This research entails investigation of ammonia, nitrate, and nitrite removal from wastewater using a proprietary blend of bacteria known as BiOWiSHTM. The degradation rates of ammonia, nitrate, and nitrite for Aqua were determined using wastewater at the San Luis Obispo Water Resource Recovery Facility (SLO WRRF). Laboratory and field experiments were conducted to test how Aqua compared with natural bacteria for removal of nitrogen compounds. Preliminary data suggested that Aqua performed nitrate removal best in SLO WRRF wastewater at the secondary clarifier. Aqua could perform anoxic and aerobic denitrification in secondary clarifier wastewater. In mineral media, Aqua removed 6.6 mg NO3-N/L/hr. In partially sterilized wastewater, Aqua removed 2.67 mg NO3-N/L/hr. Field experiments using a batch reactor suggested that Aqua aided in nitrate removal when dosed above 25 ppm in secondary clarifier wastewater. A dose of 25 ppm Aqua resulted in a 0.1 mg NO3-N/L/hr removal rate. A dose of 50 ppm Aqua resulted in a 0.15 mg NO3-N/L/hr. Aqua did not aid in ammonia or nitrate removal in sludgewash at the SLO WRRF likely due to high concentrations of nitrate and ammonia existing in the wastewater were toxic to Aqua. Aqua removed about 5 ppm more nitrate than a competitor bacteria blend in a laboratory setting. Activating Aqua to increase initial cell count before inoculation did not have any effect on removal. Providing partial aeration did not help nitrification rates and inhibited nitrate removal for Aqua. Laboratory experiments showed that Aqua did not remove nitrate in final clarifier wastewater most likely due to a limited carbon source. Aqua can perform nitrification in mineral media. Aerobic activation of Aqua inhibited denitrification. Aqua activated anoxically can perform denitrification. Using a powder with 70% microbial cultures, instead of the 1% found in Aqua, resulted in quicker nitrate removal. Inoculating as a concentrated liquid versus a dry powder did not affect nitrate removal rates. Use of trace mineral media did not affect nitrate removal rates.

The Savannah River Site (SRS), a Department of Energy facility, generated non-hazardous petroleum and radiological co-contaminated soils that did not have a disposal pathway. The purpose of this project was to generate treatment data and test the hypothesis that an engineered biological process could safely and efficiently remove petroleum co-contamination from radiological contaminated soil. Demonstration of the treatment would allow the soils to be disposed as low-level radiological materials. Although radiation and radiological contamination may, depending on the type and level, impact microbial activity and growth, the impact of low levels of radiation were not expected to impact the biodegradation of petroleum contaminated soils. Important parameters identified for successful biological treatment included oxygen mass transfer, bioavailability, temperature, microbiological capabilities, nutrients, and moisture. System design was based on a bioventing approach to control the supply of oxygen (air) based on petroleum contamination levels and type of soil being treated. Before bioremediation began, a bioreactor system was permitted, designed, constructed, and tested. An operating permit was obtained from SCDHEC, as were approvals required by the SRS. The design was based on bioventing principles and used a modified prefabricated skid-pan, which was constructed by SRNL. System operation included formulating a test plan, developing and using system sampling and monitoring methods, loading the system, starting up operations, obtaining results, modifying operation, and final disposal of the soil after the bioremediation goal was achieved. The PRCS bioreactor operated for 22 months in various configurations treating the contaminated soil to a final TPH concentration of 45 mg/kg. During operation, degradation of over 20,000 mg/kg of waste was accounted for through monitoring of carbon dioxide levels in the effluent. System operation worked best when soil temperatures were above 15 ?nd the pumps were operated continuously. The low level radiological contaminated soil was disposed in an engineered trench at SRS that accepts this type of waste. The project demonstrated that co-contaminated soils could be treated biologically to remove petroleum contamination to levels below 100 mg/kg while protecting workers and the environment from radiological contamination.

ABSTRACT A new, non-pathogenic bioaugmentation product was formulated specifically for underground use in South African mines, using local bacterial isolates. This was designed for the remediation of various hydrocarbons via biochemical breakdown by sub-surface microorganisms. The active microorganisms were isolated from hydrocarbon-polluted areas of a gold mine. Many commercially available bioaugmentation products are already in existence however, all, to our knowledge, have been developed and tested primarily for use in the northern hemisphere. None have been formulated and tested in Africa. Our series of bacterial isolates are the first to be isolated from mine soils for hydrocarbon biodegradation purposes. Such isolates have further, not previously been tested on sub-surface contamination. The safety associated with the use of such a product in a closed mine-environment is of paramount importance. Initial batch-flask experiments were conducted using a readily-available commercial bioremediation product. This was tested on simple surfactant molecules and compared to the biodegradation observed under standard waste water treatment plant conditions. The bioremediation product increased biodegradation by 6% on average. Bacteria in the product were identified by 16S rDNA gene sequence analysis and found to be homologous to potentially pathogenic Bacillus cereus, known especially to effect immunocompromised individuals, this was of particular concern in the closed mine system. South African isolates were sourced from various hydrocarbon-polluted sources, with six bacteria ultimately being selected from deep sub-surface mine soil and water samples. The ability of these isolates to biodegrade waterborne monograde engine oil was assessed via GC-FID. The isolate showing average percentage growth increase, homologous to Pseudomonas pseudoalcaligenes, was found to degrade the motor oil by 98%. The new isolates were, on average, 16% more efficient at biodegrading petroleum hydrocarbons than the commercial bioremediation product isolates. Formulation of these isolates into the first commercially-available South African developed and tested bioaugmentation product will prove a successful conclusion to this study.

Les contaminations des sols par les éléments traces métalliques constituent la deuxième contamination la plus importante au niveau mondial avec plus de 5 millions de sites contaminés répertoriés. Cette contamination est devenue globale et diffuse au sein des écosystèmes et présente des risques non négligeables pour l'environnement, l'économie agricole et la santé publique. Cd, Cu et Pb sont parmi les métaux les plus représentés et/ou les plus à risque de cette contamination. Les comportements difficilement prévisibles des métaux au sein de la matrice sol complexifient les stratégies de remédiation. Les méthodes biologiques représentent une alternative économique, écologique et efficace. Le potentiel des champignons est de plus en plus étudié dans ce domaine.Notre travail propose de mesurer la tolérance et les capacités de biosorption de 28 isolats fongiques issus de sols contaminés et de sélectionner les isolats les plus efficaces pour des essais en microcosmes de sols contaminés. Ainsi, 3 isolats ont été sélectionnés. Absidia cylindrospora tolère 1000 mg.L-1 de Cd en milieu gélosé et biosorbe plus de 50% de Cd et Pb en milieu liquide après 3 jours d'exposition. Coprinellus micaceus biosorbe 100% de Pb en milieu liquide. Enfin, Chaetomium atrobrunneum biosorbe plus de 50% de ces 3 métaux en milieu liquide, après 3 jours d'exposition.Les essais en microcosmes de sols révèlent que les capacités de colonisation de la matrice par l'isolat fongique sont essentielles afin d'avoir une influence significative sur la fraction potentiellement mobile des métaux. Ainsi, A. cylindrospora montre le meilleur potentiel des isolats testés en bioaugmentation. Au bout de 20 jours de traitement, cette espèce est capable de diminuer la fraction potentiellement disponible de Cd de 5% et de Cu de 7%. Des essais complémentaires semblent nécessaires afin d'améliorer le processus (traitement plus long avec un apport de biomasse fongique plus important) et de mieux comprendre les transferts d'ETM au sein du sol en présence de champignons. / Soil contaminations by trace metals are the second most frequent contamination in the world, counting more than 5 million polluted sites. This contamination represents a risk for the environment, the economy, the agriculture and the public health. Cd, Cu, and Pb are among the most frequent and/or toxic elements of this contamination. The hardly predictable behavior of trace metals in the soil matrix turn the remediation methods into a complex issue. Biological methods could be an economic, eco-friendly, and efficient alternative. The potential of the Fungi is more and more studied in this field.Our work aim is to evaluate the tolerance and the biosorption abilities of 28 fungi isolated from polluted soils, and to select the most efficient ones to perform microcosm's experiments of polluted soils. Thus, 3 fungal isolates have been selected. Absidia cylindrospora tolerates 1000 mg.L-1 in agar medium and biosorbs more than 50% of Cd and Pb after 3 days in liquid medium. Coprinellus micaceus biosorbs 100% of Pb in liquid medium. Finally, Chaetomium atrobrunneum biosorbs more than 50% of each metals after 3 days of exposure in liquid medium.The microcosm's experiments reveal that the colonization abilities of the isolates is crucial to enhance the influence of the fungal development on the potentially mobile metal fraction. Thus, A. cylindrospora shows the best potential among the tested isolates in bioaugmented microcosms in order to decrease the potentially mobile fraction of the metals. After 20 days, this isolate decrease the potentially mobile fraction of Cd and Cu respectively by 5 and 7%. Complementary experiments are needed to improve the process (longer experiment, higher fungal biomass) and to better understand the transfers of the metals in presence of fungal organisms.

Prior to the implementation of an enhanced bioremediation pilot study for a trichloroethene (TCE) source area at an industrial site in the United Kingdom (the Site), laboratory microcosm and column studies were performed. The purpose of this column study was to determine if TCE removal rates could be increased with the addition of partitioning electron donors and bioaugmentation with KB-1® culture. Three 1-meter continuous flow columns were constructed using aquifer solids from the Site and artificial groundwater. A TCE dense non-aqueous phase liquid (DNAPL) zone was emplaced in each column. SRS™, a commercially available emulsified vegetable oil (EVO) product, and n-butyl acetate (nBA) were evaluated as partitioning electron donors, while the third column acted as an unamended control. Both nBA and SRSTM were successfully used in previous microcosm studies with high concentrations of TCE (400 and 800 mg/L) to successfully promote the reductive dechlorination of TCE to ethene. Dechlorination of TCE to cis-1,2-dichloroethene (cis-DCE) with trace amounts of vinyl chloride (VC) and ethene, as well as sulfate reduction, were observed in the SRSTM column effluent while DNAPL was present. A dissolution enhancement factor of 2.1 was calculated. The TCE source zone was depleted after approximately 300 days of column operation. Following depletion of the TCE DNAPL, high concentration (~400 mg/L) of TCE amended artificial groundwater was pumped through the column to simulate high TCE concentrations in a plume down gradient from a source zone. Dechlorination of TCE via cis-DCE and VC to ethene was observed in the column effluent along with increases in Dehalococcoides (Dhc) counts. Sulfate concentrations increased during the plume phase while dechlorination to ethene still occurred indicating that complete dechlorination to ethene was possible in the presence of sulfate. Dechlorination of TCE to cis-DCE was observed, but neither VC nor ethene was detected in the nBA Amended column. The nBA was observed to degrade in the column to butyl alcohol and acetate, neither of which partition as strongly as nBA, and were not retained in the column. A continuous addition of nBA promoted the highest amount of cis-DCE production and sulfate reduction was also observed. Once the continuous addition was stopped, dechlorination and sulfate reduction halted indicating that electron donor retention in the column was not achieved. Dehalococcoides (Dhc) concentrations did not increase in the effluent of this column. A dissolution enhancement factor of 1.2 was calculated for the nBA column.

Prior to the implementation of an enhanced bioremediation pilot study for a trichloroethene (TCE) source area at an industrial site in the United Kingdom (the Site), laboratory microcosm and column studies were performed. The purpose of this column study was to determine if TCE removal rates could be increased with the addition of partitioning electron donors and bioaugmentation with KB-1® culture. Three 1-meter continuous flow columns were constructed using aquifer solids from the Site and artificial groundwater. A TCE dense non-aqueous phase liquid (DNAPL) zone was emplaced in each column. SRS™, a commercially available emulsified vegetable oil (EVO) product, and n-butyl acetate (nBA) were evaluated as partitioning electron donors, while the third column acted as an unamended control. Both nBA and SRSTM were successfully used in previous microcosm studies with high concentrations of TCE (400 and 800 mg/L) to successfully promote the reductive dechlorination of TCE to ethene. Dechlorination of TCE to cis-1,2-dichloroethene (cis-DCE) with trace amounts of vinyl chloride (VC) and ethene, as well as sulfate reduction, were observed in the SRSTM column effluent while DNAPL was present. A dissolution enhancement factor of 2.1 was calculated. The TCE source zone was depleted after approximately 300 days of column operation. Following depletion of the TCE DNAPL, high concentration (~400 mg/L) of TCE amended artificial groundwater was pumped through the column to simulate high TCE concentrations in a plume down gradient from a source zone. Dechlorination of TCE via cis-DCE and VC to ethene was observed in the column effluent along with increases in Dehalococcoides (Dhc) counts. Sulfate concentrations increased during the plume phase while dechlorination to ethene still occurred indicating that complete dechlorination to ethene was possible in the presence of sulfate. Dechlorination of TCE to cis-DCE was observed, but neither VC nor ethene was detected in the nBA Amended column. The nBA was observed to degrade in the column to butyl alcohol and acetate, neither of which partition as strongly as nBA, and were not retained in the column. A continuous addition of nBA promoted the highest amount of cis-DCE production and sulfate reduction was also observed. Once the continuous addition was stopped, dechlorination and sulfate reduction halted indicating that electron donor retention in the column was not achieved. Dehalococcoides (Dhc) concentrations did not increase in the effluent of this column. A dissolution enhancement factor of 1.2 was calculated for the nBA column.

Arbuscular mycorrhizal fungi (AMF) can play an important role in the phytoremediation of petroleum hydrocarbon (PH)-contaminated soil. However, little is known about the effects of AMF in combination with biostimulation via fertilization or bioaugmentation with hydrocarbonoclastic microorganisms, during phytoremediation of PH in soils. This research evaluated the influence of the AMF Glomus intraradices and inorganic fertilization on growth and physiological responses of Lolium multiflorum Lam. cv. Passarel Plus during phytoremediation of soil contaminated with Arabian medium crude oil (ACO). Also determined was the interaction of AMF with the hydrocarbonoclastic bacterium, Sphingomonas paucimobilis EPA505 (Sp), and the filamentous fungus, Cunninghamella echinulata var. elegans ATCC-36112 (Ce), on growth and selected physiological responses of L. multiflorum during phytoremediation of soil contaminated with benzo[a]pyrene (BaP) or ACO. This research provides evidence that AMF enhance the phytoremediation of petroleum hydrocarbons in soils when inoculated with L. multiflorum. The concentration of petroleum hydrocarbons in soil was a determining factor of potential benefits of AMF on L. multiflorum. Low (3000 mg·kg-1) or high (15000 mg·kg-1) concentrations of ACO resulted in limited benefits of AMF on plant growth, physiology, and degradation of ACO in soil. However, when plants were exposed to an intermediate ACO concentration in soil (6000 mg·kg-1), AMF plants had enhanced growth, physiological responses, and greater ACO-degradation than non-AMF plants. The AMF symbiosis in roots of plants was observed at all concentrations of ACO-contaminated soil. This research is one of the first reports demonstrating the benefits of AMF on the degradation of benzo[a]pyrene or ACO, alone or in combination, with the hydrocarbonoclastic microorganisms. Thus, AMF resulted in a beneficial synergism with the hydrocarbonoclastic microorganisms, particularly during ACO-degradation in the rhizosphere of L. multiflorum. Hydrocarbonoclastic microorganisms had no negative effects on AMF colonization.

Biodegradation of petroleum hydrocarbon can be an effective treatment method applied to control oil pollution in both fresh water and marine environments. Hydrocarbon degraders, both indigenous and exogenous, are responsible for utilizing petroleum hydrocarbon as their substrate for growth and energy, thereby degrading them. Biodegradation of hydrocarbons is often enhanced by bioaugmentation and biostimulation depending on the contaminated environment and the competence of the hydrocarbon degraders present. An evaluation of the performance of the biological treatment of petroleum hydrocarbon by the hydrocarbon degrading microbes at the Brayton Fire School??s 4 million gallon per day (MGD) wastewater treatment plant was the main research objective. Samples were taken for two seasons, winter (Nov 03 ?? Jan 03) and summer (Jun 04 ?? Aug 04), from each of the four treatment units: the inlet tank, equalization tank, aeration tank and the outfall tank. The population of aliphatic hydrocarbon degraders were enumerated and nutrient availability in the system were used to evaluate the effectiveness of on-going bioaugmentation and biostimulation. Monitoring of general effluent parameters was conducted to evaluate the treatment plant??s removal efficiency and to determine if effluent discharge was in compliance with the TCEQ permit. The aeration tank is an activated sludge system with no recycling. Hydrocarbon degraders are supplied at a constant rate with additional nutrient supplement. There was a significant decrease in the population of microbes that was originally fed to the system and the quantity resident in the aeration tank. Nutrient levels in the aeration tank were insufficient for the concentration of hydrocarbon degraders, even after the application of dog food as a biostimulant. The use of dog food is not recommended as a nutrient supplement. Adding dog food increases the nitrogen and phosphorus concentration in the aeration tank but the amount of carbon being added with the dog food increases the total chemical oxygen demand (COD) and biochemical oxygen demand (BOD). An increase in the concentration of total COD and BOD further increases the nitrogen and phosphorus requirement in the system. The main objective of supplying adequate nutrients to the hydrocarbon degraders would never be achieved as there would be an additional demand of nutrients to degrade the added carbon source. This research study was conducted to identify the drawbacks in the treatment plant which needs further investigation to improve efficiency.

Anaerobic digestion of agricultural wastes provides an opportunity for renewable energy production while reducing emissions of greenhouse gasses such as carbon dioxide and methane from crop and livestock production. While anaerobic digestion is possible under a wide range of temperatures and reactor configurations, it does require a stable methanogenic community composed of hydrolytic and fermentative bacteria and methanogenic archaea in order to maintain robust methane production. Research focused on characterizing and optimizing the microbial community during anaerobic digestion is increasingly exploiting DNA-based methods. In addition to providing an in-depth phylogenetic survey, these techniques permit examination of dynamic changes in α- and β-diversity during the digestion process and in response to perturbations in the system. This study used universal target amplification, next generation sequencing, and quantitative PCR to characterize the Bacteria and Archaea in digestate from thermophilic batch anaerobic digesters processing different combinations wheat ethanol stillage waste and cattle manure. The results indicated that the bacterial community was composed primarily of Firmicutes, with Proteobacteria and Bacteroidetes also numerically abundant. While less phylogenetically diverse, the archaeal community showed robust populations of both hydrogenotrophic and acetoclastic methanogens. A core microbiome present across all reactors was identified and differences in the relative abundances of the bacteria within the core community suggested significant niche overlap and metabolic redundancy in the reactors. A time-course study correlating the abundances of individual Bacteria and Archaea to methane production and volatile fatty acid catabolization identified several microorganisms hypothesized to be critical to both hydrogenotrophic and acetoclastic methanogenesis. Individual Bacteria most closely related to Clostridium spp. and Acetivibrio spp. were 10-1000-fold less abundant in reactors suffering from volatile fatty acid accumulation and inhibition of methanogenesis. Additionally, failing reactors were devoid of robust populations of acetoclastic methanogens. Microorganisms identified as critical during the time-course study were targeted for isolation in vitro and a robust methanogenic consortium consisting of at least 9 bacteria and both a hydrogenotrophic and an acetoclastic methanogen was stably propagated. Addition of this bioaugmentation consortium to digesters experiencing classic symptoms of acid crisis resulted in reduced acetate accumulation and initiation of methanogenesis. One acetoclastic methanogen, most likely a novel species from the genus Methanosarcina, showed particularly robust growth in the recovered bioaugmented reactors, increasing 100-fold in the first 7 days post-treatment. A combination of Illumina shotgun and Roche 454 paired-end sequencing chemistry was used to generate a high quality draft genome for this organism. Analysis of the annotated genome revealed diverse metabolic potential with a full complement of genes for acetoclastic, hydrogenotrophic and methylotrophic methanogenesis pathways represented. Taken as a whole, this thesis provides the foundation for using microbial community characterization to inform anaerobic digester design and operation. By identifying organisms of interest, correlating their abundance to specific biochemical functions and confirming their hypothesized functions in situ, microorganisms critical for robust methane production were acquired. The logical extension of this work is to establish monitoring tools for microorganisms identified as critical to specific performance parameters, to enumerate them in real-time, and to use that data to improve reactor operation.

Biological nutrient removal (BNR) from wastewater, and specifically nitrogen removal, is a growing concern to wastewater dischargers such as municipalities. Excess nutrients in effluent can create problems such as eutrophication, toxicity to aquatic life, and dissolved oxygen depletion in receiving waters. BNR systems have been installed in many locations with success, but their operation presents operational and financial demands greater than conventional biological treatment. Nitrogen removal is typically performed in sequential autotrophic nitrification and denitrification, which increases needed energy input, operational complexity, and therefore cost. Simultaneous nitrification-denitrification (SNdN) achieved in a single system has also been successfully implemented, however operational parameters that compromise between ideals for aerobic nitrification and anoxic denitrification result in decreased reaction rates and removal efficiencies. The application of a product that could potentially enhance SNdN reaction rates and removal efficiencies through bioaugmentation could help ease operational and financial strains. In contrast to common sequential processes, some heterotrophic Bacillus bacteria have demonstrated SNdN (Kim et al., 2005), (Zhang et al., 2011). However, their application outside of laboratory setting has yet to be established. Aqua™ is a proprietary bioaugmentation product composed of specific Bacillus strains developed by BiOWiSH® Technologies with the intent of improving aerobic, heterotrophic SNdN rates and removal efficiencies. Screening and bench-scale experiments were performed in flasks at 35° C on orbital shakers operated at a range of speeds. Primary wastewater and minimal media were used for the experiment, and inoculation was performed with both specific Bacillus strains and Aqua™. Rapid total ammonia nitrogen (TAN) removal was observed in initial screening experiments with Aqua™ in sterile wastewater. Bacillus pumilus was identified as the fastest growing organism of the Aqua™ assemblage with the greatest TAN removal 1st order rate constant (0.32/ hr.), decreasing TAN 96% within 10 hours from an initial 48.5 ppm. The orbital shaker speed that maximized TAN removal was 100 rpm, with reduction 47% and 88% more effective than both the upper (150 rpm) and lower (50 rpm) bound tested speeds, respectively. Visible floc growth centered in flasks, along with optical density data indicated cell growth and the possibility the system could support SNdN. Carbon amendments to minimal media were then evaluated, and sodium succinate improved TAN reduction by 53% compared to dextrose amended systems. This was likely because dextrose metabolism requires glycolysis to produce pyruvate for utilization in the TCA cycle for energy production; while succinate avoids glycolysis and thus is more easily utilized. In another experiment, flasks with supplemental trace minerals had a 59% higher TAN removal than the controls. Additions of supplemental vitamin solution or yeast extract improved TAN removal by 18% and 38%, respectively. Two 10-day experiments assessed Aqua™ performance in municipal primary clarifier effluent. Nitrogen balance and optical density data showed that Aqua™ dosing at 10 ppm had no effect on nitrogen removal. The second 10-day experiment increased Aqua™ dosing to 50 ppm and evaluated product activation through incubation in growth media prior to inoculation. Nitrogen balance analysis showed no effect from Aqua™ on nitrogen removal during the second 10-day experiment as well. Systems amended with dextrose saw an initial rapid TAN first order removal rate (0.25/ hr.). However, difference between control and inoculated flasks was negligible showing no effect from Aqua™. A lack of total nitrogen losses and a lack of nitrate presence during initial rapid TAN losses confirmed these losses were by assimilation into organic nitrogen. The above experiments suggest that initial success in TAN removal during screening experiments resulted from lack of competition with other microorganisms, the high 1500 ppm dose of Aqua™, and amended dextrose.