Native Bacterial Community Convergence in Augmented and Stimulated Ureolytic MICP Biocementation.

Microbially induced calcite precipitation is a biomineralization process with numerous civil engineering and ground improvement applications. In replicate soil columns, the efficacy and microbial composition of soil bioaugmented with the ureolytic bacterium Sporosarcina pasteurii were compared to a biostimulation method that enriches native ureolytic soil bacteria in situ under conditions analogous to field implementation. The selective enrichment resulting from sequential stimulation treatments strongly selected for Firmicutes (>97%), with Sporosarcina and Lysinibacillus comprising 60 to 94% of high-throughput 16S rDNA sequences in each suspended community sample. Seven species of the former and two of the latter were present in greater than 10% abundance at different times, demonstrating unexpected within-genus diversity and robustness in the suspended phase of this highly selective environment. Based on longer 16S sequences, it was inferred that augmented S. pasteurii competed poorly with natural bacteria, decreasing to below detection after nine treatments, while the native microbial community was enriched to approximately that present in the stimulated columns. These analyses were corroborated by the observed convergence in bulk ureolytic rates and calcite contents between techniques. However, a 10-fold discrepancy between the observed cell density and an activity-based estimate indicates the attached community, uncharacterized despite efforts, substantially contributes to bulk behavior.

Investigating Ammonium By-product Removal for Ureolytic Bio-cementation Using Meter-scale Experiments.

Microbially Induced Calcite Precipitation (MICP), or bio-cementation, is a promising bio-mediated technology that can improve the engineering properties of soils through the precipitation of calcium carbonate. Despite significant advances in the technology, concerns regarding the fate of produced NH4+ by-products have remained largely unaddressed. In this study, five 3.7-meter long soil columns each containing one of three different soils were improved using ureolytic bio-cementation, and post-treatment NH4+ by-product removal was investigated during the application of 525 L of a high pH and high ionic strength rinse solution. During rinsing, reductions in aqueous NH4+ were observed in all columns from initial concentrations between ≈100 mM to 500 mM to final values between ≈0.3 mM and 20 mM with higher NH4+ concentrations observed at distances furthest from the injection well. In addition, soil Vs measurements completed during rinse injections suggested that no significant changes in cementation integrity occurred during NH4+ removal. After rinsing and a 12 hour stop flow period, all column solutions achieved cumulative NH4+ removals exceeding 97.9%. Soil samples collected following rinsing, however, contained significant sorbed NH4+ masses that appeared to have a near linear relationship with surrounding aqueous NH4+ concentrations. While these results suggest that NH4+ can be successfully removed from bio-cemented soils, acceptable limits for NH4+ aqueous concentrations and sorbed NH4+ masses will likely be governed by site-specific requirements and may require further investigation and refinement of the developed techniques.

Biogeochemical Changes During Bio-cementation Mediated by Stimulated and Augmented Ureolytic Microorganisms.

Microbially Induced Calcite Precipitation (MICP) is a bio-mediated cementation process that can improve the engineering properties of granular soils through the precipitation of calcite. The process is made possible by soil microorganisms containing urease enzymes, which hydrolyze urea and enable carbonate ions to become available for precipitation. While most researchers have injected non-native ureolytic bacteria to complete bio-cementation, enrichment of native ureolytic microorganisms may enable reductions in process treatment costs and environmental impacts. In this study, a large-scale bio-cementation experiment involving two 1.7-meter diameter tanks and a complementary soil column experiment were performed to investigate biogeochemical differences between bio-cementation mediated by either native or augmented (Sporosarcina pasteurii) ureolytic microorganisms. Although post-treatment distributions of calcite and engineering properties were similar between approaches, the results of this study suggest that significant differences in ureolysis rates and related precipitation rates between native and augmented microbial communities may influence the temporal progression and spatial distribution of bio-cementation, solution biogeochemical changes, and precipitate microstructure. The role of urea hydrolysis in enabling calcite precipitation through sustained super-saturation following treatment injections is explored.

Diversity of Sporosarcina-like Bacterial Strains Obtained from Meter-Scale Augmented and Stimulated Biocementation Experiments.

Microbially Induced Calcite Precipitation (MICP) is a biomediated soil cementation process that offers an environmentally conscious alternative to conventional geotechnical soil improvement technologies. This study provides the first comparison of ureolytic bacteria isolated from sand cemented in parallel, meter-scale, MICP experiments using either biostimulation or bioaugmentation approaches, wherein colonies resembling the augmented strain ( Sporosarcina pasteurii ATCC 11859) were interrogated. Over the 13 day experiment, 47 of the 57 isolates collected were strains of Sporosarcina and the diversity of these strains was high, with 20 distinct strains belonging to 5 species identified. Although the S. pasteurii inoculant used for augmentation was recovered immediately after introduction in the augmented specimen, the strain was not recovered after 8 days in either augmented or stimulated soils, suggesting that it competes poorly with indigenous bacteria. Past studies on the physiological properties of S. pasteurii ATCC 11859 suggest that close relatives may have selective advantages under the biogeochemical conditions employed during MICP; however, the extent to which these properties apply to isolates of the current study is unknown. Whole cell urease kinetic properties were investigated for representative isolates and suggest up to 100-fold higher rates of carbonate production when compared to other biomediated processes proposed for MICP.

Soil engineering in vivo: harnessing natural biogeochemical systems for sustainable, multi-functional engineering solutions.

Carbon sequestration, infrastructure rehabilitation, brownfields clean-up, hazardous waste disposal, water resources protection and global warming-these twenty-first century challenges can neither be solved by the high-energy consumptive practices that hallmark industry today, nor by minor tweaking or optimization of these processes. A more radical, holistic approach is required to develop the sustainable solutions society needs. Most of the above challenges occur within, are supported on, are enabled by or grown from soil. Soil, contrary to conventional civil engineering thought, is a living system host to multiple simultaneous processes. It is proposed herein that 'soil engineering in vivo', wherein the natural capacity of soil as a living ecosystem is used to provide multiple solutions simultaneously, may provide new, innovative, sustainable solutions to some of these great challenges of the twenty-first century. This requires a multi-disciplinary perspective that embraces the science of biology, chemistry and physics and applies this knowledge to provide multi-functional civil and environmental engineering designs for the soil environment. For example, can native soil bacterial species moderate the carbonate cycle in soils to simultaneously solidify liquefiable soil, immobilize reactive heavy metals and sequester carbon-effectively providing civil engineering functionality while clarifying the ground water and removing carbon from the atmosphere? Exploration of these ideas has begun in earnest in recent years. This paper explores the potential, challenges and opportunities of this new field, and highlights one biogeochemical function of soil that has shown promise and is developing rapidly as a new technology. The example is used to propose a generalized approach in which the potential of this new field can be fully realized.

Vacuolate-attached filaments: highly productive Ridgeia piscesae epibionts at the Juan de Fuca hydrothermal vents.

Vacuolate sulfur bacteria with high morphological similarity to vacuolate-attached filaments previously described from shallow hydrothermal vents (White Point, CA) were found at deep-sea hydrothermal vents. These filamentous bacteria grow in dense mats that cover surfaces and potentially provide a significant source of organic carbon where they occur. Vacuolate-attached filaments were collected near vents at the Clam Bed site of the Endeavour Segment of the Juan de Fuca Ridge and from the sediment surface at Escanaba Trough on the Gorda Ridge. A phylogenetic analysis comparing their 16S rRNA gene sequences to those collected from the shallow White Point site showed that all vacuolate-attached filament sequences form a monophyletic group within the vacuolate sulfur-oxidizing bacteria clade in the gamma proteobacteria. Abundance of the attached filaments was quantified over the length of the exterior surface of the tubes of Ridgeia piscesae worms collected from the Clam Bed site at Juan de Fuca yielding a per worm average of 0.070 ± 0.018 cm3 (n = 4). In agreement with previous results for White Point filaments, anion measurements by ion chromatography showed no detectable internal nitrate concentrations above ambient seawater (n = 9). For one R. piscesae tube worm "bush" at the Easter Island vent site, potential gross epibiont productivity is estimated to be 15 to 45× the net productivity of the worms.

Using health inspection scores to assess risk in food services.

This study gathered health inspectors' opinions about appropriate weightings of critical, noncritical, and repeat violations under the current food inspection system, and developed a classification of violations for high-, medium-, and low-risk restaurants. Results showed that health inspectors thought that the appropriate weights were five points for a critical violation, one point for a noncritical violation, and double points for a repeat violation. In addition, health inspectors thought that the maximum numbers of critical violations for a high-, medium-, and low-risk category were 2.05, 3.02, and 4.83, respectively, and for noncritical violations, 4.59, 7.30, and 10.37, respectively. A paired t-test was used to compare these values with estimations based on the traditional health inspection scoring system. Results indicate that the maximum number of critical violations for medium risk and maximum numbers of noncritical violations for low-, medium-, or high-risk restaurants were significantly different between health inspectors' opinions and mathematical estimations. Health inspectors appear to be stricter than the traditional health inspection scoring system about violations, particularly repeat violations, and their importance in enforcement of food safety.

Methylotrophy in freshwater Beggiatoa alba strains.

Two freshwater strains of the gammaproteobacterium Beggiatoa alba, B18LD and OH75-2a, are able to use methanol as a sole carbon and energy source under microoxic conditions. Genes encoding a methanol dehydrogenase large-subunit homolog and four enzymes of the tetrahydromethanopterin-dependent C(1) oxidation pathway were identified in B18LD. No evidence of methanotrophy was detected.

Clients' safe food-handling knowledge and risk behavior in a home-delivered meal program.

OBJECTIVE: To determine typical handling practices of home-delivered meals, and provide appropriate handling instructions to reduce the risk of foodborne illness by improving consumer handling of home-delivered meals. DESIGN: Once permission was given by the home-delivered meal site directors, clients were provided a voluntary survey and requested by the delivery drivers to complete the self-administered questionnaire. The completed questionnaire was collected by the driver the following day. Because of the special needs of the home-delivered meal populations, the questionnaire was made as easy and convenient to answer as possible. SUBJECTS: Two hundred fifty-eight male clients (31%) and 575 female clients (69%) whose mean age was 79 years participated in the study. MAIN OUTCOME MEASURES: The respondents' safe food-handling practices, food safety knowledge, and demographic information were assessed. Average time for delivery and consumption of meals were also measured. STATISTICAL ANALYSIS: Descriptive statistics (frequency and chi(2) test) of the participants' handling of home-delivered meals, their general food safety knowledge, and demographic information were reported. Delivery time and consumption time were calculated for each subject. RESULTS: Five hundred thirty-six of 869 clients (63%) reported that they ate their meals as soon as they were delivered. Of those clients who did not eat their meals immediately, 234 (82%) stored the cold food in the refrigerator and 142 (58%) stored the hot food in the freezer. More than one-third of the clients (n=277, 35%) reported that they had leftovers and only 34 (15%) ate the leftovers within 2 hours. Significant differences among groups on the basis of a derived food safety knowledge score were observed in terms of whether or not they ate their meal immediately (P

Mitigating the risk of food handling in the home-delivered meal program.

The purpose of this cross-sectional study was to examine the length of time between packing and delivery of home-delivered meals, and the extent of foodborne illness risk to the elderly. Procedures to mitigate that risk were also evaluated. Researchers surveyed 95 drivers from home-delivered meal preparation sites in six states across the United States to determine the average length of time that passed during packing, loading, leaving, and delivery. The efficiency of various risk mitigation methods were evaluated and used to adjust the actual delivery time. Total average delivery time from packing to last delivery was 1.92 hours. This study suggests that the risk associated with the actual 1.92 hours of total delivery time could be mitigated to represent approximately 1.55 hours of effective time with proper packing and holding conditions. This methodology proposes a single measure for evaluating the effectiveness of various handling procedures associated with distributing home-delivered meals, which can be utilized to evaluate overall risk when combined with in-house preparation and client-handling behaviors.

Cultivated Beggiatoa spp. define the phylogenetic root of morphologically diverse, noncultured, vacuolate sulfur bacteria.

Within the last 10 years, numerous SSU rRNA sequences have been collected from natural populations of conspicuous, vacuolate, colorless sulfur bacteria, which form a phylogenetically cohesive cluster (large-vacuolate sulfur bacteria clade) in the gamma-Proteobacteria. Currently, this clade is composed of four named or de facto genera: all known Thioploca and Thiomargarita strains, all vacuolate Beggiatoa strains, and several strains of vacuolate, attached filaments, which bear a superficial similarity to Thiothrix. Some of these vacuolate bacteria accumulate nitrate for respiratory purposes. This clade encompasses the largest known prokaryotic cells (Thiomargarita namibiensis) and several strains that are important in the global marine sulfur cycle. Here, we report additional sequences from five pure culture strains of Beggiatoa spp., including the only two cultured marine strains (nonvacuolate), which firmly establish the root of this vacuolate clade. Each of several diverse metabolic motifs, including obligate and facultative chemolithoautotrophy, probable mixotrophy, and seemingly strict organoheterotrophy, is represented in at least one of the nonvacuolate strains that root the vacuolate clade. Because the genus designation Beggiatoa is interspersed throughout the vacuolate clade along with other recognized or de facto genera, the need for taxonomic revision is clear.

Mercury methylation from unexpected sources: molybdate-inhibited freshwater sediments and an iron-reducing bacterium.

Methylmercury has been thought to be produced predominantly by sulfate-reducing bacteria in anoxic sediments. Here we show that in circumneutral pH sediments (Clear Lake, CA) application of a specific inhibitor of sulfate-reducing bacteria at appropriate concentrations typically inhibited less than one-half of all anaerobic methylation of added divalent mercury. This suggests that one or more additional groups of microbes are active methylators in these sediments impacted by a nearby abandoned mercury mine. From Clear Lake sediments, we isolated the iron-reducing bacterium Geobacter sp. strain CLFeRB, which can methylate mercury at a rate comparable to Desulfobulbus propionicus strain 1pr3, a sulfate-reducing bacterium known to be an active methylator. This is the first time that an iron-reducing bacterium has been shown to methylate mercury at environmentally significant rates. We suggest that mercury methylation by iron-reducing bacteria represents a previously unidentified and potentially significant source of this environmental toxin in iron-rich freshwater sediments.

Novel vacuolate sulfur bacteria from the Gulf of Mexico reproduce by reductive division in three dimensions.

Large spherical sulfur bacteria, 180-375 microm in diameter, were found regularly and in abundance in surface sediments collected from hydrocarbon seeps (water depth 525-640 m) in the Gulf of Mexico. These bacteria were characterized by a thin 'shell' of sulfur globule-filled cytoplasm that surrounded a central vacuole (roughly 80% of biovolume) containing high concentrations of nitrate (average 460 mM). Approximately 800 base pairs of 16S rRNA gene sequence data, linked to this bacterium by fluorescent in situ hybridization, showed 99% identity with Thiomargarita namibiensis, previously described only from sediments collected off the coast of Namibia (Western Africa). Unlike T. namibiensis, where cells form a linear chain within a common sheath, the Gulf of Mexico strain occurred as single cells and clusters of two, four and eight cells, which were clearly the product of division in one to three planes. In sediment cores maintained at 4 degrees C, which undoubtedly experienced a diminishing flux of hydrogen sulfide over time, the Thiomargarita-like bacterium remained viable for up to 2 years. During that long period, each cell appeared to undergo (as judged by change in biovolume) one to three reductive divisions, perhaps as a dispersal strategy in the face of diminished availability of its putative electron donor.

Novel, attached, sulfur-oxidizing bacteria at shallow hydrothermal vents possess vacuoles not involved in respiratory nitrate accumulation.

Novel, vacuolate sulfur bacteria occur at shallow hydrothermal vents near White Point, Calif. There, these filaments are attached densely to diverse biotic and abiotic substrates and extend one to several centimeters into the surrounding environment, where they are alternately exposed to sulfidic and oxygenated seawater. Characterizations of native filaments collected from this location indicate that these filaments possess novel morphological and physiological properties compared to all other vacuolate bacteria characterized to date. Attached filaments, ranging in diameter from 4 to 100 microm or more, were composed of cylindrical cells, each containing a thin annulus of sulfur globule-filled cytoplasm surrounding a large central vacuole. A near-complete 16S rRNA gene sequence was obtained and confirmed by fluorescent in situ hybridization to be associated only with filaments having a diameter of 10 microm or more. Phylogenetic analysis indicates that these wider, attached filaments form within the gamma proteobacteria a monophyletic group that includes all previously described vacuolate sulfur bacteria (the genera Beggiatoa, Thioploca, and Thiomargarita) and no nonvacuolate genera. However, unlike for all previously described vacuolate bacteria, repeated measurements of cell lysates from samples collected over 2 years indicate that the attached White Point filaments do not store internal nitrate. It is possible that these vacuoles are involved in transient storage of oxygen or contribute to the relative buoyancy of these filaments.

Food service health inspectors' opinions on the reporting of inspections in the media.

Media reporting of restaurant inspection results is a topic hotly debated by food service health inspectors. A survey of Indiana health inspectors was conducted to determine attitudes, preferences, and perceptions about the impact of media reporting. The survey showed that in the geographic areas of slightly fewer than half of the respondents, inspection results were already being reported in the media. The question of whether media reporting was advisable received mixed responses, with slightly over half favoring media reporting. Respondents were more likely to favor media reporting if they were from areas where inspection results were already being reported to the media, areas with larger populations, or areas in which more restaurant inspections were being done. Newspapers were the preferred venue for media reporting.