Selective Enrichment Yields Robust Ethene-Producing Dechlorinating Cultures from Microcosms Stalled at cis-Dichloroethene

abstract: Dehalococcoides mccartyi strains are of particular importance for bioremediation due to their unique capability of transforming perchloroethene (PCE) and trichloroethene (TCE) to non-toxic ethene, through the intermediates cis-dichloroethene (cis-DCE) and vinyl chloride (VC). Despite the widespread environmental distribution of Dehalococcoides, biostimulation sometimes fails to promote dechlorination beyond cis-DCE. In our study, microcosms established with garden soil and mangrove sediment also stalled at cis-DCE, albeit Dehalococcoides mccartyi containing the reductive dehalogenase genes tceA, vcrA and bvcA were detected in the soil/sediment inocula. Reductive dechlorination was not promoted beyond cis-DCE, even after multiple biostimulation events with fermentable substrates and a lengthy incubation. However, transfers from microcosms stalled at cis-DCE yielded dechlorination to ethene with subsequent enrichment cultures containing up to 10[superscript 9] Dehalococcoides mccartyi cells mL[superscript −1]. Proteobacterial classes which dominated the soil/sediment communities became undetectable in the enrichments, and methanogenic activity drastically decreased after the transfers. We hypothesized that biostimulation of Dehalococcoides in the cis-DCE-stalled microcosms was impeded by other microbes present at higher abundances than Dehalococcoides and utilizing terminal electron acceptors from the soil/sediment, hence, outcompeting Dehalococcoides for H[subscript 2]. In support of this hypothesis, we show that garden soil and mangrove sediment microcosms bioaugmented with their respective cultures containing Dehalococcoides in high abundance were able to compete for H[subscript 2] for reductive dechlorination from one biostimulation event and produced ethene with no obvious stall. Overall, our results provide an alternate explanation to consolidate conflicting observations on the ubiquity of Dehalococcoides mccartyi and occasional stalling of dechlorination at cis-DCE; thus, bringing a new perspective to better assess biological potential of different environments and to understand microbial interactions governing bioremediation.The article is published at http://journals.plos.org/plosone/article?id=10.1371/journal.pone.010065

Microbial community structure elucidates performance of Glyceria maxima plant microbial fuel cell

The plant microbial fuel cell (PMFC) is a technology in which living plant roots provide electron donor, via rhizodeposition, to a mixed microbial community to generate electricity in a microbial fuel cell. Analysis and localisation of the microbial community is necessary for gaining insight into the competition for electron donor in a PMFC. This paper characterises the anode–rhizosphere bacterial community of a Glyceria maxima (reed mannagrass) PMFC. Electrochemically active bacteria (EAB) were located on the root surfaces, but they were more abundant colonising the graphite granular electrode. Anaerobic cellulolytic bacteria dominated the area where most of the EAB were found, indicating that the current was probably generated via the hydrolysis of cellulose. Due to the presence of oxygen and nitrate, short-chain fatty acid-utilising denitrifiers were the major competitors for the electron donor. Acetate-utilising methanogens played a minor role in the competition for electron donor, probably due to the availability of graphite granules as electron acceptors

Ecological Interactions Among Nitrate-, Perchlorate-, and Sulfate-Reducing Bacteria in Hydrogen-Fed Biofilm Reactors

abstract: Water contamination with nitrate (NO3−) (from fertilizers) and perchlorate (ClO4−) (from rocket fuel and explosives) is a widespread environmental problem. I employed the Membrane Biofilm Reactor (MBfR), a novel bioremediation technology, to treat NO3− and ClO4− in the presence of naturally occurring sulfate (SO42−). In the MBfR, bacteria reduce oxidized pollutants that act as electron acceptors, and they grow as a biofilm on the outer surface of gas-transfer membranes that deliver the electron donor (hydrogen gas, (H2). The overarching objective of my research was to achieve a comprehensive understanding of ecological interactions among key microbial members in the MBfR when treating polluted water with NO3− and ClO4− in the presence of SO42−. First, I characterized competition and co-existence between denitrifying bacteria (DB) and sulfate-reducing bacteria (SRB) when the loading of either the electron donor or electron acceptor was varied. Then, I assessed the microbial community structure of biofilms mostly populated by DB and SRB, linking structure with function based on the electron-donor bioavailability and electron-acceptor loading. Next, I introduced ClO4− as a second oxidized contaminant and discovered that SRB harm the performance of perchlorate-reducing bacteria (PRB) when the aim is complete ClO4− destruction from a highly contaminated groundwater. SRB competed too successfully for H2 and space in the biofilm, forcing the PRB to unfavorable zones in the biofilm. To better control SRB, I tested a two-stage MBfR for total ClO4− removal from a groundwater highly contaminated with ClO4−. I document successful remediation of ClO4− after controlling SO4 2− reduction by restricting electron-donor availability and increasing the acceptor loading to the second stage reactor. Finally, I evaluated the performance of a two-stage pilot MBfR treating water polluted with NO3− and ClO4−, and I provided a holistic understanding of the microbial community structure and diversity. In summary, the microbial community structure in the MBfR contributes to and can be used to explain/predict successful or failed water bioremediation. Based on this understanding, I developed means to manage the microbial community to achieve desired water-decontamination results. This research shows the benefits of looking "inside the box" for "improving the box".Dissertation/ThesisPh.D. Sustainability 201

Bioprocess Monitoring and Control

Process monitoring and control are fundamental to all processes; this holds especially for bioprocesses, due to their complex nature. Usually, bioprocesses deal with living cells, which have their own regulatory systems. It helps to adjust the cell to its environmental condition. This must not be the optimal condition that the cell needs to produce whatever is desired. Therefore, a close monitoring of the cell and its environment is essential to provide optimal conditions for production. Without measurement, no information of the current process state is obtained. In this book, methods and techniques are provided for the monitoring and control of bioprocesses. From new developments for sensors, the application of spectroscopy and modelling approaches, the estimation and observer implementation for ethanol production and the development and scale-up of various bioprocesses and their closed loop control information are presented. The processes discussed here are very diverse. The major applications are cultivation processes, where microorganisms were grown, but also an incubation process of bird’s eggs, as well as an indoor climate control for humans, will be discussed. Altogether, in 12 chapters, nine original research papers and three reviews are presented

Transient optical studies of photoinduced charge transfer in semiconductor quantum dot solar cells

Semiconductor quantum dots (also referred to as 'nanocrystals‘) are well suited as light-harvesting agents in solar cells because they are robust, have tuneable effective band gaps, and are easy to process. The research presented in this thesis is targeted towards the study of excitonic solar cells employing semiconductor nanocrystals as a light harvesting component. Gaining control of the interfacial charge transfer processes in operation in these devices forms a crucial part of any attempt to optimise their performance. In particular, the use of transient spectroscopic techniques reveals how efficient and long-lived charge separation can be achieved in these solar cell architectures. The primary focus of this research is to investigate the parameters influencing charge transfer in dye-sensitised solar cells (DSSCs) using colloidal quantum dots as light-absorbers. One aim is to study the impact of varying the thermodynamic driving forces provided for interfacial electron transfer on the yield of both the electron injection and hole regeneration reactions occurring within the DSSC; this can be achieved by varying the energetics of each component of the system (metal oxide, quantum dot and hole conductor) in turn. In addition, the interfacial morphology can be modulated by changing the passivating ligands present at the QD surface, and by modifying the structure of the redox mediator (or hole conductor). In doing so, we also attempt to improve our understanding of how charge carrier trapping in quantum dots impacts upon solar cell performance. Furthermore, new strategies towards solar cell design are presented, which show great potential as a result of their favourable photophysical properties. One of these approaches (presented in the final chapter) is to effect the in situ growth of CdS nanocrystals in a conducting polymer, a method which circumvents many of the processing issues associated with the use of nanocrystals in polymer blend solar cell architectures. It is hoped that the work presented in this thesis is used to develop design rules for the construction of semiconductor nanocrystal-based excitonic solar cells. By identifying which key parameters control the rates and yields of electron transfer at the nanocrystal interface, improvements in device efficiency can be realised. It is believed that these studies fill an important gap in our current understanding, and highlight some of the potential benefits and shortcomings of using semiconductor nanocrystals in cheap, solution-processed solar cells

Analysis Of Methanogenic Archael Populations In Anoxic Sediments Of The Marmara Sea Using Fluorescent In Situ Hybridization

Tez (Yüksek Lisans) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2006Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2006Bu çalışmada, Marmara Denizi’ndeki farklı noktalardan alınan (Gemlik Körfezi İzmit Körfezi ve Küçükçekmece) anoksik sedimentlerde metanojenik arkeal populasyonlar kültürden bağımsız moleküler metot, floresanli yerinde hibritleşme (FISH), kullanılarak sayıldı ve tanımlandı. Ayrıca sediment örneklerinin organik ve inorganik içerikleri araştırıldı ve sonuçlar sediment örneklerinin arkeal topluluk yapısıyla tarıtışıldı. Gemlik Körfezi, Izmit Körfezi (Iz17), Izmit Körfezi (Iz25) ve Küçükçekmece’den alınan sedimentlerdeki DAPI ile boyanmış toplam mikroorganizmaların sırasıyla 67%±8%, 62%±5%, 77%±11% ve 80%±7%’i universal prob kullanılarak tespit edildi. FISH sonuçlarına göre Gemlik Körfezi, Izmit Körfezi (Iz17), Izmit Körfezi (Iz25) ve Küçükçekmece’den alınan sedimentlerdeki arkeal hücrelerin göreceli bolluğu sırasıyla 32%±4%, 23%±3%, 33%±2% ve 29%±7%’dir. Gemlik Körfezi, Izmit Körfezi (Iz17), Izmit Körfezi (Iz25) ve Küçükçekmece’den alınan anoksik sedimentlerdeki toplam metanojenlerin 19%, 21%, 23% ve 20%’ si H2 kullanan metanojenlerdir buna karsi bunlarin 7%, 9%, 10% ve 11%’ i asetoklastik metanojenlerdir. Bu sonuçlar Marmara Denizi’ndeki anoksik sedimentlerde aktif bir arkeal populasyonun olduğunu ve baskın olarak H2+CO2’ye bağımlı metanojenesisin önemli seviyelerde gerçekleştiğini göstermektirIn this study, methanogenic archaeal populations in anoxic sediments from different regions of the Marmara Sea (Gemlik Bay, Izmit Bay and Kucukcekmece) were identified and quantified using a cultivation independent molecular method, Fluorescent in situ hybridization (FISH). Organic and inorganic content of the sediment samples were also investigated and the results were discussed along with the archaeal community structure of the sediment samples. 67%±8%, 62%±5%, 77%±11% and 80%±7% of DAPI stained total microorganisms in sediments from Gemlik Bay, Izmit Bay (Iz17), Izmit Bay (Iz25) and Kucukcekmece respectively were detected using universal probe in this study. According to FISH results, relative abundances of archaeal cells within the sediments from Gemlik Bay, Izmit Bay (Iz17), Izmit Bay (Iz25) and Kucukcekmece are 32%±4%, 23%±3%, 33%±2% and 29%±7% respectively. 19%, 20%, 21% and 23% of the total methanogens in anoxic sediments from Gemlik Bay, Kucukcekmece, Izmit Bay (Iz17) and Izmit Bay (Iz25) are H2 utilizing methanogens whereas 7%, 11%, 9% and 10% of them are acetoclastic methanogens. . These results revealed that there are active archaeal populations within the the anoxic sediments from the Marmara Sea, and methanogenesis occurs in the sediments at important levels , dominantly H2+CO2 dependentYüksek LisansM.Sc

Microcosm Evaluation of Natural and Biologically-enhanced Abiotic Transformation of Chlorinated Ethenes in Low Permeability Formations

Trichloroethene (TCE) is one of the most commonly found hazardous compounds at Superfund sites, especially in groundwater. Its volatility and toxicity pose a threat to drinking water safety and human health. In the past decades, research on the fate of TCE in the environment has shifted to contamination of low-permeability formations, for example fractured bedrock aquifers. TCE back-diffusion from low permeability zones and management of persistent TCE groundwater plumes caused by this long-term source zone pose major challenges for remediation. Monitored natural attenuation (MNA) is a cost-effective remediation strategy that has gradually gained acceptance by regulators. Compared to extensive active remediation of persistent TCE plumes, MNA is more attractive for its cost-effectiveness. However, acceptance of MNA as a remediation strategy depends on adequate documentation of in situ TCE degradation at meaningful rates. Collecting lines of evidence in support of TCE natural degradation is an essential component of MNA. In this study, two lines of evidence were collected for three Department of Defense sites overlain with fractured bedrock that are experiencing TCE back-diffusion. The first type of evidence involved monitoring groundwater for dissolved gas products associated with abiotic degradation (i.e., acetylene, ethene, and ethane) using a novel passive vapor diffusion (PVD) sampler. The other line of evidence was based on the use of intact rock core microcosms to estimate TCE degradation rate constants. This is the first study to employ the use of 14C-labeled TCE in intact rock core microcosms. Estimating the TCE degradation rate constants was accomplished with numerical modeling

Electricity generation by living plants in a plant microbial fuel cell

Society is facing local and global challenges to secure needs of people. One of those needs is the increasing demand of energy. Currently most energy is generated by conversion of fossil fuels. The major drawback of using fossil fuels is pollution of the environment by emission of carbon dioxide, nitrogen oxides, sulfur dioxide, volatile organic compounds, heavy metals, and fine particles. Furthermore fossil fuels are not renewable in a time scale in the order of decades. The microbial solar cell (MSC) is a new collective name of biotechnological systems that integrate photosynthetic and electrochemically active organisms to generate electricity in a clean and renewable manner. Among the MSCs, the plant microbial fuel cell (PMFC) that employs higher plants, is the most promising MSCs. In PMFCs, plant roots provide substrate for electrochemically active bacteria in the anode by the loss of organic compounds. In natural environments plant roots loose organic compound by diffusion through the cell membrane, or release organic compounds in order to acquire necessary nutrient. In both cases these organic compounds are an energy source for micro-organisms. In the PMFC these lost or released organic compounds are partly utilized by electrochemically active bacteria. During the oxidation of these organic compounds s electrochemically active bacteria transfer electrons to the anode electrode and produce protons and carbon dioxide. The electrons flow via a power harvester to the cathode compartment where the electrons are consumed by typically oxygen reduction. The aim of this thesis was to characterize the PMFC biologically and electrochemically and to improve the design towards higher applicable power outputs. The approach of this thesis was to understand processes in the PMFC which limit electrical power generation and use these findings to improve electrical power generation and the applicability of the PMFC design.</p

Synthesis and Chemistry of Kinamycins and Related Antibiotics

The kinamycin antitumor antibiotics, discovered in Japan in the early 1970s as secondary metabolites of the soil bacterium Streptomyces murayamaensis, were at first believed to be derivatives of the N-cyanobenzo[b]carbazole ring system. In 1994, studies in this laboratory revealed that the initial structural assignment was incorrect. The true structures of the kinamycins are based on a novel diazobenzo[b]fluorene ring system, a fused 6-6-5-6 carbon skeleton bearing an unusually stable diazo moiety along with a para-quinone and other functionalities. Additionally, this group revised the structure of isoprekinamycin (IPK), a metabolite from Streptomyces murayamaensis previously considered to be a fully aromatized diazobenzo[b]fluorene. IPK was shown to be an isomeric diazobenzo[a]fluorene possessing a fused 6-5-6-6 carbon skeleton and incorporating an ortho-quinonediazide moiety. These observations stimulated much research elsewhere in regard to the synthesis and biological activity of these structurally novel natural products. Among the notable discoveries in other groups was the isolation and characterization of the lomaiviticins, metabolites of the marine bacterium Micromonospora lomaivitiensis that are dimeric diazobenzo[b]fluorene analogues, which are even more potent than the kinamycins as anticancer and antibacterial agents. The present project was designed to develop new synthetic methods to improve access to the diazobenzo[b]fluorenes, with a focus on (1R,2R,3R,4S)-11-diazo-1,2,3,4,9-pentahydroxy-2-methyl- 3,4-dihydro-1H-benzo[b]fluorene-5,10-(2H,11H)-dione, also called kinamycin F. The present project was also designed to carry out experimental and theoretical studies to gain insights into the structures and chemical properties of kinamycins, to better understand their biological properties and to identify how such properties might be optimized through specific structural alterations. A synthetic study was carried out on 2-methyl-1,4-naphthoquinone as a model for a possible biomimetic generation of the highly oxygenated D-ring of the kinamycins as found in kinamycin F. Epoxidation of the model quinone, followed by stereoselective reduction of both keto-carbonyl groups and ring opening of the epoxide with acetate as the nucleophile in a novel process involving tetramethylammonium triacetoxyborohydride provided (1R*,2R*,3R*,4S*)-2-methyl-1,2,3,4- tetrahydronaphthalene-1,2,3,4-tetraol in good yield. Comparison of the proton NMR characteristics of the model tetrol with those of the D-ring of kinamycins led to the conclusion that kinamycin F, unlike other kinamycins with some of their D-ring oxygen(s) bearing acyl groups, prefers a D-ring conformation in which the hydroxyl group that is nearest the diazo group is in a pseudo-equatorial orientation such that the C-O bond is approximately parallel with the diazo group. Ab initio molecular orbital calculations at the RHF 6-31G level led to the conclusion, supported by experimental measurements of diazo IR stretching frequencies, that the diazo group of kinamycin F has an enhanced diazonium ion character in this favoured conformation. This observation is of potential significance since the electrophilicity of the diazo group may play a role in the mode-of-action of the kinamycins, and since there is evidence to suggest that the other kinamycins may undergo conversion into kinamycin F in vivo before exerting their biological effect. A strategy for applying the results of the model study to the total synthesis of kinamycin F is disclosed. In addition, the construction of 6-hydroxy-8-methoxy-3-methyl-7,12-dioxo-7,12- dihydrotetraphen-4-yl methanesulfonate from readily available starting materials is described and suggestions as to how this compound might serve as a key intermediate in the biomimetic synthesis of kinamycin F are provided. A critical analysis of this synthetic strategy to the kinamycins in contrast with several other approaches that have been reported by other groups during the course of this thesis research is presented. Additionally it is pointed out that this synthetic method could provide (1S,2R,3R,4R)-5-diazo-1,2,3,4,8-pentahydroxy-3-methyl-1,2,3,4-tetrahydrotetraphene-6,7,12(5H)- trione as a key intermediate, which might well represent a novel analogue of the kinamycins with potentially intrinsic anticancer and antibacterial activity of its own, since this compound possesses a 6-6-6-6 carbon skeleton containing an ortho-quinonediazide that could serve as an unique hybrid between the 6-6-5-6 diazobenzo[b]fluorene and the 6-5-6-6 diazobenzo[a]fluorene systems. A semi-synthetic method for generating kinamycin F from other natural kinamycins by applying a modified Zemplen deacylation condition is reported. Electrospray mass spectrometry was employed to identify products from interaction of kinamycin F with glutathione on a very small scale. Kinamycin F was found to form a covalent adduct with this thiol that is ubiquitous in mammalian cells. A discussion of the potential biological significance of this process as well as possible interactions with other biologically important thiols in specific potential target proteins is provided. A systematic ab initio molecular orbital analysis at the RHF 6-31 G level of the influence of substituents in the aromatic D-ring of isoprekinamycin was also carried out. The results have led to the suggestion of specific structural alterations that might be employed to fine tune the electrophilicity of the diazo group, which might affect the biological activity of such compounds. Despite the very high potency of the lomaiviticins as anticancer and antibacterial agents, progress towards badly needed practical drugs in these areas has been frustrated by a lack of access to adequate quantities of these complex secondary metabolites either through in vitro fermentation or total synthesis at the moment. In the hope that a prediction of the three dimensional properties of the lomaiviticins might inspire the design and synthesis of simpler analogues with comparable biological activities, a systematic ab initio molecular orbital study at the RHF 6-31G level was undertaken. In the end, predictions of the most likely conformations of lomaiviticins A and B were achieved and are provided as potential starting points for medicinal chemists to design simpler but equally potent and much more accessible analogues