Anaerobic microbial communities and their potential for bioenergy production in heavily biodegraded petroleum reservoirs.

Most of the oil in low temperature, non-uplifted reservoirs is biodegraded due to millions of years of microbial activity, including via methanogenesis from crude oil. To evaluate stimulating additional methanogenesis in already heavily biodegraded oil reservoirs, oil sands samples were amended with nutrients and electron acceptors, but oil sands bitumen was the only organic substrate. Methane production was monitored for over 3000 days. Methanogenesis was observed in duplicate microcosms that were unamended, amended with sulfate or that were initially oxic, however methanogenesis was not observed in nitrate-amended controls. The highest rate of methane production was 0.15 µmol CH4 g-1 oil d-1 , orders of magnitude lower than other reports of methanogenesis from lighter crude oils. Methanogenic Archaea and several potential syntrophic bacterial partners were detected following the incubations. GC-MS and FTICR-MS revealed no significant bitumen alteration for any specific compound or compound class, suggesting that the very slow methanogenesis observed was coupled to bitumen biodegradation in an unspecific manner. After 3000 days, methanogenic communities were amended with benzoate resulting in methanogenesis rates that were 110-fold greater. This suggests that oil-to-methane conversion is limited by the recalcitrant nature of oil sands bitumen, not the microbial communities resident in heavy oil reservoirs.

Pea-protein alginate encapsulation adversely affects development of clinical signs of Citrobacter rodentium-induced colitis in mice treated with probiotics.

The efficacy of two strains of Lactobacillus probiotics (Lactobacillus rhamnosus R0011 and Lactobacillus helveticus R0052) immobilized in microcapsules composed of pea protein isolate (PPI) and alginate microcapsules was assessed using a mouse model of Citrobacter rodentium-induced colitis. Accordingly, 4-week-old mice were fed diets supplemented with freeze-dried probiotics (group P), probiotic-containing microcapsules (group PE) (lyophilized PPI-alginate microcapsules containing probiotics), or PPI-alginate microcapsules containing no probiotics (group E). Half of the mice (controls, groups P, PE, and E) received C. rodentium by gavage 2 weeks after initiation of feeding. Daily monitoring of disease symptoms (abnormal behavior, diarrhea, etc.) and body weights was undertaken. Histopathological changes in colonic and cecal tissues, cytokine expression levels, and pathogen and probiotic densities in feces were examined, and the microbial communities of the distal colon mucosa were characterized by 16S rRNA sequencing. Infection with C. rodentium led to marked progression of infectious colitis, as revealed by symptomatic and histopathological data, changes in cytokine expression, and alteration of composition of mucosal communities. Probiotics led to changes in most of the disease markers but did not have a significant impact on cytokine profiles in infected animals. On the basis of cytokine expression analyses and histopathological data, it was evident that encapsulation materials (pea protein and calcium alginate) contributed to inflammation and worsened a set of symptoms in the cecum. These results suggest that even though food ingredients may be generally recognized as safe, they may in fact contribute to the development of an inflammatory response in certain animal disease models.

Draft Genome Sequence of Polaromonas eurypsychrophila AER18D-145, Isolated from a Uranium Tailings Management Facility in Northern Saskatchewan, Canada.

The 4.8-Mbp draft genome sequence of Polaromonas eurypsychrophila AER18D-145, isolated from a uranium tailings management facility, is reported. The sequence may provide insights into the mechanisms of the hypertolerance of this strain to extreme conditions and help determine its potential for bioremediation applications.

Prediction of bacterial functional diversity in clay microcosms.

Microorganisms in clay barriers could affect the long-term performance of waste containers in future deep geological repositories (DGR) for used nuclear fuel through production of corrosive metabolites (e.g., sulfide), which is why clay materials are highly compacted: to reduce both physical space and access to water for microorganisms to grow. However, the highly compacted nature of clays and the resulting low activity or dormancy of microorganisms complicate the extraction of biomarkers (i.e., PLFA, DNA etc.) from such barriers for predictive analysis of microbial risks. In order to overcome these challenges, we have combined culture- and 16S rRNA gene amplicon sequencing-based approaches to describe the functional diversity of microorganisms in several commercial clay products, including two different samples of Wyoming type MX-80 bentonite (Batch 1 and Batch 2), the reference clay for a future Canadian DGR, and Avonlea type Canaprill, a clay sample for comparison. Microorganisms from as-received bentonites were enriched in anoxic 10% w/v clay microcosms for three months at ambient temperature with addition of 10% hydrogen along with presumable indigenous organics and sulfate in the clay. High-throughput sequencing of 16S rRNA gene fragments indicated a high abundance of Gram-positive bacteria of the phylum Firmicutes (82%) in MX-80 Batch 1 incubations. Bacterial libraries from microcosms with MX-80 Batch 2 were enriched with Firmicutes (53%) and Chloroflexi (43%). Firmicutes also significantly contributed (<15%) to the bacterial community in Canaprill clay microcosm, which was dominated by Gram-negative Proteobacteria (>70%). Sequence analysis revealed presence of the bacterial families Peptostreptococcaceae, Clostridiaceae, Peptococcaceae, Bacillaceae, Enterobacteriaceae, Veillonellaceae, Tissierellaceae and Planococcaceae in MX-80 Batch 1 incubations; Bacillaceae, along with unidentified bacteria of the phylum Chloroflexi, in MX-80 Batch 2 clay microcosms, and Pseudomonadaceae, Hydrogenophilaceae, Bacillaceae, Desulfobacteraceae, Desulfobulbaceae, Peptococcaceae, Pelobacteraceae, Alcaligenaceae, Rhodospirillaceae in Canaprill microcosms. Exploration of potential metabolic pathways in the bacterial communities from the clay microcosms suggested variable patterns of sulfur cycling in the different clays with the possible prevalence of bacterial sulfate-reduction in MX-80 bentonite, and probably successive sulfate-reduction/sulfur-oxidation reactions in Canaprill microcosms. Furthermore, analysis of potential metabolic pathways in the bentonite enrichments suggested that bacteria with acid-producing capabilities (i.e., fermenters and acetogens) together with sulfide-producing prokaryotes might perhaps contribute to corrosion risks in clay systems. However, the low activity or dormancy of microorganisms in highly compacted bentonites as a result of severe environmental constraints (e.g., low water activity and high swelling pressure in the confined bentonite) in situ would be expected to largely inhibit bacterial activity in highly compacted clay-based barriers in a future DGR.

Draft Genome Sequence of Arthrobacter sp. Strain 260, Isolated from a Uranium Tailings Management Facility in Northern Saskatchewan, Canada.

The 3.9-Mbp draft genome sequence of Arthrobacter sp. strain 260, which was isolated from a uranium tailings management facility, is reported. The sequence may help determine the bioremediation potential of this strain and facilitate further research aimed at a better understanding of the hypertolerance of this genus to extreme conditions.

Microscale and molecular analyses of river biofilm communities treated with microgram levels of cerium oxide nanoparticles indicate limited but significant effects.

Cerium oxide (CeO2) nanoparticles are used as in-fuel catalysts and in manufacturing processes, creating a potential for release to aquatic environments. Exposures at 1 and 10 µg/L CeO2-nanoparticles were made to assess effects during the development of river biofilm communities. Scanning transmission x-ray microscopy (STXM) indicated extensive sorption of nanoparticles to the community and co-localization with lipid moieties. Following 8 weeks of development, polycarbonate coupons were removed from the reactors and used for molecular analyses, denaturing gradient gel electrophoresis analysis (DGGE-16S rRNA) and 16S rRNA amplicon sequencing. Microscopic imaging of the biofilm communities (bacterial, photosynthetic biomass, exopolymer composition, thickness, protozoan numbers), as well as carbon substrate utilization fingerprinting was performed. There was a trend toward reduced photosynthetic biomass, but no significant effects of CeO2 exposure were found on photosynthetic and bacterial biomass or biofilm thickness. Sole carbon source utilization analyses indicated increased utilization of 10 carbon sources in the carbohydrate, carboxylic acid and amino acids categories related to CeO2 exposures; however, predominantly, no significant effects (p < 0.05) were detected. Measures of microbial diversity, lectin binding affinities of exopolymeric substances and results of DGGE analyses, indicated significant changes to community composition (p < 0.05) with CeO2 exposure. Increased binding of the lectin Canavalia ensiformis was observed, consistent with changes in bacterial-associated polymers. Whereas, no significant changes were observed in binding to residues associated with algal and cyanobacterial exopolymers. 16S rRNA amplicon sequencing of community DNA indicated changes in diversity and shifts in community composition; however, these did not trend with increasing CeO2 exposure. Counting of protozoans in the biofilm communities indicated no significant effects on this trophic level. Thus, based on biomass and functional measures, CeO2 nanoparticles did not appear to have significant effects; however, there was evidence of selection pressure resulting in significant changes in microbial community composition.

Microbiology to help solve our energy needs: methanogenesis from oil and the impact of nitrate on the oil-field sulfur cycle.

Our society depends greatly on fossil fuels, and the environmental consequences of this are well known and include significant increases of the CO(2) concentration in the earth's atmosphere. Although microbiology has traditionally played only a minor role in fossil-fuel extraction, two novel key discoveries indicate that this may change. First, the realization that oil components can be converted to methane and CO(2) by methanogenic consortia in the absence of electron acceptors (oxygen, nitrate, sulfate) explains how much of the world's oil has been biodegraded in situ. In addition to inorganic nutrients, only water is needed for these methanogenic conversions. Hence, continued methanogenic biodegradation may have shaped the heavy-oil reservoirs that are so prevalent today. The potential to exploit these reactions, for example, by in situ gasification, is currently being actively investigated. Second, injection of nitrate in oil and gas fields can lower sulfide concentrations. High sulfide concentrations, caused by the action of sulfate-reducing bacteria (SRB), are associated with increased risk of corrosion, reservoir plugging (through precipitated sulfides), and human safety. Nitrate injection into an oil field stimulates subsurface heterotrophic nitrate-reducing bacteria (hNRB) and nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB). Nitrite, formed by these NRB by partial reduction of nitrate, is a strong and specific SRB inhibitor. Nitrate injection has, therefore, promise in positively controlling the oil-field sulfur cycle. There is now more interest in and potential to apply petroleum microbiology than there has been in the past, allowing microbiologists to contribute to a sustainable energy future.

Bacterial diversity and production of sulfide in microcosms containing uncompacted bentonites.

AIMS: This study examined the diversity and sulfide-producing activity of microorganisms in microcosms containing commercial clay products (e.g., MX-80, Canaprill and National Standard) similar to materials which are currently considered for use in the design specifications for deep geologic repositories (DGR) for spent nuclear fuel. METHODS AND RESULTS: In anoxic microcosms incubated for minimum of 60 days with 10 g l-1 NaCl, sulfide production varied with temperature, electron donor and bentonite type. Maximum specific sulfide production rates of 0.189 d-1, 0.549 d-1 and 0.157 d-1 occurred in lactate-fed MX-80, Canaprill and National Standard microcosms, respectively. In microcosms with 50 g l-1 NaCl, sulfide production was inhibited. Denaturing gradient gel electrophoresis (DGGE) profiling of microcosms revealed the presence of bacterial classes Clostridia, Bacilli, Gammaproteobacteria, Deltaproteobacteria, Actinobacteria, Sphingobacteriia and Erysipelotrichia. Spore-forming and non-spore-forming bacteria were confirmed in microcosms using high-throughput 16S rRNA gene sequencing. Sulfate-reducing bacteria of the genus Desulfosporosinus predominated in MX-80 microcosms; whereas, Desulfotomaculum and Desulfovibrio genera contributed to sulfate-reduction in National Standard and Canaprill microcosms. CONCLUSIONS: Commercial clays microcosms harbour a sparse bacterial population dominated by spore-forming microorganisms. Detected sulfate- and sulfur-reducing bacteria presumably contributed to sulfide accumulation in the different microcosm systems. SIGNIFICANCE AND IMPACT OF STUDY: The use of carbon-supplemented, clay-in-water microcosms offered insights into the bacterial diversity present in as-received clays, along with the types of metabolic and sulfidogenic reactions that might occur in regions of a DGR (e.g., interfaces between the bulk clay and host rock, cracks, fissures, etc.) that fail to attain target parameters necessary to inhibit microbial growth and activity.

Draft Genome Sequences of Biofilm-Forming and Non-Biofilm-Forming Nontyphoidal Salmonella enterica Serovars.

The genetic basis for biofilm formation among nontyphoidal salmonellae (NTS) remains poorly understood. This draft genome submission provides initial insights on the genetic differences between biofilm-forming and non-biofilm-forming clinical and environmental NTS serovars.

Geobacillus jurassicus sp. nov., a new thermophilic bacterium isolated from a high-temperature petroleum reservoir, and the validation of the Geobacillus species.

Four thermophilic, spore-forming bacterial strains, DS1(T), DS2, 46 and 49, were isolated from the high-temperature Dagang oilfield, located in China. The strains were identified by using the polyphasic taxonomy approach. These were aerobic, gram-positive, rod-shaped, moderately thermophilic (with an optimum growth temperature of 60-65 degrees C), chemoorganotrophic bacteria capable of growing on various sugars, carboxylic acids and crude oil. Two strains, DS1(T) and DS2, were capable of growing on individual saturated hydrocarbons. The G + C content of the DNA of strains DS1(T) and DS2 was 54.5 and 53.8 mol%, respectively. The phylogenetic analysis of the 16S rDNA of strains DS1(T) and DS2 showed that they form a separate cluster within the genus Geobacillus. The cellular fatty acids of the isolates were dominated by iso-15:0, iso-16:0 and iso-17:0 acids, which are the typical fatty acids of bacteria from the genus Geobacillus. The DNA-DNA hybridization study and the comparative analysis of the morphological and chemotaxonomic characteristics of strains DS1(T) and DS2 showed that they differ from the previously described Geobacillus species and belong to a new species, which was called Geobacillus jurassicus. DS1(T) (=VKM B2301(T), = DSM 15726(T)) is the type strain of this species. According to both DNA-DNA reassociation studies and 16S rDNA sequence analysis, two other strains, 46 and 49, were assigned to the species G. stearothermophilus. In this paper, we provide evidence that the new combinations G. stearothermophilus, G. thermoleovorans, G. kaustophilus, G. thermoglucosidasius and G. thermodenitrificans may be considered to be valid.