Microbial Insights (United States)

The ecological and genetic changes that underlie evolution of host-microbe interactions remain elusive, primarily due to challenges in disentangling the variables that alter microbiome composition. To understand the impact of host habitat, host genetics and evolutionary history on microbial community structure, we examined gut microbiomes of river- and three cave-adapted morphotypes of the Mexican tetra, Astyanax mexicanus, in their natural environments and under controlled laboratory conditions. We found that lab-reared fish exhibited increased microbiome richness and distinct composition compared to their wild counterparts, underscoring the significant influence of habitat. Most notably, however, we found that morphotypes reared on the same diet throughout life developed distinct microbiomes suggesting that genetic loci resulting from cavefish adaptation shape microbiome composition. We observed stable differences in Fusobacteriota abundance between morphotypes and demonstrate that this could be used as a trait for quantitative trait loci mapping to uncover the genetic basis of microbial community structure.

Biostimulation to promote reductive dechlorination is widely practiced, but the value of adding an exogenous nitrogen (N) source (e.g., NH4+) during treatment is unclear. This study investigates the effect of NH4+ availability on organohalide-respiring Dehalococcoides mccartyi (Dhc) growth and reductive dechlorination in enrichment cultures derived from groundwater (PW4) and river sediment (TC) impacted with chlorinated ethenes. In PW4 cultures, the addition of NH4+ increased cis-1,2-dichloroethene (cDCE)-to-ethene dechlorination rates about 5-fold (20.6 ± 1.6 versus 3.8 ± 0.5 μM Cl- d-1), and the total number of Dhc 16S rRNA gene copies were about 43-fold higher in incubations with NH4+ ((1.8 ± 0.9) × 108 mL-1) compared to incubations without NH4+ ((4.1 ± 0.8) × 107 mL-1). In TC cultures, NH4+ also stimulated cDCE-to-ethene dechlorination and Dhc growth. Quantitative polymerase chain reaction (qPCR) revealed that Cornell-type Dhc capable of N2 fixation dominated PW4 cultures without NH4+, but their relative abundance decreased in cultures with NH4+ amendment (i.e., 99 versus 54% of total Dhc). Pinellas-type Dhc incapable of N2 fixation were responsible for cDCE dechlorination in TC cultures, and diazotrophic community members met their fixed N requirement in the medium without NH4+. Responses to NH4+ were apparent at the community level, and N2-fixing bacterial populations increased in incubations without NH4+. Quantitative assessment of Dhc nitrogenase genes, transcripts, and proteomics data linked Cornell-type Dhc nifD and nifK expression with fixed N limitation. NH4+ additions also demonstrated positive effects on Dhc in situ dechlorination activity in the vicinity of well PW4. These findings demonstrate that biostimulation with NH4+ can enhance Dhc reductive dechlorination rates; however, a "do nothing" approach that relies on indigenous diazotrophs can achieve similar dechlorination end points and avoids the potential for stalled dechlorination due to inhibitory levels of NH4+ or transformation products (i.e., nitrous oxide).

Abstract An in‐well sediment incubator (ISI) was developed to investigate the stability and dynamics of sediment‐associated microbial communities to prevailing subsurface oxidizing or reducing conditions. Herein we describe the use of these devices at the Old Rifle Uranium Mill Tailings Remedial Action (UMTRA) site. During a seven‐month period in which oxidized Rifle Aquifer background sediment (RABS) were deployed in previously biostimulated wells under iron‐reducing conditions, cell densities of known iron‐reducing bacteria, including Geobacteraceae, increased significantly, showing the microbial community response to local subsurface conditions. Phospholipid fatty acid (PLFA) profiles of RABS following in situ deployment were strikingly similar to those of adjacent sediment cores, suggesting ISI results could be extrapolated to the native material of the test plots. Results for ISI deployment with laboratory‐reduced sediments showed only slight changes in community composition and pointed toward the ability of the ISI to monitor microbial community stability and response to subsurface conditions. © 2009 Wiley Periodicals, Inc.

A laboratory incubation experiment was conducted with uranium-contaminated subsurface sediment to assess the geochemical and microbial community response to ethanol amendment. A classical sequence of terminal electron-accepting processes (TEAPs) was observed in ethanol-amended slurries, with NO3- reduction, Fe(III) reduction, SO4(2-) reduction, and CH4 production proceeding in sequence until all of the added 13C-ethanol (9 mM) was consumed. Approximately 60% of the U(VI) content of the sediment was reduced during the period of Fe(III) reduction. No additional U(VI) reduction took place during the sulfate-reducing and methanogenic phases of the experiment Only gradual reduction of NO3-, and no reduction of U(VI), took place in ethanol-free slurries. Stimulation of additional Fe(III) or SO4(2-) reduction in the ethanol-amended slurries failed to promote further U(VI) reduction. Reverse transcribed 16S rRNA clone libraries revealed major increases in the abundance of organisms related to Dechloromonas, Geobacter, and Herbaspirillum in the ethanol-amended slurries. Phospholipid fatty acids (PLFAs) indicative of Geobacter showed a distinct increase in the amended slurries, and analysis of PLFA 13C/12C ratios confirmed the incorporation of ethanol into these PLFAs. A increase in the abundance of 13C-labeled PLFAs indicative of Desulfobacter, Desulfotomaculum, and Desulfovibrio took place during the brief period of sulfate reduction that followed the Fe(III) reduction phase. Our results show that major redox processes in ethanol-amended sediments can be reliably interpreted in terms of standard conceptual models of TEAPs in sediments. However, the redox speciation of uranium is complex and cannot be explained based on simplified thermodynamic considerations.

The objective of this study was to establish a national relative moldiness index for homes in the United States. As part of the Housing and Urban Development's American Healthy Homes Survey, dust samples were collected by vacuuming 2 m in the bedrooms plus 2 m in the living rooms from a nationally representative 1096 homes in the United States using the Mitest sampler. Five milligrams of sieved (300 mum pore, nylon mesh) dust was analyzed by mold-specific quantitative polymerase chain reaction for the 36 indicator species in 1096 samples. On the basis of this standardized national sampling and analysis, an "Environmental Relative Moldiness Index" was created with values ranging from about -10 to 20 or above (lowest to highest). The Environmental Relative Moldiness Index scale may be useful for home mold-burden estimates in epidemiological studies.

Changing regulations to lower disinfectant byproducts in drinking water is forcing utilities to switch disinfection from chlorine to monochloramine. It is generally unknown whether this will impact positively or negatively on the microbiological quality of drinking water. A utility in Florida, using water with relatively high organic carbon levels from deep wells in several wellfields, made the decision to change its disinfection regime from chlorine to chloramine in order to meet the new regulations. To assess the impacts of such a change on the microbiology of its water supplies, it undertook a number of studies before and after the change. In particular, the presence of the opportunistic pathogens Legionella and Mycobacterium, and also the composition of drinking-water biofilms, were examined. A preliminary synthesis and summary of these results are presented here. Legionella species were widely distributed in source waters and in the distribution system when chlorine was the disinfectant. In some samples they seemed to be among the dominant biofilm bacteria. Following the change to monochloramine, legionellae were not detected in the distribution system during several months of survey; however, they remained detectable at point of use, although with less species diversity. A variety of mycobacteria (21 types) were widely distributed in the distribution system when chlorine was the disinfectant, but these seemed to increase in dominance after chloramination was instituted. At point of use, only four species of mycobacteria were detected. Other changes occurring with chloramination included (a) an altered biofilm composition, (b) increased numbers of total coliforms and heterotrophs and (c) nitrification of water storage tanks. The results suggested that consideration should be given to the microbiological effects of changing disinfection regimes in drinking-water and distribution system biofilms.

Modern molecular methods such as lipid biomarker analysis (LBA) and PCRDenaturing Gradient Gel Electrophoresis (DGGE) of rDNA or functional genes have enabled sensitive and quantitative assessment of in situ microbial communities that is independent of isolation and culture of the microbes. Application of artificial neural network analysis (ANN) to biomarker data has demonstrated a powerful means to characterize soil, marine and deep subsurface microbial communities. This analysis has provided accurate detection of bioavailability, bioremediation effectiveness, and rational end points for remediation based on the community microbial ecology. Sequential high pressure/temperature extraction greatly shortens sample-processing time and increases the recovery of lipid biomass from soils. Sequential extraction by supercritical CO2 and/or accelerated solvent extraction (ACE) lyses cells and allows sequential recovery of neutral lipids, then polar lipids, and facilitates recovery of DNA. The extracted residue is then derivatized in situ and re-extracted with supercritical CO2 or hexane with ACE to yield dipicolinic acid originating from bacterial spores and ester-linked hydroxy fatty acids derived from the lipopolysaccharide of Gram-negative bacteria. Once extracted, components in each fraction are separated by capillary high performance liquid chromatography (HPLC) with solvent systems compatible with electrospray ionization and analyzed by tandem quadrupole mass spectrometry (HPLC/ESI/MS/MS) and capillary gas chromatography/mass spectrometry (GC/MS). HPLC utilizing electrospray ionization (ESI) provides the potential to analyze intact lipid biomarkers and provides greater insights into the viable biomass, community composition and physiological status than with previous techniques. Use of HPLC/ESI/MS/MS enables analysis of respiratory quinones, diacylglycerols, and sterols from the neutral lipids. Also, the structures of intact phospholipids that have been collisionally dissociated can be determined at sensitivities (for synthetic molecules) of ≈ 90 attomol/μL (concentration of total lipids in a single E. coli) Moreover HPLC/ESI/MS/MS also enables analysis/detection of the isoprenoid tetraethers of the Archaea, Ornithine lipids of Gram-negative bacteria signaling for bio-available phosphate, and the lysyl esters of phosphatidyl glycerol of Gram-positive bacteria. This expanded LBA not only facilitates recovery of DNA for highly specific PCR amplification but provides deeper insight into predicting in situ metabolic activities without disturbance artifacts. In addition, the analysis is based on the responses to microniche environments—the Holy Grail of microbial ecology.

Heating or freezing leads to loss in infectivity of oocysts of Cryptosporidium parvum toward neonatal BALB/c mice and is reflected in the profile of the polar lipid fatty acids. Upon loss of infectivity, the ratio of polar lipid to neutral lipid fatty acid decreased and the relative proportions of 18:1n-9 also decreased; proportions of 18:2n-6 and 20:5n-6 increased, whereas the proportions of 16:0 remained constant with freezing. During these investigations, a novel fatty acid, 10-OH 18:0, was discovered in the glycolipid fraction. The identification of a fatty acid unique to species of Cryptosporidium was thought to provide a specific biomarker for this organism. Cryptosporidium also demonstrated fluctuations in absolute quantities of 10-OH 18:0 with events that lead to loss of infectivity. This led to the presumed correlation of this biomarker with infectious Cryptosporidium. The 10-OH 18:0 was putatively localized at the sn-2 position of phosphatidylethanolamine. High-performance liquid chromatography/electrospray ionization mass spectrometry revealed that the 10-OH 18:0 existed principally in the free fatty acid form. Herein, we establish that the free fatty acid 10-OH 18:0 was, in actuality, an artifact of the procedures for sample preparation.

A 10-hydroxy stearic acid-containing lipid from Cryptosporidium parvum was purified by thin-layer chromatography and analyzed by infrared spectroscopy, fast-atom bombardment mass spectrometry, 1H and 31P nuclear magnetic resonance spectroscopy, and was identified as phosphatidyl-ethanolamine.