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On-line Raman spectroscopic study of cytochromes’ redox state of biofilms in microbial fuel cells
Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.ORCID iD: 0000-0002-3386-701x
Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.ORCID iD: 0000-0002-1600-8424
Luleå University of Technology, Department of Engineering Sciences and Mathematics, Fluid and Experimental Mechanics.ORCID iD: 0000-0003-3268-1691
Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.ORCID iD: 0000-0003-0079-5950
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2019 (English)In: Molecules, ISSN 1431-5157, E-ISSN 1420-3049, Vol. 24, no 3, article id 646Article in journal (Refereed) Published
Abstract [en]

Bio-electrochemical systems such as microbial fuel cells and microbial electrosynthesis cells depend on efficient electron transfer between the microorganisms and the electrodes. Understanding the mechanisms and dynamics of the electron transfer is important in order to design more efficient reactors, as well as modifying microorganisms for enhanced electricity production. Geobacter are well known for their ability to form thick biofilms and transfer electrons to the surfaces of electrodes. Currently, there are not many “on-line” systems for monitoring the activity of the biofilm and the electron transfer process without harming the biofilm. Raman microscopy was shown to be capable of providing biochemical information, i.e., the redox state of C-type cytochromes, which is integral to external electron transfer, without harming the biofilm. In the current study, a custom 3D printed flow-through cuvette was used in order to analyze the oxidation state of the C-type cytochromes of suspended cultures of three Geobacter sulfurreducens strains (PCA, KN400 and ∆pilA). It was found that the oxidation state is a good indicator of the metabolic state of the cells. Furthermore, an anaerobic fluidic system enabling in situ Raman measurements was designed and applied successfully to monitor and characterize G. sulfurreducens biofilms during electricity generation, for both a wild strain, PCA, and a mutant, ∆S. The cytochrome redox state, monitored by the Raman peak areas, could be modulated by applying different poise voltages to the electrodes. This also correlated with the modulation of current transferred from the cytochromes to the electrode. The Raman peak area changed in a predictable and reversible manner, indicating that the system could be used for analyzing the oxidation state of the proteins responsible for the electron transfer process and the kinetics thereof in-situ. 

Place, publisher, year, edition, pages
MDPI, 2019. Vol. 24, no 3, article id 646
Keywords [en]
Cytochrome-C, Geobacter sulfurreducens, Microbial fuel cell, Omc, Raman spectroscopy
National Category
Bioprocess Technology Applied Mechanics
Research subject
Biochemical Process Engineering; Experimental Mechanics
Identifiers
URN: urn:nbn:se:ltu:diva-73003DOI: 10.3390/molecules24030646ISI: 000458934000270PubMedID: 30759821Scopus ID: 2-s2.0-85061525740OAI: oai:DiVA.org:ltu-73003DiVA, id: diva2:1291689
Note

Validerad;2019;Nivå 2;2019-02-26 (svasva)

Available from: 2019-02-26 Created: 2019-02-26 Last updated: 2023-09-05Bibliographically approved
In thesis
1. Microbial Fuel cells, applications and biofilm characterization
Open this publication in new window or tab >>Microbial Fuel cells, applications and biofilm characterization
2019 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Since the 1900’s it has been known that microorganisms are capable of generating electrical power through extracellular electron transfer by converting the energy found organic compounds (Potter, 1911). Microbial fuel cells (MFCs) has garnered more attention recently, and have shown promise in several applications, including wastewater treatment (Yakar et al., 2018), bioremediation (Rosenbaum & Franks, 2014), biosensors (ElMekawy et al., 2018) desalination (Zhang et al., 2018) and as an alternative renewable energy source in remote areas (Castro et al., 2014). In MFCs catalytic reactions of microorganisms oxidize an electron donor through extracellular electron transfer to the anode, under anaerobic conditions, with the cathode exposed to an electron acceptor, facilitating an electrical current (Zhuwei, Haoran & Tingyue, 2007; Lovley, 2006). For energy production in remote areas a low cost and easily accessible feed stock is required for the MFCs. Sweet sorghum is a drought tolerant feedstock with high biomass and sugar yields, good water-use efficiency, established production systems and the potential for genetic improvements. Because of these advantages sweet sorghum stalks were proposed as an attractive feedstock (Rooney et al., 2010; Matsakas & Christakopoulos, 2013). Dried sweet sorghum stalks were, therefore, tested as a raw material for power generation in a MFC, with anaerobic sludge from a biogas plant as inoculum (Sjöblom et al., 2017a).

Using sorghum stalks the maximum voltage obtained was 546±10 mV, the maximum power and current density of 131±8 mW/m2 and 543±29 mA/m2 respectively and the coulombic efficiency was 2.2±0.5%. The Ohmic resistances were dominant, at an internal resistance of 182±17 Ω, calculated from polarization data. Furthermore, hydrolysis of the dried sorghum stalks did not improve the performance of the MFC but slightly increased the total energy per gram of substrate. During the MFC operation, the sugars were quickly fermented to formate, acetate, butyrate, lactate and propionate with acetate and butyrate being the key acids during electricity generation.

Efficient electron transfer between the microorganisms and the electrodes is an essential aspect of bio-electrochemical systems such as microbial fuel cells. In order to design more efficient reactors and to modify microorganisms, for enhanced electricity production, understanding the mechanisms and dynamics of the electron transport chain is important. It has been found that outer membrane C-type cytochromes (OMCs) (including omcS and omcZ discussed in this study) play a key role in the electron transport chain of Geobacter sulfurreducens, a well-known, biofilm forming, electro-active microorganism  (Millo et al., 2011; Lovley, 2008). It was found that Raman microscopy is capable of providing biochemical information, i.e., the redox state of c-type cytochromes (cyt-C) without damaging the microbial biofilm, allowing for in-situ observation.

Raman microscopy was used to observe the oxidation state of OMCs in a suspended culture, as well as in a biofilm of an MFC. First, the oxidation state of the OMCs of suspended cultures from three G. sulfurreducens strains (PCA, KN400 and ΔpilA) was analyzed. It was found that the oxidation state can also be used as an indicator of the metabolic state of the cells, and it was confirmed that PilA, a structural pilin protein essential for long range electron transfer, is not required for external electron transfer. Furthermore, we designed a continuous, anaerobic MFC enabling in-situ Raman measurements of G. sulfurreducens biofilms during electricity generation, while poised using a potentiostat, in order to monitor and characterize the biofilm. Two strains were used, a wild strain, PCA, and a mutant, ΔOmcS. The cytochrome redox state, observed through the Raman spectra, could be altered by applying different poise voltages to the electrodes. This change was indirectly proportional to the modulation of current transferred from the cytochromes to the electrode. This change in Raman peak area was reproducible and reversible, indicating that the system could be used, in-situ, to analyze the oxidation state of proteins responsible for the electron transfer process and the kinetics thereof.

Place, publisher, year, edition, pages
Luleå: Luleå University of Technology, 2019. p. 32
Series
Licentiate thesis / Luleå University of Technology, ISSN 1402-1757
Keywords
MFC, Microbial fuel cell, Raman microscopy, BES, Geobacter sulfurreducens
National Category
Other Environmental Biotechnology Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-73938 (URN)978-91-7790-398-7 (ISBN)978-91-7790-399-4 (ISBN)
Presentation
2019-06-19, E632, Luleå University of Technology, E building, Luleå, 14:00 (English)
Opponent
Supervisors
Funder
Swedish Research Council, 2014-05906
Available from: 2019-05-15 Created: 2019-05-14 Last updated: 2023-09-05Bibliographically approved
2. Sound, Light and Electricity: as applications and analysis techniques to study metabolic effect and biofilm characterization of Geobacter sulfurreducens
Open this publication in new window or tab >>Sound, Light and Electricity: as applications and analysis techniques to study metabolic effect and biofilm characterization of Geobacter sulfurreducens
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Electricity

Bio-electrochemical systems such as microbial fuel cells (MFCs) and microbial electrolysis cells have shown promise in wastewater treatment, bioremediation, desalination, carbon sequestration and as an alternative, renewable energy source. MFCs produces electricity via anaerobic oxidation of substrates with the subsequent extracellular electron transfer to an electrode. A wide variety of feedstocks have been researched, including various artificial and real wastewater sources as well as lignocellulosic material. Sweet sorghum, has been identified as a possible feedstock for electricity production in MFCs, using an anaerobic sludge inoculum, due to its high sugar content. To study sweet sorghum as an MFC feedstock a standard two chamber H-cell MFC was used, with an anaerobic sludge inoculum (Boden Biogas). A maximum voltage of 546±10 mV was obtained, and a maximum power and current density of 131±8 mW/m2 and 543±29 mA/m2 respectively. The substrate concentrations were monitored during the MFC operation, and the sugars were quickly fermented to volatile fatty acids which were then consumed during electricity generation. The power output was essentially independent of the substrate profile, with little difference between different VFAs. A more direct way was therefore needed to monitor the growth of an MFC biofilm as well as the effect of various substrates on extracellular electron transfer (EET).

Light

One option for the direct monitoring of a biofilm is to use Raman spectroscopy to monitor the redox status of the biofilm, since Raman can be used to detect the redox state heme groups. Therefore, resonance Raman spectroscopy was chosen to monitor the cytochrome redox of Geobacter sulfurreducens, is a well know electroactive microorganism commonly found in mixed culture MFCs. G. sulfurreducensis able to produce thick, conductive biofilms as well as high current densities in MFCs. Due to the large variety of cytochromes present in G. sulfurreducens, it has various intricate and adaptable EET pathways, which makes the characterization of the essential EET components difficult. Due to the resonance of the cytochromes found in G. sulfurreducens it is possible to measure the redox state of the biofilm using resonance Raman spectroscopy. This was used for on-line monitoring of various G. sulfurreducens mutants during MFC operation (including the wild type PCA, the ii enhanced KN400 strain capable of higher current densities, and two deficient strains missing key cytochromes involved in the EET, i.e. ΔOmcS and ΔpilA). From this, the applicability of resonance Raman spectroscopy was shown to provide a non-destructive analytical tool for the in-situ monitoring of the oxidation state of proteins responsible for the EET process and the dynamics thereof. Resonance Raman with short integration times was further used, along with a dynamic model, to describe the dynamics of the EET pathways in the wild type as well as in an OmcS deficient strain during a stepped chronoamperometry measurement. This showed a significant difference in EET dynamics between ΔOmcS and the wild type, which was not detectible in the chronoamperometry data alone. The ΔOmcS biofilm showed a linearly decreasing trend in the reduced cytochrome concentration. This was likely caused by the saturation of a limiting mediator, resulting in an oxidation rate that was independent of the mediator concentration. The ΔOmcS biofilms response could, however, be better modelled using an empirical zeroth order model. This analytical method could prove valuable for the establishment of G. sulfurreducens as a chassis microorganism, allowing one to observe the effect of genetic modification on EET mechanisms.

Sound

Furthermore, to see if an abiotic factor such as sound can affect the functions in bacterial cells, we selected to study the effect of ultrasound on the growth of G. sulfurreducens. G. sulfurreducens is a key candidate for the development of a chassis organism in bioelectrochemical systems, and an external abiotic method of affecting growth or metabolite production could be extremely beneficial. For this, a well-defined sonobioreactor was developed and modelled to study the effect of ultrasound on G. sulfurreducens. This resulted in a significant increase in malate production during the exponential phase of planktonic growth (11 mmol when sonicated vs the 5 mmol control). Transcriptomics was then used to determine the reason for the observed increase. Although there was a large variance in the samples, this was possibly linked to the overexpression of glycosyltransferases, which are known to play a role in membrane stability and bind malate. Finally, a low-cost modification, which modifies a standard 3D printer into a bio-printer was developed to print artificial biofilms for bio-electrochemical systems. This was then used to print an artificial biofilm of G. sulfurreducens, significantly reducing the time required to produce an established biofilm

Place, publisher, year, edition, pages
Luleå: Luleå University of Technology, 2020. p. 55
Series
Doctoral thesis / Luleå University of Technology 1 jan 1997 → …, ISSN 1402-1544
Keywords
MFC, Microbial fuel cell, Raman microscopy, BES, Geobacter sulfurreducens
National Category
Biological Sciences Industrial Biotechnology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-80314 (URN)978-91-7790-628-5 (ISBN)978-91-7790-629-2 (ISBN)
Public defence
2020-09-29, F1031, Luleå University of Technology, 10:00 (English)
Opponent
Supervisors
Funder
Swedish Research Council, 2017-04867Vattenfall AB, 2014-05906
Available from: 2020-08-10 Created: 2020-08-05 Last updated: 2023-09-05Bibliographically approved

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Krige, AdolfSjöblom, MagnusRamser, KerstinChristakopoulos, PaulRova, Ulrika

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