Matching Items (7)
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- All Subjects: Photosynthetic bacteria
- Creators: Krajmalnik-Brown, Rosa
Description
Reductive dechlorination by members of the bacterial genus Dehalococcoides is a common and cost-effective avenue for in situ bioremediation of sites contaminated with the chlorinated solvents, trichloroethene (TCE) and perchloroethene (PCE). The overarching goal of my research was to address some of the challenges associated with bioremediation timeframes by improving the rates of reductive dechlorination and the growth of Dehalococcoides in mixed communities. Biostimulation of contaminated sites or microcosms with electron donor fails to consistently promote dechlorination of PCE/TCE beyond cis-dichloroethene (cis-DCE), even when the presence of Dehalococcoides is confirmed. Supported by data from microcosm experiments, I showed that the stalling at cis-DCE is due a H2 competition in which components of the soil or sediment serve as electron acceptors for competing microorganisms. However, once competition was minimized by providing selective enrichment techniques, I illustrated how to obtain both fast rates and high-density Dehalococcoides using three distinct enrichment cultures. Having achieved a heightened awareness of the fierce competition for electron donor, I then identified bicarbonate (HCO3-) as a potential H2 sink for reductive dechlorination. HCO3- is the natural buffer in groundwater but also the electron acceptor for hydrogenotrophic methanogens and homoacetogens, two microbial groups commonly encountered with Dehalococcoides. By testing a range of concentrations in batch experiments, I showed that methanogens are favored at low HCO3 and homoacetogens at high HCO3-. The high HCO3- concentrations increased the H2 demand which negatively affected the rates and extent of dechlorination. By applying the gained knowledge on microbial community management, I ran the first successful continuous stirred-tank reactor (CSTR) at a 3-d hydraulic retention time for cultivation of dechlorinating cultures. I demonstrated that using carefully selected conditions in a CSTR, cultivation of Dehalococcoides at short retention times is feasible, resulting in robust cultures capable of fast dechlorination. Lastly, I provide a systematic insight into the effect of high ammonia on communities involved in dechlorination of chloroethenes. This work documents the potential use of landfill leachate as a substrate for dechlorination and an increased tolerance of Dehalococcoides to high ammonia concentrations (2 g L-1 NH4+-N) without loss of the ability to dechlorinate TCE to ethene.
ContributorsDelgado, Anca Georgiana (Author) / Krajmalnik-Brown, Rosa (Thesis advisor) / Cadillo-Quiroz, Hinsby (Committee member) / Halden, Rolf U. (Committee member) / Rittmann, Bruce E. (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2013
Description
Microbial electrochemical cells (MXCs) are promising platforms for bioenergy production from renewable resources. In these systems, specialized anode-respiring bacteria (ARB) deliver electrons from oxidation of organic substrates to the anode of an MXC. While much progress has been made in understanding the microbiology, physiology, and electrochemistry of well-studied model ARB such as Geobacter and Shewanella, tremendous potential exists for MXCs as microbiological platforms for exploring novel ARB. This dissertation introduces approaches for selective enrichment and characterization of phototrophic, halophilic, and alkaliphilic ARB. An enrichment scheme based on manipulation of poised anode potential, light, and nutrient availability led to current generation that responded negatively to light. Analysis of phototrophically enriched communities suggested essential roles for green sulfur bacteria and halophilic ARB in electricity generation. Reconstruction of light-responsive current generation could be successfully achieved using cocultures of anode-respiring Geobacter and phototrophic Chlorobium isolated from the MXC enrichments. Experiments lacking exogenously supplied organic electron donors indicated that Geobacter could produce a measurable current from stored photosynthate in the dark. Community analysis of phototrophic enrichments also identified members of the novel genus Geoalkalibacter as potential ARB. Electrochemical characterization of two haloalkaliphilic, non-phototrophic Geoalkalibacter spp. showed that these bacteria were in fact capable of producing high current densities (4-8 A/m2) and using higher organic substrates under saline or alkaline conditions. The success of these selective enrichment approaches and community analyses in identifying and understanding novel ARB capabilities invites further use of MXCs as robust platforms for fundamental microbiological investigations.
ContributorsBadalamenti, Jonathan P (Author) / Krajmalnik-Brown, Rosa (Thesis advisor) / Garcia-Pichel, Ferran (Committee member) / Rittmann, Bruce E. (Committee member) / Torres, César I (Committee member) / Vermaas, Willem (Committee member) / Arizona State University (Publisher)
Created2013
Description
Contamination by chlorinated ethenes is widespread in groundwater aquifers, sediment, and soils worldwide. The overarching objectives of my research were to understand how the bacterial genus Dehalococcoides function optimally to carry out reductive dechlorination of chlorinated ethenes in a mixed microbial community and then apply this knowledge to manage dechlorinating communities in the hydrogen-based membrane biofilm reactor (MBfR). The MBfR is used for the biological reduction of oxidized contaminants in water using hydrogen supplied as the electron donor by diffusion through gas-transfer fibers. First, I characterized a new anaerobic dechlorinating community developed in our laboratory, named DehaloR^2, in terms of chlorinated ethene turnover rates and assessed its microbial community composition. I then carried out an experiment to correlate performance and community structure for trichloroethene (TCE)-fed microbial consortia. Fill-and-draw reactors inoculated with DehaloR^2 demonstrated a direct correlation between microbial community function and structure as the TCE-pulsing rate was increased. An electron-balance analysis predicted the community structure based on measured concentrations of products and constant net yields for each microorganism. The predictions corresponded to trends in the community structure based on pyrosequencing and quantitative PCR up to the highest TCE pulsing rate, where deviations to the trend resulted from stress by the chlorinated ethenes. Next, I optimized a method for simultaneous detection of chlorinated ethenes and ethene at or below the Environmental Protection Agency maximum contaminant levels for groundwater using solid phase microextraction in a gas chromatograph with a flame ionization detector. This method is ideal for monitoring biological reductive dechlorination in groundwater, where ethene is the ultimate end product. The major advantage of this method is that it uses a small sample volume of 1 mL, making it ideally suited for bench-scale feasibility studies, such as the MBfR. Last, I developed a reliable start-up and operation strategy for TCE reduction in the MBfR. Successful operation relied on controlling the pH-increase effects of methanogenesis and homoacetogenesis, along with creating hydrogen limitation during start-up to allow dechlorinators to compete against other microorgansims. Methanogens were additionally minimized during continuous flow operation by a limitation in bicarbonate resulting from strong homoacetogenic activity.
ContributorsZiv-El, Michal (Author) / Rittmann, Bruce E. (Thesis advisor) / Krajmalnik-Brown, Rosa (Thesis advisor) / Halden, Rolf U. (Committee member) / Arizona State University (Publisher)
Created2012
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 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.
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
Description
Creating sustainable alternatives to fossil fuel resources is one of the greatest
challenges facing mankind. Solar energy provides an excellent option to alleviate modern dependence on fossil fuels. However, efficient methods to harness solar energy are still largely lacking. Biomass from photosynthetic organisms can be used as feedstock to produce traditional fuels, but must be produced in great quantities in order to meet the demands of growing populations. Cyanobacteria are prokaryotic photosynthetic microorganisms that can produce biomass on large scales using only sunlight, carbon dioxide, water, and small amounts of nutrients. Thus, Cyanobacteria are a viable option for sustainable production of biofuel feedstock material. Photobioreactors (PBRs) offer a high degree of control over the temperature, aeration, and mixing of cyanobacterial cultures, but cannot be kept sterile due to the scales necessary to meet domestic and global energy demands, meaning that heterotrophic bacteria can grow in PBRs by oxidizing the organic material produced and excreted by the Cyanobacteria. These heterotrophic bacteria can positively or negatively impact the performance of the PBR through their interactions with the Cyanobacteria. This work explores the microbial ecology in PBR cultures of the model cyanobacterium Synechocystis sp. PCC6803 (Synechocystis) using microbiological, molecular, chemical, and engineering techniques. I first show that diverse phylotypes of heterotrophic bacteria can associate with Synechocystis-based PBRs and that excluding them may be impossible under typical PBR operating conditions. Then, I demonstrate that high-throughput sequencing can reliably elucidate the structure of PBR microbial communities without the need for pretreatment to remove Synechocystis 16S rRNA genes, despite the high degree of polyploidy found in Synechocystis. Next, I establish that the structure of PBR microbial communities is strongly influenced by the microbial community of the inoculum culture. Finally, I show that maintaining available phosphorus in the culture medium promotes the production and enrichment of Synechocystis biomass in PBRs by reducing the amount of soluble substrates available to heterotrophic bacteria. This work presents the first analysis of the structure and function of microbial communities associated with Synechocystis-based PBRs.
challenges facing mankind. Solar energy provides an excellent option to alleviate modern dependence on fossil fuels. However, efficient methods to harness solar energy are still largely lacking. Biomass from photosynthetic organisms can be used as feedstock to produce traditional fuels, but must be produced in great quantities in order to meet the demands of growing populations. Cyanobacteria are prokaryotic photosynthetic microorganisms that can produce biomass on large scales using only sunlight, carbon dioxide, water, and small amounts of nutrients. Thus, Cyanobacteria are a viable option for sustainable production of biofuel feedstock material. Photobioreactors (PBRs) offer a high degree of control over the temperature, aeration, and mixing of cyanobacterial cultures, but cannot be kept sterile due to the scales necessary to meet domestic and global energy demands, meaning that heterotrophic bacteria can grow in PBRs by oxidizing the organic material produced and excreted by the Cyanobacteria. These heterotrophic bacteria can positively or negatively impact the performance of the PBR through their interactions with the Cyanobacteria. This work explores the microbial ecology in PBR cultures of the model cyanobacterium Synechocystis sp. PCC6803 (Synechocystis) using microbiological, molecular, chemical, and engineering techniques. I first show that diverse phylotypes of heterotrophic bacteria can associate with Synechocystis-based PBRs and that excluding them may be impossible under typical PBR operating conditions. Then, I demonstrate that high-throughput sequencing can reliably elucidate the structure of PBR microbial communities without the need for pretreatment to remove Synechocystis 16S rRNA genes, despite the high degree of polyploidy found in Synechocystis. Next, I establish that the structure of PBR microbial communities is strongly influenced by the microbial community of the inoculum culture. Finally, I show that maintaining available phosphorus in the culture medium promotes the production and enrichment of Synechocystis biomass in PBRs by reducing the amount of soluble substrates available to heterotrophic bacteria. This work presents the first analysis of the structure and function of microbial communities associated with Synechocystis-based PBRs.
ContributorsZevin, Alexander Simon (Author) / Rittmann, Bruce E. (Thesis advisor) / Krajmalnik-Brown, Rosa (Thesis advisor) / Vermaas, Willem Fj (Committee member) / Arizona State University (Publisher)
Created2015
Description
Large-scale cultivation of photosynthetic microorganisms for the production of biodiesel and other valuable commodities must be made more efficient. Recycling the water and nutrients acquired from biomass harvesting promotes a more sustainable and economically viable enterprise. This study reports on growing the cyanobacterium Synechocystis sp. PCC 6803 using permeate obtained from concentrating the biomass by cross-flow membrane filtration. I used a kinetic model based on the available light intensity (LI) to predict biomass productivity and evaluate overall performance.
During the initial phase of the study, I integrated a membrane filter with a bench-top photobioreactor (PBR) and created a continuously operating system. Recycling permeate reduced the amount of fresh medium delivered to the PBR by 45%. Biomass production rates as high as 400 mg-DW/L/d (9.2 g-DW/m2/d) were sustained under constant lighting over a 12-day period.
In the next phase, I operated the system as a sequencing batch reactor (SBR), which improved control over nutrient delivery and increased the concentration factor of filtered biomass (from 1.8 to 6.8). I developed unique system parameters to compute the amount of recycled permeate in the reactor and the actual hydraulic retention time during SBR operation. The amount of medium delivered to the system was reduced by up to 80%, and growth rates were consistent at variable amounts of repeatedly recycled permeate. The light-based model accurately predicted growth when biofilm was not present. Coupled with mass ratios for PCC 6803, these predictions facilitated efficient delivery of nitrogen and phosphorus. Daily biomass production rates and specific growth rates equal to 360 mg-DW/L/d (8.3 g/m2/d) and 1.0 d-1, respectively, were consistently achieved at a relatively low incident LI (180 µE/m2/s). Higher productivities (up to 550 mg-DW/L/d) occurred under increased LI (725 µE/m2/s), although the onset of biofilm impeded modeled performance.
Permeate did not cause any gradual growth inhibition. Repeated results showed cultures rapidly entered a stressed state, which was followed by widespread cell lysis. This phenomenon occurred independently of permeate recycling and was not caused by nutrient starvation. It may best be explained by negative allelopathic effects or viral infection as a result of mixed culture conditions.
During the initial phase of the study, I integrated a membrane filter with a bench-top photobioreactor (PBR) and created a continuously operating system. Recycling permeate reduced the amount of fresh medium delivered to the PBR by 45%. Biomass production rates as high as 400 mg-DW/L/d (9.2 g-DW/m2/d) were sustained under constant lighting over a 12-day period.
In the next phase, I operated the system as a sequencing batch reactor (SBR), which improved control over nutrient delivery and increased the concentration factor of filtered biomass (from 1.8 to 6.8). I developed unique system parameters to compute the amount of recycled permeate in the reactor and the actual hydraulic retention time during SBR operation. The amount of medium delivered to the system was reduced by up to 80%, and growth rates were consistent at variable amounts of repeatedly recycled permeate. The light-based model accurately predicted growth when biofilm was not present. Coupled with mass ratios for PCC 6803, these predictions facilitated efficient delivery of nitrogen and phosphorus. Daily biomass production rates and specific growth rates equal to 360 mg-DW/L/d (8.3 g/m2/d) and 1.0 d-1, respectively, were consistently achieved at a relatively low incident LI (180 µE/m2/s). Higher productivities (up to 550 mg-DW/L/d) occurred under increased LI (725 µE/m2/s), although the onset of biofilm impeded modeled performance.
Permeate did not cause any gradual growth inhibition. Repeated results showed cultures rapidly entered a stressed state, which was followed by widespread cell lysis. This phenomenon occurred independently of permeate recycling and was not caused by nutrient starvation. It may best be explained by negative allelopathic effects or viral infection as a result of mixed culture conditions.
ContributorsThompson, Matthew (Author) / Rittmann, Bruce E. (Thesis advisor) / Fox, Peter (Committee member) / Krajmalnik-Brown, Rosa (Committee member) / Arizona State University (Publisher)
Created2015
Description
DehaloR^2 is a previously characterized, trichloroethene (TCE)-dechlorinating culture and contains bacteria from the known dechlorinating genus, Dehalococcoides. DehaloR^2 was exposed to three anthropogenic contaminants, Triclocarban (TCC), tris(2-chloroethyl) phosphate (TCEP), and 1,1,1-trichloroethane (TCA) and two biogenic-like halogenated compounds, 2,6-dibromophenol (2,6-DBP) and 2,6-dichlorophenol (2,6-DCP). The effects on TCE dechlorination ability due to 2,6-DBP and 2,6-DCP exposures were also investigated. DehaloR^2 did not dechlorinate TCC or TCEP. After initial exposure to TCA, half of the initial TCA was dechlorinated to 1,1-dichloroethane (DCA), however half of the TCA remained by day 100. Subsequent TCA and TCE re-exposure showed no reductive dechlorination activity for both TCA and TCE by 120 days after the re-exposure. It has been hypothesized that the microbial TCE-dechlorinating ability was developed before TCE became abundant in groundwater. This dechlorinating ability would have existed in the microbial metabolism due to previous exposure to biogenic halogenated compounds. After observing the inability of DehaloR^2 to dechlorinate other anthropogenic compounds, DehaloR^2 was then exposed to two naturally occurring halogenated phenols, 2,6-DBP and 2,6-DCP, in the presence and absence of TCE. DehaloR^2 debrominated 2,6-DBP through the intermediate 2-bromophenol (2-BP) to the end product phenol faster in the presence of TCE. DehaloR^2 dechlorinated 2,6-DCP to 2-CP in the absence of TCE; however, 2,6-DCP dechlorination was incomplete in the presence of TCE. Additionally, when 2,6-DBP was present, complete TCE dechlorination to ethene occurred more quickly than when TCE was present without 2,6-DBP. However, when 2,6-DCP was present, TCE dechlorination to ethene had not completed by day 55. The increased dehalogenation rate of 2,6-DBP and TCE when present together compared to conditions containing only 2,6-DBP or only TCE suggests a possible synergistic relationship between 2,6-DBP and TCE, while the decreased dechlorination rate of 2,6-DCP and TCE when present together compared to conditions containing only 2,6-DCP or only TCE suggests an inhibitory effect.
ContributorsKegerreis, Kylie (Author) / Krajmalnik-Brown, Rosa (Thesis advisor) / Halden, Rolf U. (Committee member) / Torres, César I (Committee member) / Arizona State University (Publisher)
Created2012