<p>Microorganisms, particularly the Bacteria, are differentially impacted by metal toxicities, and will respond very quickly to changes in their environment, making them ideal bioindicators of environmental health. In this study, we evaluated the sediment-associated bacterial diversity of fifty-seven samples collected from twenty-four anthropogenically and endogenously metal impacted, geographically distinct sites in the Colorado Mineral Belt, and elucidated the factors that correlated with observed differences in the bacterial community structure. Overall, the geochemistry of all sites distinguished anthropogenic from endogenous sources of metal impact. Anthropogenic samples, on average, had higher concentrations of total recoverable and dissolved sodium and magnesium, and lower concentrations of aluminum and zinc, compared to the endogenous samples. Bacterial communities from both anthropogenically and endogenously metal impacted sites were characterized using Illumina high-throughput amplicon sequencing of the V4 region of the 16S rRNA gene. Overall, bacterial communities were remarkably diverse, with endogenously metal impacted sediments having higher diversity compared to anthropogenic sediments. The <i>Actinobacteria </i> and <i>Betaproteobacteria</i> dominated anthropogenic samples, and the <i>Acidobacteria</i> and <i>Deltaproteobacteria </i> dominated endogenous samples. Clustering of bacterial communities based on membership and structure (presence/absence and relative abundance of particular taxa, respectively) also distinguished samples based on their source of metal impact. Analysis of similarity (ANOSIM) tests indicated a significant difference between bacterial community structure based on source of metal impact (weighted UniFrac R_ANOSIM = 0.746, p = 0.001). Mantel tests indicated that total recoverable magnesium concentrations accounted for &sim;54% of variance in community structure of all bacterial communities in the study. Dissolved aluminum concentrations accounted for &sim;71% of the variation in all communities with an anthropogenic source of metal, and dissolved aluminum concentrations also accounted for &sim;41% of the variation in bacterial communities with endogenous sources of metal impact. </p><p> This study provides one of the first direct comparisons between microbial community structures of sediments based on source of metal impact. This study is also one of the first comprehensive characterizations of bacterial communities from naturally occurring iron fen systems. </p>

<p> High elevation, mountainous regions have a high concentration of mining activities and resulting acid mine drainage (AMD) that is typically acidic and often contains elevated concentrations of metals. The impacts of AMD on denitrifying microbial communities is not well understood, despite these organisms&rsquo; central role in the nitrogen cycle, contribution to greenhouse gas production, and potential to provide ecosystem services through the mitigation of nitrogen pollution. This study examined denitrifying microbes across four regions within the Iron Springs Mining District (13 sites over four time-points) located in Southwest Colorado at high elevation that receive AMD or naturally-occurring acid rock drainage (ARD). Denitrification functional gene sequences (<i> nirS</i> and <i>nirK</i> coding for nitrite reductase) had a high number of observed OTUs (260 for <i>nirS</i> and 253 for <i> nirK</i>) and were observed at sites with pH as low as 3.2, dissolved oxygen as low as 1.0 mg/L, and metals >10 mg/L (including aluminum, iron, manganese, and zinc). A majority of the <i>nirK</i> and <i> nirS</i> OTUs (> 60%) were present in only one sampling region. Approximately 8% of the <i>nirK</i> and <i>nirS</i> OTUs had a more cosmopolitan distribution with presence in three or more regions. Phylogenetically related OTUs were found across sites with very different chemistry. The total <i>nirS</i> community structure was correlated to iron, conductivity, sodium, and calcium, which may suggest that these factors play an important role in shaping the <i>nirS</i> community. Overall, these findings improve upon our understanding of the potential for denitrification within an ecosystem impacted by AMD and provide a foundation for future research to understand the rates and physiology of these denitrifying organisms.</p><p>

Two strains of Clostridium hungatei sp. nov., a cellulolytic, N$\sb2$-fixing bacterium were isolated from samples of soil rich in decaying plant material. On the basis of comparisons of their morphological, physiological and phylogenetic characteristics, and of their G + C mol % content, with those of other Clostridium species, it was concluded that the two strains (designated strain AD and strain B3B) were representatives of a novel species of Clostridium. C. hungatei produces an extracellular cellulase complex which exhibits cellulase (i.e., Avicelase, carboxymethylcellulase) and xylanase activities. The cellulase-xylanase complex biosynthesis, in C. hungatei, was induced when cells of strain AD were grown with soluble products of cellulose hydrolysis (e.g., cellobiose) as the carbon and energy source. Induction of cellulase-xylanase complex, above a low constitutive level, occurred when the cellobiose concentration was 0.1% (wt/vol) or lower, but not at higher concentrations. Induction above the constitutive level was not observed when other soluble sugars (e.g., D-mannose) served as carbon and energy source. Cellulase activity of the cellulase-xylanase complex produced by C. hungatei is inhibited by cellobiose (at concentrations as low as 0.02%, wt/vol). Growth kinetics, cellulose degradation studies and enzyme assays revealed that supernatant fluid samples of C. hungatei cultures growing under N$\sb2$-fixing conditions, or at low dilution rates (in a chemostat limited by carbon and energy source) in the presence of NH$\sb4$Cl showed higher cellulase activity per cell mass produced as compared to cultures growing in the presence of combined nitrogen or at higher dilution rates. In nature, the ability to regulate the cellulase-xylanase complex synthesis, and the ability to enhance cellulase activity per cell mass produced under conditions of higher energy demand (e.g., N$\sb2$ fixation) may give C. hungatei an advantage over other bacteria living in the same environment. A N$\sb2$-fixing stable coculture, consisting of C. hungatei strain AD and a facultatively anaerobic, non-cellulolytic bacterium (strain CU-1), was used as a model to determine whether extracellular cellulases produced by cellulolytic bacteria may serve as nitrogen sources for non-N$\sb2$-fixing bacteria growing in heterogeneous microbial communities. Inasmuch as supernatant fluids of cocultures showed lower cellulase and xylanase activities as compared to clostridial monoculture supernatants, and supernatant fluids of strain CU-1 showed proteolytic activity when grown in the presence of sources of amino acids (e.g., C. hungatei's cellulase system), and of the basis of other evidence it was concluded that, in anaerobic environments deficient in combined nitrogen, extracellular cellulases may serve as nitrogen sources for non-N$\sb2$-fixing bacteria. In addition, strain CU-1 grew in monoculture in a chemically-defined cellobiose-containing medium to which a gel-filtration purified strain AD cellulase preparation was added as the only nitrogen source. Using a cellulolytic methanogenic coculture, composed of C. hungatei strain AD, and a strain of H$\sb2$-consuming methanogen (e.g., Methanobacterium formicicum strain WH), the effects of interspecies H$\sb2$ transfer on growth, cellulose degradation and product formation were studied. The results indicated that interspecies H$\sb2$ transfer enhances growth rate and affects product formation by the cellulolytic clostridium, and indirectly stimulates cellulose degradation.

Dissimilatory Fe(III) reduction is one of the most significant metabolisms in sedimentary environments. Molecular and geochemical studies conducted on uranium contaminated sediments suggested that Fe(III) reducing bacteria could be stimulated to reduce U(VI) in uranium contaminated subsurface sediments. This is particularly significant because U(VI) is highly soluble, and therefore mobile, in most groundwaters whereas the reduced form of uranium, U(IV), is generally insoluble and precipitates from most groundwaters. Therefore, stimulating microbial reduction of U(VI) could potentially stop the spread of uranium contamination in the subsurface and, if the bioremediation process is engineered properly, concentrate uranium into a discrete zone for subsequent recovery. The anaerobic electrode of sediment fuel cells is another sedimentary environment where dissimilatory Fe(III) reducing bacteria predominate. Molecular and culturing studies have shown that dissimilatory Fe(III) reducing microorganisms are directly involved in electricity production by sediment fuel cells. These findings should help with the development and optimization of several environmentally relevant tools; i.e. microbial fuel cells that convert waste to useable energy forms. Further evaluation of the dissimilatory Fe(III) reducing microbial communities associated with these two important sedimentary environments suggested that a specific group of organisms, the Geobacteraceae, are primarily responsible for uranium removal from subsurface sediments and electron transfer to an electrode. In fact, two organisms, strains A1 and A2, were recovered from the surface of an electrode and are able to quantitatively transfer electrons available from the complete oxidation of organic acids to an electrode surface. A detailed evaluation of pure cultures of Geobaceraceae species indicated that the Geobacteraceae are a phylogenetically and physiologically distinct family within the δ-subdivision of Proteobacteria. This unique physiology may allow Geobacteraceae species to be most competitive in these sedimentary environments. Another family of dissimilatory metal reducing microorganisms, the Desulfobulbaceae, were also found to be associated with the current-harvesting anodes of marine sediment fuel cells. Further evaluation of a member of this family, Desulfobulbus propionicus, indicated that this organism is also capable of dissimilatory Fe(III) reduction and is able to directly transfer electrons to an electrode. Thus, Desulfobulbaceae species may also contribute to energy production in marine sediment fuel cells.

The in situ concentrations and repeating unit characterization of poly(hydroxyalkanoates) (PHAs) were examined in stratified photosynthetic microbial mats from Great Sippewissett Salt Marsh, Massachusetts and the Ebro Delta, Spain. In the cyanobacteria-dominated green layer material the mole % ratio of hydroxybutyrate (HB) repeating units to hydroxyvalerate (HV) units was generally 1HB:1HV. In the purple sulfur bacteria-dominated pink material the relationship was typically 1HB:2HV. When total PHA content was normalized to organic carbon content there was little seasonal variation in the PHA levels. However, a diel cycle of varying PHA levels was evident at all sites. Overnight, PHA accumulated to about 1 1/2 to 2 times the amount that had been present the previous evening. Over the course of the next daylight period, those levels declined to what they had been the previous evening. Exogenous acetate, lactate, and propionate induced 2 to 5-fold increases in PHA content when applied in the daylight, but had no effect on PHA content when applied at night. Intact microbial mat slabs, incubated in the light for 6 h in H 14CO3– amended seawater, incorporated 58% of the initial radiolabel. The cyano/green material incorporated 4 times more H14CO3– than did the PSB/pink material. The mats were fractionated into the major molecular pools of (1) low molecular weight (lmw) material, (2) proteins/nucleic acids, (3) PRA, and (4) glycogen. After the initial labeling period in the light, the 14C incorporated into the green layer material was partitioned as follows: 61% in lmw material, 20% in proteins/nucleic acids, 20% in glycogen, and less than 1% in PHA. After the initial labeling period in light the 14C incorporated into pink layer material was partitioned as follows: 61% in lmw material, 14% in proteins/nucleic acids, 25% in glycogen, and less than 1% in PHA. When mat that was labeled in the light was transferred to darkness, there was a marked now of 14C from the glycogen fraction to the PHA fraction, particularly in pink material where 13% of the incorporated 14C was detected after 20 h of darkness. There was no incorporation of label into the PHA of mats that were continuously incubated in the light.

Davis Mine, an abandoned pyrite mine in western Massachusetts, was my study model for acid mine drainage (AMD). Acidic waters from AMD cause the dissolution of other resident minerals increasing the concentration of heavy metals in these environments. Therefore, bacteria isolated from AMD exhibit high levels of tolerance to heavy metals. The objective of this work was to isolate and characterize acidophilic, aerobic, chemoorganotrophic bacteria from AMD that are tolerant to high concentrations of heavy metal divalent cations. First, I analyzed the microbial community present at aerobic sediments obtained at the opening of the former main shaft at Davis Mine. This sediment was used as inoculum for enrichments of bacteria in a mixture of heavy metals. Enrichments and isolated bacteria were characterized by tolerance to heavy metals, presence of extrachromosomal DNA, and presence of known metal tolerance-related genes. In addition, community structure and phylogenetic identities were determined. The diversity analysis of the sediment revealed that the microbial diversity at Davis Mine at the level of Bacterial classes is comparable to other aerobic sediments from AMD sites. The majority of the sequences were related to yet uncultured bacteria obtained from other mine sites or acidic soils making it difficult to describe accurately the microbial community structure. Chemoorganotrophic, heavy metal tolerant bacteria were successfully isolated from Davis Mine. The majority of the strains were closely related to the acidophilic genera Acidocella and Thiomonas. In addition, I isolated bacteria related to Burkholderia which was not previously isolated from AMD. The heavy metal tolerance profiles of 21 strains revealed that high tolerance was correlated to the addition of heavy metals to the pre-cultivation medium as well as the presence of a 480 kb plasmid. I suggest that the heavy metal resistances observed are the result of more than on type of mechanism that are induced by the presence of heavy metals in the culture medium.

Over the last two decades, minimization of excess sludge generation within the activated sludge process has been studied. Among several net sludge reduction technologies, the anaerobic side-stream reactor (ASSR) process is of particular interest because it has shown significant sludge reduction without causing negative effects on operational performances. This study focused on the verification of the ASSR process, the mechanisms of excess sludge reduction, and the development of a new process using high rate ASSR. The earlier part of this research found that a bench-scale ASSR with 10 day solids retention time (SRT) led to about 60% less sludge yield than conventional activated sludge, without causing negative effects on the main activated sludge process, i.e., sludge settling and effluent properties. This sludge yield result indicated that incorporation of an anaerobic side-stream reactor into activated sludge was a valid sludge reduction process and was much more effective than any other conventional methods (anaerobic digestion or aerobic digestion). New methods to estimate SRT and observed sludge yield for the ASSR process were also proposed during this stage of research. The later part of this research investigated the interaction between ASSR and activated sludge and the role of extracellular polymeric substances (EPS) and enzymes in sludge flocs to reveal the mechanisms of excess sludge reduction in the ASSR process. It was observed that much of the organic matter, particularly the EPS, was solublized in the ASSR and readily degraded in the main activated sludge reactor as the previous study showed. By accounting for the mass of sludge in the reactors, it was determined that half of the sludge reduction occurred directly in the ASSR while the other half of the sludge was degraded in the aeration basin. From an intensive side-by-side reactor study, it was found that the amount of the released material from ASSR was not proportional to overall sludge reduction, indicating that the success of the ASSR process is not solely dependent on the extent of hydrolysis or anaerobic sludge degradation in the ASSR but on the recirculation of the whole sludge between aerobic conditions (activated sludge) and anaerobic conditions (ASSR). This sludge recirculation reduced the accumulation of excessive EPS fractions within the flocs, allowing for balanced EPS fractions even under extremely long SRT conditions, and thus resulting in effective flocculation and sludge settling. Overall, the ASSR process kept the sludge refreshed in spite of the extremely long SRT due to the extremely minimal sludge wasting. This concept is proposed in this research as Sludge Refreshment. Preliminary research and examination of literature reviews during this doctoral research led us to develop a new hypothesis that deflocculation and subsequent sludge hydrolysis occur more effectively under the short period of anaerobic digestion and that recirculation of this sludge back to the aeration basin could lead to even more effective excess sludge reduction. To verify this hypothesis, an anaerobic batch study was conducted and various schemes of the ASSR process (different SRTs and temperatures) were operated side-by-side in the laboratory. The results from the anaerobic batch tests showed that maximum solubilization of key floc cations, extracellular polymeric substances, and enzyme activity occurred within 2 days of anaerobic digestion, regardless of temperature. The results from the reactor study showed that activated sludge with a 2.5-day-SRT ASSR, generated the lowest sludge yield among the studied systems. All these results indicate that an activated sludge process with a short-SRT (termed high rate) ASSR could result in greater solids reduction during wastewater treatment. In summary, this research found that the ASSR process is valid for effective sludge reduction in biological wastewater treatment. The study of a novel high rate ASSR process also expanded insight into sludge flocs and allow a better understanding of the fate of EPS in aerobic and anaerobic repeating conditions. This process should be considered to be a very effective method for sludge reduction which also maintains good operational performance for the activated sludge process.

<p> Vibrio bacteria are emerging pathogens responsible for 80,000 illnesses and 100 deaths in the United States each year. Infections are directly linked to the marine environment and are acquired by consuming contaminated seafood or exposing wounds during aquatic activities. Florida has the highest national incidence of vibriosis, with 20% of its cases reported from the Indian River Lagoon region, a popular recreation destination. This study utilized a combination of cultivation and molecular techniques to investigate the local distribution of V. vulnificus, V. parahaemolyticus and V. cholerae in this local waterway. </p><p> The targeted species were found in an array of samples which may facilitate their transmission to humans. Overall, these bacteria were abundant in estuarine sediments (Vp: 2,439 CFU/g, Vv: 303 CFU/g, Vc: 176 CFU/g), on the sharp edges of oyster shells (Vp: 82 CFU/cm, Vv: 102 CFU/cm, Vc: 41 CFU/cm), and in the water column (Vp: 3.78 CFU/ml, Vv: 5.51 CFU/ml, Vc: 2.46 CFU/ml). Vibrio also pose a hazard to recreational anglers as they were recovered from fish (Vp: 61%, Vv: 55%, Vc: 30%), live bait shrimp (Vp: 80%, Vv: 37%, Vc: 0%) and hooks (Vp: 32%, Vv: 18%, Vc: 0%). Additionally, a molecular analysis of the V. vulnificus virulence revealed that the local population was dominated by disease-causing (vcgC) strains, which may explain why wound-related infections are common in this region. </p><p> Vibrio occurrence varied both spatially and temporally due to their relationship with salinity and temperature. These bacteria exhibited a strong negative correlation with salinity, being particularly abundant near freshwater discharge locations. Due to Florida&rsquo;s year-round warm climate, these species were found to be permanent members of the local microbial community. Seasonal peaks in abundance occurred between August and October, a period which corresponds with the warmest water temperatures as well as frequent rainfall. Predictive models were constructed based on these parameters to provide a better understanding of how, when and where Vibrio spp. may be encountered by humans. This information is important for both water management and healthcare initiatives, with an overall goal of improving local recreational safety.</p>

Bioremediation is becoming a widely accepted solution for cleaning up contaminated sediment, water, and soil. The associated microbial, chemical, and geologic principles are still in their relative infancy compared to other common remediation strategies. The list of contaminants that are susceptible to biotransformation has grown to encompass metals as well as organic compounds. The goal of this research was to develop anaerobic bioremediation strategies for two compounds—the fuel oxygenate methyl tert-butyl ether (MTBE) and the heavy metal uranium. The potential for anaerobic degradation of MTBE as well as its metabolite tert-butyl alcohol (TBA) was tested in aquifer and freshwater aquatic sediment. Aquifer sediment amended with Fe (III) oxide plus the electron shuttling compound humic acid degraded 50 mg/l MTBE to levels below detection. The humic acid analog anthraquinone-2,6-disulfonate (AQDS) also stimulated MTBE degradation. Aquifer sediment without Fe (III) plus the electron shuttle did not degrade MTBE. Freshwater aquatic sediment converted uniformly labeled [14C]-MTBE to 14CO2 and over time. Adding Fe (III) and electron shuttling compounds did not significantly increase the rate and extent of MTBE mineralization. TBA was also rapidly degraded in the aquatic sediment in the absence of any amendments. Both 14CO2 and 14CH4 were produced when sediments were incubated with uniformly labeled [14C]-TBA. Microbial U(VI) reduction was stimulated in uranium-contaminated aquifer sediment upon the addition of acetate. U(VI) reduction was concurrent with Fe (III) reduction and preceded sulfate reduction. In sediments that also contained nitrate, nitrate first had to be completely reduced prior to the onset of U(VI) reduction. U(VI) reduction was solely a biological process; abiotic interaction with Fe (II), sulfide, or electron shuttles did not affect U(VI) in solution. Nitrate added to sediment that contained microbially reduced U(VI) re-oxidized U(IV) to U(VI) and Fe (II) to Fe (III). Over 80% of the initial reduced U(IV) was recovered as U(VI). Pure culture studies with Geobacter metallireducens confirmed that the most likely mechanism was biological oxidation of Fe (II) to Fe (III) with abiotic U(IV) oxidation via Fe (III) produced. Finally, two novel Fe (III)-reducing bacterial species were isolated from these sediments—Desulfitobacterium metallireducens and Geoferax ferrireducens.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2007. / Source: Dissertation Abstracts International, Volume: 68-11, Section: B, page: 7583. Adviser: Eberhard Morgenroth. Includes bibliographical references (leaves 167-174) Available on microfilm from Pro Quest Information and Learning.