid extracts from samples treated in the lab with the BioDry method indicated that molecular integrity was protected in samples stored at room temperature for up to 30 days. Analysis of 16S/18S rRNA genes for presence/absence and relative abundance of microorganisms using both 454-pyrosequencing and TRFLP profiling revealed statistically indistinguishable communities from control samples that were frozen in liquid nitrogen immediately after collection. Seawater and river water biomass samples collected with a portable BioDry "field unit", constructed from off-the-shelf materials and a battery-operated pumping system, also displayed high levels of community rRNA preservation, despite a slight decrease in nucleic acid recovery over the course of storage for 30 days. Functional mRNA and protein pools from the field samples were also effectively conserved with BioDry, as assessed by respective RT-PCR amplification and western blot of ribulose-1-5-bisphosphate carboxylase/oxygenase. Collectively, these results demonstrate that BioDry can adequately preserve a suite of biomolecules from aquatic biomass at ambient temperatures for up to a month, giving it great potential for high resolution sampling in remote locations or on autonomous platforms where space and power are limited.

Identification of Bacteria Synthesizing Ribosomal RNA in Response to Uranium Addition During Biostimulation at the Rifle, CO Integrated Field Research Site.

Understanding which organisms are capable of reducing uranium at historically contaminated sites provides crucial information needed to evaluate treatment options and outcomes. One approach is determination of the bacteria which directly respond to uranium addition. In this study, uranium amendments were made to groundwater samples from a site of ongoing biostimulation with acetate. The active microbes in the planktonic phase were deduced by monitoring ribosomes production via RT-PCR. The results indicated several microorganisms were synthesizing ribosomes in proportion with uranium amendment up to 2 µM. Concentrations of U (VI) >2 µM were generally found to inhibit ribosome synthesis. Two active bacteria responding to uranium addition in the field were close relatives of Desulfobacter postgateii and Geobacter bemidjiensis. Since RNA content often increases with growth rate, our findings suggest it is possible to rapidly elucidate active bacteria responding to the addition of uranium in field samples and provides a more targeted approach to stimulate specific populations to enhance radionuclide reduction in contaminated sites.

Spatial distribution of an uranium-respiring betaproteobacterium at the Rifle, CO field research site.

The Department of Energy's Integrated Field-Scale Subsurface Research Challenge Site (IFRC) at Rifle, Colorado was created to address the gaps in knowledge on the mechanisms and rates of U(VI) bioreduction in alluvial sediments. Previous studies at the Rifle IFRC have linked microbial processes to uranium immobilization during acetate amendment. Several key bacteria believed to be involved in radionuclide containment have been described; however, most of the evidence implicating uranium reduction with specific microbiota has been indirect. Here, we report on the cultivation of a microorganism from the Rifle IFRC that reduces uranium and appears to utilize it as a terminal electron acceptor for respiration with acetate as electron donor. Furthermore, this bacterium constitutes a significant proportion of the subsurface sediment community prior to biostimulation based on TRFLP profiling of 16S rRNA genes. 16S rRNA gene sequence analysis indicates that the microorganism is a betaproteobacterium with a high similarity to Burkholderia fungorum. This is, to our knowledge, the first report of a betaproteobacterium capable of uranium respiration. Our results indicate that this microorganism occurs commonly in alluvial sediments located between 3-6 m below ground surface at Rifle and may play a role in the initial reduction of uranium at the site.

Crenarchaeal heterotrophy in salt marsh sediments.

Mesophilic Crenarchaeota (also known as Thaumarchaeota) are ubiquitous and abundant in marine habitats. However, very little is known about their metabolic function in situ. In this study, salt marsh sediments from New Jersey were screened via stable isotope probing (SIP) for heterotrophy by amending with a single (13)C-labeled compound (acetate, glycine or urea) or a complex (13)C-biopolymer (lipids, proteins or growth medium (ISOGRO)). SIP incubations were done at two substrate concentrations (30-150 µM; 2-10 mg ml(-1)), and (13)C-labeled DNA was analyzed by terminal restriction fragment length polymorphism (TRFLP) analysis of 16S rRNA genes. To test for autotrophy, an amendment with (13)C-bicarbonate was also performed. Our SIP analyses indicate salt marsh crenarchaea are heterotrophic, double within 2-3 days and often compete with heterotrophic bacteria for the same organic substrates. A clone library of (13)C-amplicons was screened to find matches to the (13)C-TRFLP peaks, with seven members of the Miscellaneous Crenarchaeal Group and seven members from the Marine Group 1.a Crenarchaeota being discerned. Some of these crenarchaea displayed a preference for particular carbon sources, whereas others incorporated nearly every (13)C-substrate provided. The data suggest salt marshes may be an excellent model system for studying crenarchaeal metabolic capabilities and can provide information on the competition between crenarchaea and other microbial groups to improve our understanding of microbial ecology.

Bacterial genome replication at subzero temperatures in permafrost.

Microbial metabolic activity occurs at subzero temperatures in permafrost, an environment representing ∼25% of the global soil organic matter. Although much of the observed subzero microbial activity may be due to basal metabolism or macromolecular repair, there is also ample evidence for cellular growth. Unfortunately, most metabolic measurements or culture-based laboratory experiments cannot elucidate the specific microorganisms responsible for metabolic activities in native permafrost, nor, can bulk approaches determine whether different members of the microbial community modulate their responses as a function of changing subzero temperatures. Here, we report on the use of stable isotope probing with (13)C-acetate to demonstrate bacterial genome replication in Alaskan permafrost at temperatures of 0 to -20 °C. We found that the majority (80%) of operational taxonomic units detected in permafrost microcosms were active and could synthesize (13)C-labeled DNA when supplemented with (13)C-acetate at temperatures of 0 to -20 °C during a 6-month incubation. The data indicated that some members of the bacterial community were active across all of the experimental temperatures, whereas many others only synthesized DNA within a narrow subzero temperature range. Phylogenetic analysis of (13)C-labeled 16S rRNA genes revealed that the subzero active bacteria were members of the Acidobacteria, Actinobacteria, Chloroflexi, Gemmatimonadetes and Proteobacteria phyla and were distantly related to currently cultivated psychrophiles. These results imply that small subzero temperature changes may lead to changes in the active microbial community, which could have consequences for biogeochemical cycling in permanently frozen systems.

Replicability of bacterial communities in denitrifying bioreactors as measured by PCR/T-RFLP analysis.

Bioreactors hold great promise for treating graywater in an advanced life support system for space applications. However, questions remain regarding the reproducibility and reliability of biological systems for long-term use. Although there have been numerous studies on ground-based biological systems, most studies focus on a single reactor or a simple (single carbon) waste stream. There have been very few studies on microbial communities in replicate reactors using a nonsterile, complex waste stream. In this report, we describe the characterization of five replicate denitrifying reactors receiving a complex feed, including urine and limb washes from donors at Johnson Space Center over a 100-day period. Denitrifying conditions were employed because of the ease in adding a terminal electron acceptor to the bioreactor. Bacterial populations were tracked by 16S rRNA and nosZ genes T-RFLP analysis to target the total and denitrifying microbial communities. The results demonstrated reproducible biological communities with nearly identical performance that slowly changed with time and exhibited low variability with respect to the bacterial community (T-RFLP peak area) in all reactors. These results suggest that, when designed for replication, bioreactors are not stochastic systems exhibiting chaotic behavior, but are biological systems that can be highly reproducible and reliable.