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Intimate coupling of Ti2 photocatalysis and biodegradation (ICPB) offers potential for degrading biorecalcitrant and toxic organic compounds much better than possible with conventional wastewater treatments. This study reports on using a novel sponge-type, Ti2-coated biofilm carrier that shows significant adherence of Ti2 to its exterior and the ability to accumulate

Intimate coupling of Ti2 photocatalysis and biodegradation (ICPB) offers potential for degrading biorecalcitrant and toxic organic compounds much better than possible with conventional wastewater treatments. This study reports on using a novel sponge-type, Ti2-coated biofilm carrier that shows significant adherence of Ti2 to its exterior and the ability to accumulate biomass in its interior (protected from UV light and free radicals). First, this carrier was tested for ICPB in a continuous-flow photocatalytic circulating-bed biofilm reactor (PCBBR) to mineralize biorecalcitrant organic: 2,4,5-trichlorophenol (TCP). Four mechanisms possibly acting of ICPB were tested separately: TCP adsorption, UV photolysis/photocatalysis, and biodegradation. The carrier exhibited strong TCP adsorption, while photolysis was negligible. Photocatalysis produced TCP-degradation products that could be mineralized and the strong adsorption of TCP to the carrier enhanced biodegradation by relieving toxicity. Validating the ICPB concept, biofilm was protected inside the carriers from UV light and free radicals. ICPB significantly lowered the diversity of the bacterial community, but five genera known to biodegrade chlorinated phenols were markedly enriched. Secondly, decolorization and mineralization of reactive dyes by ICPB were investigated on a refined Ti2-coated biofilm carrier in a PCBBR. Two typical reactive dyes: Reactive Black 5 (RB5) and Reactive Yellow 86 (RY86), showed similar first-order kinetics when being photocatalytically decolorized at low pH (~4-5), which was inhibited at neutral pH in the presence of phosphate or carbonate buffer, presumably due to electrostatic repulsion from negatively charged surface sites on Ti2, radical scavenging by phosphate or carbonate, or both. In the PCBBR, photocatalysis alone with Ti2-coated carriers could remove RB5 and COD by 97% and 47%, respectively. Addition of biofilm inside macroporous carriers maintained a similar RB5 removal efficiency, but COD removal increased to 65%, which is evidence of ICPB despite the low pH. A proposed ICPB pathway for RB5 suggests that a major intermediate, a naphthol derivative, was responsible for most of the residual COD. Finally, three low-temperature sintering methods, called O, D and DN, were compared based on photocatalytic efficiency and Ti2 adherence. The DN method had the best Ti2-coating properties and was a successful carrier for ICPB of RB5 in a PCBBR.
ContributorsLi, Guozheng (Author) / Rittmann, Bruce E. (Thesis advisor) / Halden, Rolf (Committee member) / Krajmalnik-Brown, Rosa (Committee member) / Arizona State University (Publisher)
Created2011
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Description
Biological soil crusts (BSCs), topsoil microbial assemblages typical of arid land ecosystems, provide essential ecosystem services such as soil fertilization and stabilization against erosion. Cyanobacteria and lichens, sometimes mosses, drive BSC as primary producers, but metabolic activity is restricted to periods of hydration associated with precipitation. Climate models for the

Biological soil crusts (BSCs), topsoil microbial assemblages typical of arid land ecosystems, provide essential ecosystem services such as soil fertilization and stabilization against erosion. Cyanobacteria and lichens, sometimes mosses, drive BSC as primary producers, but metabolic activity is restricted to periods of hydration associated with precipitation. Climate models for the SW United States predict changes in precipitation frequency as a major outcome of global warming, even if models differ on the sign and magnitude of the change. BSC organisms are clearly well adapted to withstand desiccation and prolonged drought, but it is unknown if and how an alteration of the precipitation frequency may impact community composition, diversity, and ecosystem functions. To test this, we set up a BSC microcosm experiment with variable precipitation frequency treatments using a local, cyanobacteria-dominated, early-succession BSC maintained under controlled conditions in a greenhouse. Precipitation pulse size was kept constant but 11 different drought intervals were imposed, ranging between 416 to 3 days, during a period of 416 days. At the end of the experiments, bacterial community composition was analyzed by pyrosequencing of the 16s rRNA genes in the community, and a battery of functional assays were used to evaluate carbon and nitrogen cycling potentials. While changes in community composition were neither marked nor consistent at the Phylum level, there was a significant trend of decreased diversity with increasing precipitation frequency, and we detected particular bacterial phylotypes that responded to the frequency of precipitation in a consistent manner (either positively or negatively). A significant trend of increased respiration with increasingly long drought period was detected, but BSC could recover quickly from this effect. Gross photosynthesis, nitrification and denitrification remained essentially impervious to treatment. These results are consistent with the notion that BSC community structure adjustments sufficed to provide significant functional resilience, and allow us to predict that future alterations in precipitation frequency are unlikely to result in severe impacts to BSC biology or ecological relevance.
ContributorsMyers, Natalie Kristine (Author) / Garcia-Pichel, Ferran (Thesis advisor) / Hall, Sharon (Committee member) / Turner, Benjamin (Committee member) / Krajmalnik-Brown, Rosa (Committee member) / Arizona State University (Publisher)
Created2013
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Description
In this work, the vapor transport and aerobic bio-attenuation of compounds from a multi-component petroleum vapor mixture were studied for six idealized lithologies in 1.8-m tall laboratory soil columns. Columns representing different geological settings were prepared using 20-40 mesh sand (medium-grained) and 16-minus mesh crushed granite (fine-grained). The contaminant vapor

In this work, the vapor transport and aerobic bio-attenuation of compounds from a multi-component petroleum vapor mixture were studied for six idealized lithologies in 1.8-m tall laboratory soil columns. Columns representing different geological settings were prepared using 20-40 mesh sand (medium-grained) and 16-minus mesh crushed granite (fine-grained). The contaminant vapor source was a liquid composed of twelve petroleum hydrocarbons common in weathered gasoline. It was placed in a chamber at the bottom of each column and the vapors diffused upward through the soil to the top where they were swept away with humidified gas. The experiment was conducted in three phases: i) nitrogen sweep gas; ii) air sweep gas; iii) vapor source concentrations decreased by ten times from the original concentrations and under air sweep gas. Oxygen, carbon dioxide and hydrocarbon concentrations were monitored over time. The data allowed determination of times to reach steady conditions, effluent mass emissions and concentration profiles. Times to reach near-steady conditions were consistent with theory and chemical-specific properties. First-order degradation rates were highest for straight-chain alkanes and aromatic hydrocarbons. Normalized effluent mass emissions were lower for lower source concentration and aerobic conditions. At the end of the study, soil core samples were taken every 6 in. Soil moisture content analyses showed that water had redistributed in the soil during the experiment. The soil at the bottom of the columns generally had higher moisture contents than initial values, and soil at the top had lower moisture contents. Profiles of the number of colony forming units of hydrocarbon-utilizing bacteria/g-soil indicated that the highest concentrations of degraders were located at the vertical intervals where maximum degradation activity was suggested by CO2 profiles. Finally, the near-steady conditions of each phase of the study were simulated using a three-dimensional transient numerical model. The model was fit to the Phase I data by adjusting soil properties, and then fit to Phase III data to obtain compound-specific first-order biodegradation rate constants ranging from 0.0 to 5.7x103 d-1.
ContributorsEscobar Melendez, Elsy (Author) / Johnson, Paul C. (Thesis advisor) / Andino, Jean (Committee member) / Forzani, Erica (Committee member) / Krajmalnik-Brown, Rosa (Committee member) / Kavazanjian, Edward (Committee member) / Arizona State University (Publisher)
Created2012
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Description
With global warming becoming a more serious problem and mankind's alarming dependency on fossil fuels, the need for a sustainable and environmentally friendly fuel source is becoming more important. Biofuels produced from photosynthetic microorganisms like algae or cyanobacteria offer a carbon neutral replacement for petroleum fuel sources; however, with the

With global warming becoming a more serious problem and mankind's alarming dependency on fossil fuels, the need for a sustainable and environmentally friendly fuel source is becoming more important. Biofuels produced from photosynthetic microorganisms like algae or cyanobacteria offer a carbon neutral replacement for petroleum fuel sources; however, with the technology and information available today, the amount of biomass that would need to be produced is not economically feasible. In this work, I examined a possible factor impacting the growth of a model cyanobacterium, Synechocystis sp. PCC6803, which is heterotrophic bacteria communities accompanying the cyanobacteria. I experimented with three variables: the type of heterotrophic bacteria strain, the initial concentration of heterotrophic bacteria, and the addition of a carbon source (glucose) to the culture. With experimental information, I identified if given conditions would increase Synechocystis growth and thus increase the yield of biomass. I found that under non-limiting growth conditions, heterotrophic bacteria do not significantly affect the growth of Synechocystis or the corresponding biomass yield. The initial concentration of heterotrophic bacteria and the added glucose also did not affect the growth of Synechocystis. I did see some nutrient recycling from the heterotrophic bacteria as the phosphate levels in the growth medium were depleted, which was apparent from prolonged growth phase and higher levels of reactive phosphate in the media.
ContributorsCahill, Brendan Robert (Author) / Rittmann, Bruce (Thesis director) / Krajmalnik-Brown, Rosa (Committee member) / W. P. Carey School of Business (Contributor) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2015-12
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Description

Widespread use of halogenated organic compounds for commercial and industrial purposes makes halogenated organic pollutants (HOPs) a global challenge for environmental quality. Current wastewater treatment plants (WWTPs) are successful at reducing chemical oxygen demand (COD), but the removal of HOPs often is poor. Since HOPs are xenobiotics, the biodegradation of

Widespread use of halogenated organic compounds for commercial and industrial purposes makes halogenated organic pollutants (HOPs) a global challenge for environmental quality. Current wastewater treatment plants (WWTPs) are successful at reducing chemical oxygen demand (COD), but the removal of HOPs often is poor. Since HOPs are xenobiotics, the biodegradation of HOPs is usually limited in the WWTPs. The current methods for HOPs treatments (e.g., chemical, photochemical, electrochemical, and biological methods) do have their limitations for practical applications. Therefore, a combination of catalytic and biological treatment methods may overcome the challenges of HOPs removal.This dissertation investigated a novel catalytic and biological synergistic platform to treat HOPs. 4-chlorophenol (4-CP) and halogenated herbicides were used as model pollutants for the HOPs removal tests. The biological part of experiments documented successful co-oxidation of HOPs and analog non-halogenated organic pollutants (OPs) (as the primary substrates) in the continuous operation of O2-based membrane biofilm reactor (O2-MBfR). In the first stage of the synergistic platform, HOPs were reductively dehalogenated to less toxic and more biodegradable OPs during continuous operation of a H2-based membrane catalytic-film reactor (H2-MCfR). The synergistic platform experiments demonstrated that OPs generated in the H2-MCfR were used as the primary substrates to support the co-oxidation of HOPs in the subsequent O2-MBfR. Once at least 90% conversation of HOPs to OPs was achieved in the H2-MCfR, the products (OPs to HOPs mole ratio >9) in the effluent could be completely mineralized through co-oxidation in O2-MBfR. By using H2 gas as the primary substrate, instead adding the analog OP, the synergistic platform greatly reduced chemical costs and carbon-dioxide emissions during HOPs co-oxidation.

ContributorsLuo, Yihao (Author) / Rittmann, Bruce (Thesis advisor) / Krajmalnik-Brown, Rosa (Committee member) / Torres, Cesar (Committee member) / Arizona State University (Publisher)
Created2022
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Description
In this work, secretion of free fatty acids (FFAs) and ω-hydroxy FFAs wasachieved in the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis), and FFAs were detected by a novel fluorescence assay. Current methods of detecting FFA concentrations, including HPLC-based and GC-based methods or enzyme-based kits, have hindered research advancement due to their laborious

In this work, secretion of free fatty acids (FFAs) and ω-hydroxy FFAs wasachieved in the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis), and FFAs were detected by a novel fluorescence assay. Current methods of detecting FFA concentrations, including HPLC-based and GC-based methods or enzyme-based kits, have hindered research advancement due to their laborious and/or expensive nature. The work herein establishes a novel, rapid, fluorescence-based assay for detecting total FFA concentrations secreted by Synechocystis FFA secretion strains. The novel FFA-detection assay demonstrates the efficacy of using Nile Red as a fluorescent reporter for laurate or palmitate at concentrations up to 500 µM in the presence of cationic surfactants. Total FFA concentrations in Synechocystis supernatants quantified by the novel, Nile Red fluorescence-based assay are demonstrated herein to be highly correlative to total FFA concentrations quantified by LC-MS; this correlation was seen in supernatant samples of wild type Synechocystis and Synechocystis FFA secretion strains, both in 96-well plates and 30-mL, aerated culture tubes. This work also establishes the expression of a cytochrome P450 fusion enzyme, CYP153A-CPRmut, or a monooxygenase system from Pseudomonas putida GPo1, AlkBGT, in FFA secretion strains of Synechocystis for the generation of ω-hydroxy laurate from laurate. After finding greatly increased ω-hydroxylation activity of CYP153A-CPRmut with concurrent superoxide dismutase and catalase overexpression, 55 or 1.5 µM of ω-hydroxy laurate were produced over five days by Synechocystis strains expressing CYP153A-CPRmut or AlkBGT, respectively. As further indication of the presence of reactive oxygen species affecting ω-hydroxy laurate production with Synechocystis strains expressing CYP153A-CPRmut, concentrations of ω-hydroxy laurate in the supernatant increased over two-fold in the presence of 250 µM of the anti-oxidant, methionine, in bench-scale cultures and in 96-well plate cultures. Additionally, a mutation at the 55th amino acid position in AlkB (tryptophan to cysteine; AlkBW55C), resulted in a more than two-fold shift in AlkB’s substrate preference from decanoate towards the desired substrate, laurate. As a result, Synechocystis expressing AlkBW55C could produce 5.9 µM ω-hydroxy laurate and 2.0 µM dodecanedioic acid over five days of growth.
ContributorsAshe, Christopher (Author) / Vermaas, Willem Fj (Thesis advisor, Committee member) / Wang, Xuan (Committee member) / Nielsen, David R (Committee member) / Misra, Rajeev (Committee member) / Arizona State University (Publisher)
Created2023
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Description
Widespread use of chlorinated solvents for commercial and industrial purposes makes co-occurring contamination by 1,1,1-trichloroethane (TCA), trichloroethene (TCE), and 1,4-dioxane (1,4-D) a serious problem for groundwater. TCE and TCA often are treated by reductive dechlorination, while 1,4-D resists reductive treatment. Aerobic bacteria are able to oxidize 1,4-D, but

Widespread use of chlorinated solvents for commercial and industrial purposes makes co-occurring contamination by 1,1,1-trichloroethane (TCA), trichloroethene (TCE), and 1,4-dioxane (1,4-D) a serious problem for groundwater. TCE and TCA often are treated by reductive dechlorination, while 1,4-D resists reductive treatment. Aerobic bacteria are able to oxidize 1,4-D, but the biological oxidation of 1,4-D could be inhibited TCA, TCE, and their reductive transformation products. To overcome the challenges from co-occurring contamination, I propose a two-stage synergistic system. First, anaerobic reduction of the chlorinated hydrocarbons takes place in a H2-based hollow-fiber “X-film” (biofilm or catalyst-coated film) reactor (MXfR), where “X-film” can be a “bio-film” (MBfR) or an abiotic “palladium-film” (MPfR). Then, aerobic removal of 1,4-D and other organic compounds takes place in an O2-based MBfR. For the reductive part, I tested reductive bio-dechlorination of TCA and TCE simultaneously in an MBfR. I found that the community of anaerobic bacteria can rapidly reduce TCE to cis-dichloroethene (cis-DCE), but further reductions of cis-DCE to vinyl chloride (VC) and VC to ethene were inhibited by TCA. Also, it took months to grow a strong biofilm that could reduce TCA and TCE. Another problem with reductive dechlorination in the MBfR is that mono-chloroethane (MCA) was not reduced to ethane. In contrast, a film of palladium nano-particles (PdNPs), i.e., an MPfR, could the simultaneous reductions of TCA and TCE to mainly ethane, with only small amounts of intermediates: 1,1-dichloroethane (DCA) (~3% of total influent TCA and TCE) and MCA (~1%) in continuous operation. For aerobic oxidation, I enriched an ethanotrophic culture that could oxidize 1,4-D with ethane as the primary electron donor. An O2-based MBfR, inoculated with the enriched ethanotrophic culture, achieved over 99% 1,4-D removal with ethane as the primary electron donor in continuous operation. Finally, I evaluated two-stage treatment with a H2-based MPfR followed by an O2-MBfR. The two-stage system gave complete removal of TCA, TCE, and 1,4-D in continuous operation.
ContributorsLuo, Yihao (Author) / Rittmann, Bruce E. (Committee member) / Krajmalnik-Brown, Rosa (Committee member) / Zhou, Chen (Committee member) / Arizona State University (Publisher)
Created2018
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Description
Synechocystis sp. PCC 6803 is a readily transformable cyanobacteria used to study cyanobacterial genetics, as well as production of biofuels, polyesters, and other industrial chemicals. Free fatty acids are precursors to biofuels which are used by Synechocystis cells as a means of energy storage. By genetically modifying the cyanobacteria to

Synechocystis sp. PCC 6803 is a readily transformable cyanobacteria used to study cyanobacterial genetics, as well as production of biofuels, polyesters, and other industrial chemicals. Free fatty acids are precursors to biofuels which are used by Synechocystis cells as a means of energy storage. By genetically modifying the cyanobacteria to expel these chemicals, costs associated with retrieving the products will be reduced; concurrently, the bacteria will be able to produce the products at a higher concentration. This is achieved by adding genes encoding components of the Escherichia coli AcrAB-TolC efflux system, part of the resistance-nodulation-division (RND) transporter family, to Synechocystis sp. PCC 6803. AcrAB-TolC is a relatively promiscuous multidrug efflux pump that is noted for expelling a wide range of substrates including dyes, organic solvents, antibiotics, and free fatty acids. Adding components of the AcrAB-TolC multidrug efflux pump to a previously created high free fatty acid producing strain, SD277, allowed cells to move more free fatty acids to the extracellular environment than did the parent strain. Some of these modifications also improved tolerance to antibiotics and a dye, rhodamine 6G. To confirm the function of this exogenous efflux pump, the genes encoding components of the AcrAB-TolC efflux pump were also added to Synechocystis sp. PCC 6803 and shown to grow on a greater concentration of various antibiotics and rhodamine 6G. Various endogenous efflux systems have been elucidated, but their usefulness in expelling products currently generated in Synechocystis is limited. Most of the elucidated pumps in the cyanobacteria are part of the ATP-binding cassette superfamily. The knowledge of the resistance-nodulation-division (RND) family transporters is limited. Two genes in Synechocystis sp. PCC 6803, slr2131 and sll0180 encoding homologs to the genes that encode acrB and acrA, respectively, were removed and the modifications resulted in changes in resistance to various antibiotics and a dye and also had an impact on free fatty acid secretion. Both of these deletions were complemented independently with the homologous E. coli gene and the resulting cyanobacteria strains had some of the inherent resistance restored to chloramphenicol and free fatty acid secretion was modified when compared to the wild-type and a high free fatty acid producing strain.
ContributorsBellefleur, Matthew Paul Allen (Author) / Curtiss, III, Roy (Thesis advisor) / Nielsen, David R (Committee member) / Wang, Xuan (Committee member) / Rittmann, Bruce E. (Committee member) / Arizona State University (Publisher)
Created2018
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Description
Photosynthesis converts sunlight to biomass at a global scale. Among the photosynthetic organisms, cyanobacteria provide an excellent model to study how photosynthesis can become a practical platform of large-scale biotechnology. One novel approach involves metabolically engineering the cyanobacterium Synechocystis sp. PCC 6803 to excrete laurate, which is harvested

Photosynthesis converts sunlight to biomass at a global scale. Among the photosynthetic organisms, cyanobacteria provide an excellent model to study how photosynthesis can become a practical platform of large-scale biotechnology. One novel approach involves metabolically engineering the cyanobacterium Synechocystis sp. PCC 6803 to excrete laurate, which is harvested directly.

This work begins by defining a working window of light intensity (LI). Wild-type and laurate-excreting Synechocystis required an LI of at least 5 µE/m2-s to sustain themselves, but are photo-inhibited by LI of 346 to 598 µE/m2-s.

Fixing electrons into valuable organic products, e.g., biomass and excreted laurate, is critical to success. Wild-type Synechocystis channeled 75% to 84% of its fixed electrons to biomass; laurate-excreting Synechocystis fixed 64 to 69% as biomass and 6.6% to 10% as laurate. This means that 16 to 30% of the electrons were diverted to non-valuable soluble products, and the trend was accentuated with higher LI.

How the Ci concentration depended on the pH and the nitrogen source was quantified by the proton condition and experimentally validated. Nitrate increased, ammonium decreased, but ammonium nitrate stabilized alkalinity and Ci. This finding provides a mechanistically sound tool to manage Ci and pH independently.

Independent evaluation pH and Ci on the growth kinetics of Synechocystis showed that pH 8.5 supported the fastest maximum specific growth rate (µmax): 2.4/day and 1.7/day, respectively, for the wild type and modified strains with LI of 202 µE/m2-s. Half-maximum-rate concentrations (KCi) were less than 0.1 mM, meaning that Synechocystis should attain its µmax with a modest Ci concentration (≥1.0 mM).

Biomass grown with day-night cycles had a night endogenous decay rate of 0.05-1.0/day, with decay being faster with higher LI and the beginning of dark periods. Supplying light at a fraction of daylight reduced dark decay rate and improved overall biomass productivity.

This dissertation systematically evaluates and synthesizes fundamental growth factors of cyanobacteria: light, inorganic carbon (Ci), and pH. LI remains the most critical growth condition to promote biomass productivity and desired forms of biomass, while Ci and pH now can be managed to support optimal productivity.
ContributorsNguyen, Binh Thanh (Author) / Rittmann, Bruce E. (Thesis advisor) / Krajmalnik-Brown, Rosa (Committee member) / Westerhoff, Paul (Committee member) / Arizona State University (Publisher)
Created2015
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Description
This study reports on benzene and toluene biodegradation under different dissolved oxygen conditions, and the goal of this study is to evaluate and model their removal.

Benzene and toluene were tested for obligate anaerobic degradation in batch reactors with sulfate as the electron acceptor. A group of sulfate-reducing bacteria capable

This study reports on benzene and toluene biodegradation under different dissolved oxygen conditions, and the goal of this study is to evaluate and model their removal.

Benzene and toluene were tested for obligate anaerobic degradation in batch reactors with sulfate as the electron acceptor. A group of sulfate-reducing bacteria capable of toluene degradation was enriched after 252 days of incubation. Those cultures, originated from anaerobic digester, were able to degrade toluene coupled to sulfate reduction with benzene coexistence, while they were not able to utilize benzene. Methanogens also were present, although their contribution to toluene biodegradation was not defined.

Aerobic biodegradation of benzene and toluene by Pseudomonas putida F1 occurred, and biomass production lagged behind substrate loss and continued after complete substrate removal. This pattern suggests that biodegradation of intermediates, rather than direct benzene and toluene transformation, caused bacterial growth. Supporting this explanation is that the calculated biomass growth from a two-step model basically fit the experimental biomass results during benzene and toluene degradation with depleted dissolved oxygen.

Catechol was tested for anaerobic biodegradation in batch experiments and in a column study. Sulfate- and nitrate-reducing bacteria enriched from a wastewater treatment plant hardly degraded catechol within 20 days. However, an inoculum from a contaminated site was able to remove 90% of the initial 16.5 mg/L catechol, and Chemical Oxygen Demand was oxidized in parallel. Catechol biodegradation was inhibited when nitrite accumulated, presumably by a toxic catechol-nitrite complex.

The membrane biofilm reactor (MBfR) offers the potential for biodegrading benzene in a linked aerobic and anaerobic pathway by controlling the O2 delivery. At an average benzene surface loading of 1.3 g/m2-day and an average hydraulic retention time of 2.2 day, an MBfR supplied with pure O2 successfully achieved 99% benzene removal at steady state. A lower oxygen partial pressure led to decreased benzene removal, and nitrate removal increased, indicating multiple mechanisms, including oxygenation and nitrate reduction, were involved in the system being responsible for benzene removal. Microbial community analysis indicated that Comamonadaceae, a known aerobic benzene-degrader and denitrifier, dominated the biofilm at the end of operation.
ContributorsLiu, Zhuolin (Author) / Rittmann, Bruce E. (Thesis advisor) / Krajmalnik-Brown, Rosa (Committee member) / Fox, Peter (Committee member) / Arizona State University (Publisher)
Created2015