Campylobacterota dominate the microbial communities in a tropical karst subterranean estuary, with implications for cycling and export of nitrogen to coastal waters.

Subterranean estuaries (STEs), the zones in which seawater and subsurface groundwater mix, are recognized as hotspots for biogeochemical reactions; however, little is known of the microbial communities that control many of those reactions. This study investigated the potential functions of microbes inhabiting a cenote and an offshore submarine spring (Pargos) in the near-coastal waters of the Yucatan Peninsula, Mexico. The inland cenote (Cenote Siete Bocas; C7B) is characterized by a chemocline that is host to an array of physicochemical gradients associated with microbial activities. The chemocline includes an increasing gradient in sulfide concentrations with depth and a decreasing gradient in nitrate concentrations. The microbial community within the chemocline was dominated by Sulfurimonas and Sulfurovum of the Campylobacteria, which are likely responsible for sulfide oxidation coupled with nitrate reduction. Although C7B has not been directly connected with Pargos Spring, water discharging from the spring has physicochemical characteristics and microbial community structures similar to C7B, strongly suggesting biogeochemical processing in the STE impacts groundwater composition prior to discharge. This work yields insight into the microbial communities and biogeochemical reactions in STEs in karstic aquifers and provides evidence for the importance of Campylobacteria in controlling nitrate concentrations exported to marine springs.

Azithromycin and Ciprofloxacin Can Promote Antibiotic Resistance in Biosolids and Biosolids-Amended Soils.

Spread of biosolids-borne antibiotic resistance is a growing public and environmental health concern. Herein, we conducted incubation experiments involving biosolids, which are byproducts of sewage treatment processes, and biosolids-amended soil. Quantitative reverse transcription-PCR (RT-qPCR) was employed to assess responses of select antibiotic resistance genes (ARGs) and mobile elements to environmentally relevant concentrations of two biosolids-borne antibiotics, azithromycin (AZ) and ciprofloxacin (CIP). Additionally, we examined sequence distribution of gyrA (encoding DNA gyrase; site of action of CIP) to assess potential shifts in genotype. Increasing antibiotic concentrations generally increased the transcriptional activities of qnrS (encoding CIP resistance) and ermB and mefE (encoding AZ resistance). The transcriptional activity of intl1, a marker of class 1 integrons, was unaffected by CIP or AZ concentrations, but biosolids amendment increased intl1 activity in the soil by 4 to 5 times, which persisted throughout incubation. While the dominant gyrA sequences found herein were unrelated to known CIP-resistant genotypes, the increasing CIP concentrations significantly decreased the diversity of genes encoding the DNA gyrase A subunit, suggesting changes in microbial community structures. This study suggests that biosolids harbor transcriptionally active ARGs and mobile elements that could survive and spread in biosolids-amended soils. However, more research is warranted to investigate these trends under field conditions. IMPORTANCE Although previous studies have indicated that biosolids may be important spreaders of antibiotics and antibiotic resistance genes (ARGs) in environments, the potential activities of ARGs or their responses to environmental parameters have been understudied. This study highlights that certain biosolids-borne antibiotics can induce transcriptional activities of ARGs and mobile genetic elements in biosolids and biosolids-amended soil, even when present at environmentally relevant concentrations. Furthermore, these antibiotics can alter the structure of microbial populations expressing ARGs. Our findings indicate the bioavailability of the antibiotics in biosolids and provide evidence that biosolids can promote the activities and dissemination of ARGs and mobile genes in biosolids and soils that receive contaminated biosolids, thus, underscoring the importance of investigating anthropogenically induced antibiotic resistance in the environment under real-world scenarios.

Periphyton and Flocculent Materials Are Important Ecological Compartments Supporting Abundant and Diverse Mercury Methylator Assemblages in the Florida Everglades.

Mercury (Hg) methylation in the Florida Everglades is of great environmental concern because of its adverse effects on human and wildlife health through biomagnification in aquatic food webs. Periphyton and flocculant materials (floc) overlaying peat soil are important ecological compartments producing methylmercury (MeHg) in this ecosystem. These compartments retain higher concentrations of MeHg than did soil at study sites across nutrient and/or sulfate gradient(s). To better understand what controls Hg methylation in these compartments, the present study explored the structures and abundances of Hg methylators using genes hgcAB as biomarkers. The hgcA sequences indicated that these compartments hosted a high diversity of Hg methylators, including Deltaproteobacteria, Chloroflexi, Firmicutes, and Methanomicrobia, with community compositions that differed between these habitats. The copy numbers of hgcAB quantified by quantitative PCR revealed that floc and soil supported higher numbers of Hg methylators than periphyton in the Everglades ecosystem. The abundance of Hg methylators was strongly positively correlated with concentrations of carbon and nutrients (e.g., phosphorus and nitrogen) according to redundancy analysis. Strong correlations were also observed among numbers of sulfate reducers, methanogens, and the dominant hgcAB-carrying groups, suggesting that hgcAB would spread primarily through the growth of those assemblages. The abundances of Hg methylators were weakly negatively correlated to MeHg concentrations, suggesting that the size of this population would not solely determine the final concentrations of MeHg in the ecological compartments studied. This study extends the knowledge regarding the distribution of diverse potential mercury methylators in different environmental compartments in a wetland of national concern.IMPORTANCE Methylmercury is a potent neurotoxin that impacts the health of humans and wildlife. Most mercury in wetlands such as the Florida Everglades enters as inorganic mercury via atmospheric deposition, some of which is transformed to the more toxic methylmercury through the activities of anaerobic microorganisms. We investigated the numbers and phylogenetic diversity of hgcAB, genes that are linked to mercury methylation, in the soil, floc, and periphyton in areas of the Everglades with different sulfate and nutrient concentrations. Soil harbored relatively high numbers of cells capable of methylating mercury; however, little detectable methylmercury was present in soil. The greatest concentrations of methylmercury were found in floc and periphyton. The dominant methylators in those compartments included methanogens and Syntrophobacteriales This work provides significant insight into the microbial processes that control methylation and form the basis for accumulation through the food chain in this important environment.

Methanogens Are Major Contributors to Nitrogen Fixation in Soils of the Florida Everglades.

The objective of this study was to investigate the interaction of the nitrogen (N) cycle with methane production in the Florida Everglades, a large freshwater wetland. This study provides an initial analysis of the distribution and expression of N-cycling genes in Water Conservation Area 2A (WCA-2A), a section of the marsh that underwent phosphorus (P) loading for many years due to runoff from upstream agricultural activities. The elevated P resulted in increased primary productivity and an N limitation in P-enriched areas. Results from quantitative real-time PCR (qPCR) analyses indicated that the N cycle in WCA-2A was dominated by nifH and nirK/S, with an increasing trend in copy numbers in P-impacted sites. Many nifH sequences (6 to 44% of the total) and nifH transcript sequences (2 to 49%) clustered with the methanogenic Euryarchaeota, in stark contrast to the proportion of core gene sequences representing Archaea (≤0.27% of SSU rRNA genes) for the WCA-2A microbiota. Notably, archaeal nifH gene transcripts were detected at all sites and comprised a significant proportion of total nifH transcripts obtained from the unimpacted site, indicating that methanogens are actively fixing N2 Laboratory incubations with soils taken from WCA-2A produced nifH transcripts with the production of methane from H2 plus CO2 and acetate as electron donors and carbon sources. Methanogenic N2 fixation is likely to be an important, although largely unrecognized, route through which fixed nitrogen enters the anoxic soils of the Everglades and may have significant relevance regarding methane production in wetlands.IMPORTANCE Wetlands are the most important natural sources of the greenhouse gas methane, and much of that methane emanates from (sub)tropical peatlands. Primary productivity in these peatlands is frequently limited by the availability of nitrogen or phosphorus; however, the response to nutrient limitations of microbial communities that control biogeochemical cycling critical to ecosystem function may be complex and may be associated with a range of processes, including methane production. We show that many, if not most, of the methanogens in the peatlands of the Florida Everglades possess the nifH gene and actively express it for N2 fixation coupled with methanogenesis. These findings indicate that archaeal N2 fixation would play crucial role in methane emissions and overall N cycle in subtropical wetlands suffering N limitation.

Distribution, activities, and interactions of methanogens and sulfate-reducing prokaryotes in the Florida Everglades.

To gain insight into the mechanisms controlling methanogenic pathways in the Florida Everglades, the distribution and functional activities of methanogens and sulfate-reducing prokaryotes (SRPs) were investigated in soils (0 to 2 or 0 to 4 cm depth) across the well-documented nutrient gradient in the water conservation areas (WCAs) caused by runoff from the adjacent Everglades Agricultural Area. The methyl coenzyme M reductase gene (mcrA) sequences that were retrieved from WCA-2A, an area with relatively high concentrations of SO4 (2-) (≥39 µM), indicated that methanogens inhabiting this area were broadly distributed within the orders Methanomicrobiales, Methanosarcinales, Methanocellales, Methanobacteriales, and Methanomassiliicoccales. In more than 3 years of monitoring, quantitative PCR (qPCR) using newly designed group-specific primers revealed that the hydrogenotrophic Methanomicrobiales were more numerous than the Methanosaetaceae obligatory acetotrophs in SO4 (2-)-rich areas of WCA-2A, while the Methanosaetaceae were dominant over the Methanomicrobiales in WCA-3A (with relatively low SO4 (2-) concentrations; ≤4 µM). qPCR of dsrB sequences also indicated that SRPs are present at greater numbers than methanogens in the WCAs. In an incubation study with WCA-2A soils, addition of MoO4 (2-) (a specific inhibitor of SRP activity) resulted in increased methane production rates, lower apparent fractionation factors [αapp; defined as (amount of δ(13)CO2 + 1,000)/(amount of δ(13)CH4 + 1,000)], and higher Methanosaetaceae mcrA transcript levels compared to those for the controls without MoO4 (2-). These results indicate that SRPs play crucial roles in controlling methanogenic pathways and in shaping the structures of methanogen assemblages as a function of position along the nutrient gradient.

Diversity and distribution of actinobacterial aromatic ring oxygenase genes across contrasting soil properties.

The diversity of a gene family encoding Actinobacterial aromatic ring oxygenases (AAROs) was detected by the PCR-cloning approach using a newly designed PCR primer set. The distribution of AAROs was investigated in 11 soils representing different land management and vegetation zones and was correlated with several geochemical parameters including pH, organic matter (OM), total Kjeldahl nitrogen (TKN), and nitrogen oxides (NO(x)-N: mostly NO3(-)-N). The distribution of individual clades encoding enzymes with potentially different substrates were correlated with different environmental factors, suggesting differential environmental controls on the distribution of specific enzymes as well as sequence diversity. For example, individual clades associated with phthalate dioxygenases were either strongly negatively correlated with pH, or not correlated with pH but showed strong positive correlation with organic carbon content. A large number of clones clustering in a clade related to PAH oxygenases were positively correlated with pH and nitrogen, but not with organic matter. This analysis may yield insight into the ecological forces driving the distribution of these catabolic genes.

Syntrophs dominate sequences associated with the mercury methylation-related gene hgcA in the water conservation areas of the Florida Everglades.

The mechanisms and rates of mercury methylation in the Florida Everglades are of great concern because of potential adverse impacts on human and wildlife health through mercury accumulation in aquatic food webs. We developed a new PCR primer set targeting hgcA, a gene encoding a corrinoid protein essential for Hg methylation across broad phylogenetic boundaries, and used this primer set to study the distribution of hgcA sequences in soils collected from three sites along a gradient in sulfate and nutrient concentrations in the northern Everglades. The sequences obtained were distributed in diverse phyla, including Proteobacteria, Chloroflexi, Firmicutes, and Methanomicrobia; however, hgcA clone libraries from all sites were dominated by sequences clustering within the order Syntrophobacterales of the Deltaproteobacteria (49 to 65% of total sequences). dsrB mRNA sequences, representing active sulfate-reducing prokaryotes at the time of sampling, obtained from these sites were also dominated by Syntrophobacterales (75 to 89%). Laboratory incubations with soils taken from the site low in sulfate concentrations also suggested that Hg methylation activities were primarily mediated by members of the order Syntrophobacterales, with some contribution by methanogens, Chloroflexi, iron-reducing Geobacter, and non-sulfate-reducing Firmicutes inhabiting the sites. This suggests that prokaryotes distributed within clades defined by syntrophs are the predominant group controlling methylation of Hg in low-sulfate areas of the Everglades. Any strategy for managing mercury methylation in the Everglades should consider that net mercury methylation is not limited to the action of sulfate reduction.

23S rRNA gene-based enterococci community signatures in Lake Pontchartrain, Louisiana, USA, following urban runoff inputs after Hurricane Katrina.

Little is known about the impacts of fecal polluted urban runoff inputs on the structure of enterococci communities in estuarine waters. This study employed a 23S rRNA gene-based polymerase chain reaction (PCR) assay with newly designed genus-specific primers, Ent127F-Ent907R, to determine the possible impacts of Hurricane Katrina floodwaters via the 17th Street Canal discharge on the community structure of enterococci in Lake Pontchartrain. A total of 94 phylotypes were identified through the restriction fragment length polymorphism (RFLP) screening of 494 clones while only 8 phylotypes occurred among 88 cultivated isolates. Sequence analyses of representative phylotypes and their temporal and spatial distribution in the lake and the canal indicated the Katrina floodwater input introduced a large portion of Enterococcus flavescens, Enterococcus casseliflavus, and Enterococcus dispar into the lake; typical fecal groups Enterococcus faecium, Enterococcus durans, Enterococcus hirae, and Enterococcus mundtii were detected primarily in the floodwater-impacted waters. This study provides a global picture of enterococci in estuarine waters impacted by Hurricane Katrina-derived urban runoff. It also demonstrates the culture-independent PCR approach using 23S rRNA gene as a molecular marker could be a good alternative in ecological studies of enterococci in natural environments to overcome the limitation of conventional cultivation methods.

Characterization of diverse heterocyclic amine-degrading denitrifying bacteria from various environments.

Although, there have been many published bacterial strains aerobically degrading the heterocyclic amine compounds, only one strain to date has been reported to degrade pyrrolidine under denitrifying conditions. In this study, denitrifying bacteria degrading pyrrolidine and piperidine were isolated from diverse geological and ecological origins through selective enrichment procedures. Based on the comparative sequence results of 16S rRNA genes, 30 heterocyclic amine-degrading isolates were grouped into ten distinct phylotypes belonging to the genera Thauera, Castellaniella, Rhizobium, or Paracoccus of the phylum Proteobacteria. The representative isolates of individual phylotypes were characterized by phylogenetic, phenotypic and chemotaxonomical traits, and dissimilatory nitrite reductase gene (nirK and nirS). All isolates completely degraded pyrrolidine and piperidine under both aerobic and anaerobic conditions. The anaerobic degradations were coupled to nitrate reduction. A metabolic pathway for the anaerobic degradation of pyrrolidine was proposed on the basis of enzyme activities implicated in pyrrolidine metabolism from three isolates. The three key pyrrolidine-metabolizing enzymes pyrrolidine dehydrogenase, gamma-aminobutyrate/alpha-ketoglutarate aminotransferase, and succinic semialdehyde dehydrogenase, were induced by heterocyclic amines under denitrifying conditions. They were also induced in cells grown aerobically on heterocyclic amines, suggesting that the anaerobic degradation of pyrrolidine shares the pathway with aerobic degradation.

Response of microbial populations regulating nutrient biogeochemical cycles to oiling of coastal saltmarshes from the Deepwater Horizon oil spill.

Microbial communities play vital roles in the biogeochemistry of nutrients in coastal saltmarshes, ultimately controlling water quality, nutrient cycling, and detoxification. We determined the structure of microbial populations inhabiting coastal saltmarsh sediments from northern Barataria Bay, Louisiana, USA to gain insight into impacts on the biogeochemical cycles affected by Macondo oil from the 2010 Deepwater Horizon well blowout two years after the accident. Quantitative PCR directed toward specific functional genes revealed that oiled marshes were greatly diminished in the population sizes of diazotrophs, denitrifiers, nitrate-reducers to ammonia, methanogens, sulfate-reducers and anaerobic aromatic degraders, and harbored elevated numbers of alkane-degraders. Illumina 16S rRNA gene sequencing indicated that oiling greatly changed the structure of the microbial communities, including significant decreases in diversity. Oil-driven changes were also demonstrated in the structure of two functional populations, denitrifying and sulfate reducing prokaryotes, using nirS and dsrB as biomarkers, respectively. Collectively, the results from 16S rRNA and functional genes indicated that oiling not only markedly altered the microbial community structures, but also the sizes and structures of populations involved in (or regulating) a number of important nutrient biogeochemical cycles in the saltmarshes. Alterations such as these are associated with potential deterioration of ecological services, and further studies are necessary to assess the trajectory of recovery of microbial-mediated ecosystem functions over time in oiled saltmarsh sediment.

Bacterial diversity of an acidic Louisiana groundwater contaminated by dense nonaqueous-phase liquid containing chloroethanes and other solvents.

Bacterial concentration and diversity was assessed in a moderately acidic (pH 5.1) anaerobic groundwater contaminated by chlorosolvent-containing DNAPL at a Superfund site located near Baton Rouge, Louisiana. Groundwater analysis revealed a total aqueous-phase chlorosolvent concentration exceeding 1000 mg L(-1), including chloroethanes, vinyl chloride, 1,2-dichloropropane, and hexachloro-1,3-butadiene as the primary contaminants. Direct counting of stained cells revealed more than 3 x 10(7) cells mL(-1) in the groundwater, with 58% intact and potentially viable. Universal and 'Dehalococcoides'-specific 16S rRNA gene libraries were created and analyzed. Universal clones were grouped into 18 operational taxonomic units (OTUs), which were dominated by low-G+C Gram-positive bacteria (62%) and included several as yet uncultured or undescribed organisms. Several unique 16S rRNA gene sequences closely related to Dehalococcoides ethenogenes were detected. Anaerobically grown isolates (168 in total) were also sequenced. These were phylogenetically grouped into 18 OTUs, of which only three were represented in the clone library. Phylogenetic analysis of isolates and the clone sequences revealed close relationships with dechlorinators, fermenters, and hydrogen producers. Despite acidic conditions and saturation or near-saturation chlorosolvent concentrations, the data presented here demonstrate that large numbers of novel bacteria are present in groundwater within the DNAPL source zone, and the population appears to contain bacterial components necessary to carry out reductive dechlorination.

Propionicicella superfundia gen. nov., sp. nov., a chlorosolvent-tolerant propionate-forming, facultative anaerobic bacterium isolated from contaminated groundwater.

A novel strain, designated as BL-10(T), was characterized using a polyphasic approach after isolation from groundwater contaminated by a mixture of chlorosolvents that included 1,1,2-trichloroethane, 1,2-dichloroethane, and vinyl chloride. Stain BL-10(T) is a facultatively anaerobic bacterium able to ferment glucose to form propionate, acetate, formate, lactate, and succinate. Fermentation occurred in the presence of 1,2-dichloroethane and 1,1,2-trichloroethane at concentrations to at least 9.8 and 5.9 mM, respectively. Cells are Gram-positive, rod-shaped, non-motile, and do not form spores. Oxidase and catalase are not produced and nitrate reduction did not occur in PYG medium. Menaquinone MK-9 is the predominant respiratory quinone and meso-diaminopimelic acid is present in the cell wall peptidoglycan layer. Major cellular fatty acids are C(15:0), iso C(16:0), and anteiso C(15:0). Genomic DNA G + C content is 69.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed strain BL-10(T) to fall within the radiation of genera Propionicimonas and Micropruina. On the basis of the results obtained in this study, it is proposed that strain BL-10(T) should be classified as a novel taxon, for which the name Propionicicella superfundia gen. nov., sp. nov. is proposed. The type strain of Propionicicella superfundia is BL-10(T) (=ATCC BAA-1218(T), =LMG 23096(T)).

Distributions, abundances and activities of microbes associated with the nitrogen cycle in riparian and stream sediments of a river tributary.

River tributaries are ecologically important environments that function as sinks of inorganic nitrogen. To gain greater insight into the nitrogen cycle (N-cycle) in these environments, the distributions and activities of microbial populations involved in the N-cycle were studied in riparian and stream sediments of the Santa Fe River (SFR) tributaries located in northern Florida, USA. Riparian sediments were characterized by much higher organic matter content, and extracellular enzyme activities, including cellobiohydrolase, ß-d-glucosidase, and phenol oxidase than stream sediments. Compared with stream sediments, riparian sediments exhibited significantly higher activities of nitrification, denitrification, dissimilatory nitrate reduction to ammonia (DNRA) and anaerobic ammonia oxidation; correspondingly, with higher copies of amoA (a biomarker for enumerating nitrifiers), nirS and nirK (for denitrifiers), and nrfA (for DNRA bacteria). Among N-cycle processes, denitrification showed the highest activities and the highest concentrations of the corresponding gene (nirK and nirS) copy numbers. In riparian sediments, substantial nitrification activities (6.3 mg-N kg soil-1d-1 average) and numbers of amoA copies (7.3 × 107 copies g soil-1 average) were observed, and nitrification rates correlate with denitrification rates. The guild structures of denitrifiers and nitrifiers in riparian sediments differed significantly from those found in stream sediments, as revealed by analysis of nirS and archaeal amoA sequences. This study shows that riparian sediments serve as sinks for inorganic nitrogen loads from non-point sources of agricultural runoff, with nitrification and denitrification associated with elevated levels of carbon and nitrogen contents and extracellular enzyme activities.

Biocatalytic desulfurization of diesel oil in an air-lift reactor with immobilized Gordonia nitida CYKS1 cells.

A new type of air-lift reactor with immobilized Gordonia nitida CYKS1 cells on a fibrous support was designed and used for the biocatalytic desulfurization (BDS) of diesel oil. Its performance was evaluated at different phase ratios of the oil to the aqueous medium (or oil phase fractions) and different sucrose concentrations. When the reaction mixture contained 10% diesel oil (v/v), 61-67% of sulfur was removed as the sulfur content decreased from 202-250 to 76-90 mg L(-1) in 72 h. The sulfur content did not decrease any further because the remaining sulfur compounds were recalcitrant to BDS. During the desulfurization, the strain CYKS1 consumed hydrocarbons in the diesel oil, mainly n-alkanes with 10-26 carbons, as carbon source even though an easily available carbon source, sucrose, was supplied.

Isolation and characterization of novel halotolerant and/or halophilic denitrifying bacteria with versatile metabolic pathways for the degradation of trimethylamine.

Four denitrifying bacteria capable of degrading trimethylamine under both aerobic and denitrifying conditions were newly isolated from coastal sediments and wastewater contaminated by marine water. All strains were in alpha-Proteobacteria. Strain GP43 was classified as a member of genus Paracoccus, and strain PH32, PH34 and GRP21 were novel organisms with remote phylogenetic position from other genus alpha-Proteobacteria. Among these four strains were the halophilic strains PH32, PH34 and GRP21, which did not grow in the absence of sodium chloride in culture medium. Cells grown under denitrifying conditions possessed trimethylamine dehydrogenase while cells grown aerobically possessed two different enzymes for oxidation of trimethylamine, trimethylamine dehydrogenase and trimethylamine monooxygenase. The newly isolated strain PH32, PH34 and GRP21 may be the first halophilic bacteria to degrade trimethylamine under denitrifying conditions.

Isolation and characterization of a novel Pseudomonas sp., strain YG1, capable of degrading pyrrolidine under denitrifying conditions.

A denitrifying bacterium, strain YG1, capable of degrading pyrrolidine under denitrifying conditions, was isolated. On the basis of phenotypic and phylogenetic characteristics, it was identified as a member of the genus Pseudomonas. During the anaerobic degradation of pyrrolidine, YG1 reduced a stoichiometric amount of nitrate to nitrogen gas, demonstrating that the degradation of pyrrolidine is coupled with respiratory nitrate reduction. YG1 also degraded pyrrolidine with a higher degradation rate under aerobic conditions than under denitrifying conditions.

An anaerobic continuous-flow fixed-bed reactor sustaining a 3-chlorobenzoate-degrading denitrifying population utilizing versatile electron donors and acceptors.

An anaerobic continuous-flow fixed-bed column reactor capable of degrading 3-chlorobenzoate (3-CBA) under denitrifying conditions was established, and its rate reached 2.26 mM d(-1). The denitrifying population completely degraded 3-CBA when supplied at 0.1-0.54 mM, but its activity was partly suppressed when 3-CBA was supplied at 0.89 mM. Nitrate was concomitantly consumed throughout the operation of the reactor, the amount of which was similar to or up to 35% higher than the theoretical stoichiometric value that was calculated by assuming that 3-CBA degradation is coupled with denitrification. Batch incubation experiments proved that nitrate is strictly required for 3-CBA degradation in the absence of molecular oxygen. The population also degraded 3-CBA aerobically. Benzoate and 4-CBA were degraded under denitrifying conditions as well as 3-CBA, but 2-CBA was not. Considering that the previously reported denitrifying 3-CBA-degrading cultures do not exhibit 4-CBA degradation under denitrifying conditions, nor aerobic 3-CBA degradation [FEMS Microbiol. Lett. 144 (1996) 213, Appl. Environ. Microbiol. 66 (2000) 3446], the microbial population developed in this experiment was physiologically versatile with respect to the utilization of both electron donors and electron acceptors.

Anaerobic degradation of pyrrolidine and piperidine coupled with nitrate reduction.

Biodegradability of secondary amines (pyrrolidine, piperidine, piperazine, morpholine, and thiomorpholine) under anaerobic conditions was examined in microbial consortia from six different environmental sites. The consortia degraded pyrrolidine and piperidine under denitrifying conditions. Enrichment cultures were established by repeatedly sub-culturing the consortia on pyrrolidine or piperidine in the presence of nitrate. The enrichments strictly required nitrate for the anaerobic degradation and utilized pyrrolidine or piperidine as a carbon, nitrogen, and energy source for their anaerobic growths. The anaerobic degradation of pyrrolidine and piperidine reduced nitrate to nitrogen gas, indicating that these anaerobic degradations were coupled with a respiratory nitrate reduction.

Description of Azospira restricta sp. nov., a nitrogen-fixing bacterium isolated from groundwater.

A novel, Gram-negative bacterial strain, SUA2(T), isolated from groundwater, was characterized using a polyphasic approach. Cells are Gram-negative, non-spore-forming, straight to curved rods with a single polar flagellum. Strain SUA2(T) is oxidase- and catalase-positive and is able to fix nitrogen. Poly-beta-hydroxybutyrate storage granules are produced. Dominant fatty acids when grown in R2A and VM ethanol media for 72 h at 37 degrees C are C(16 : 0), C(16 : 1)omega7c, C(17 : 0) cyclo, C(10 : 0) 3-OH, C(18 : 1) omega 7c, C(12 : 0) and C(15 : 0). DNA G+C content is 67.9 mol%. Phenotypic and phylogenetic data indicate that strain SUA2(T) is related to, but clearly differentiated from Azospira oryzae. Strain SUA2(T) is thus proposed as a novel species of the genus Azospira with the name Azospira restricta sp. nov. The description of the genus Azospira is emended to include the characteristics of this novel species. The type strain of Azospira restricta is SUA2(T) (=NRRL B-41660(T)=DSM 18626(T)=LMG 23819(T)).

Brooklawnia cerclae gen. nov., sp. nov., a propionate-forming bacterium isolated from chlorosolvent-contaminated groundwater.

Two novel facultatively anaerobic bacterial strains, BL-34(T) and BL-35, isolated from groundwater contaminated by a mixture of chlorosolvents were characterized using a polyphasic approach. The two strains exhibited essentially identical taxonomic features except for a vitamin B(12) requirement by strain BL-35 for optimal growth. Phylogenetically, the isolates were affiliated with members of the family Propionibacteriaceae and were placed in a phylogenetic branch adjacent to, but distinct from, those of the genera Propionimicrobium, Propionibacterium, Luteococcus, Propioniferax and Tessaracoccus. The cells of the novel strains were Gram-positive, non-motile, non-spore-forming pleomorphic rods. They produced catalase but not oxidase, and nitrate reduction did not occur in peptone/yeast extract/glucose medium. Propionate and acetate were the predominant products of glucose fermentation. Fermentation occurred in the presence of 1,2-dichloroethane and 1,1,2-trichloroethane at concentrations up to at least 9.8 mM. The genomic DNA G+C content was 67.5-67.9 mol%. Menaquinone MK-9(H(4)) was the predominant respiratory quinone and meso-diaminopimelic acid was present in the cell-wall peptidoglycan layer. The major cellular fatty acids were C(15 : 0) and anteiso-C(15 : 0). On the basis of the results obtained in this study, strains BL-34(T) and BL-35 should be classified within a novel taxon, for which the name Brooklawnia cerclae gen. nov., sp. nov. is proposed. The type strain of Brooklawnia cerclae is BL-34(T) (=LMG 23248(T)=NRRL B-41418(T)). An additional strain, BL-35 (=LMG 23249=NRRL B-41419), was also characterized.