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

_{Ultrasound has been vastly applied in different areas such as medical imaging and the food industry, but not much attempt has been made to investigate its influence on microorganisms and its potential to be applied in microbial biotechnology. Previous studies show the potential of acoustic waves to increase biomass yield, production of secondary metabolites and enhanced enzyme-catalyzed reactions, probably due to improved mass transfer and cell retention. The influence of audible waves, however, is not properly elucidated, and more studies are needed to understand how audible waves affect living cells. As most of the applied equipment in biological fields are originally designed for other purposes, there has been limited control of frequency, input power and pressure distribution in biological reactor system. So far, the lack of well-defined sono(bio)reactors that uniformly distribute sound waves has prevented more systematic studies on how acoustics affect biological systems. To obtain in-depth knowledge in this specific field, a sonobioreactor was designed that enables control of the pressure distribution. The viability of a model lignocellulose degrading fungus, Fusarium oxysporum, was studied at different intensities of resonance frequency. The online growth measurement showed that sonication did not adversely affect the cells up to 6 W and the best growth was recorded at 4W. In addition, SEM analysis showed that sound waves can disrupt the mycelium and the higher the applied input power, the greater the number of these breaks. Therefore, in the next parts of the thesis, the influence of sonication on production of lignocellulose degrading enzymes was studied. Prior to studying the effect of ultrasound on lignocellulose enzyme activity, a series of experiments were conducted to identify effective cellulase and xylanase inducers. In these trials, inducers such as cellooligosaccharides, xylooligosacharides, sophorose and lactose were tested and the induced enzymatic activities were measured. Besides, the influence of consumed carbon source (sucrose vs glycerol) for biomass production on cellulase activity was also investigated. The obtained result showed that the efficiency of the inducer depends on the carbon source. Specifically, when using sucrose for the production of fungal biomass, cellooligosaccharides with a higher degree of polymerization (cellotetraose) were better inducers of endoglucanase, exoglucanase, cell-bound and extracellular β-glucosidase, while when using glycerol, cellobiose resulted in an enhanced induction of endoglucanase and exoglucanase, while cellohexaose promoted both β-glucosidases. When it comes to xylanase induction, it was found that the chain size of xylooligosacharides plays an important role with the highest induction occurring with xylotetraose. Finally, transcriptomics analysis was done for both cellulases and xylanases induction, by using cellobiose and xylotetraose respectively, to understand the mechanism of induction of biomass degrading enzymes by revealing differentially expressed genes or transcription factors. The bioinformatics analysis showed that by adding each inducer, a series of carbohydrate degrading enzymes, transcription factors, transporters, as well as genes necessary for translation are differentially expressed. In the final part, the effect of ultrasound and audible sound on the induction of cellulase was studied by using cellobiose as inducer. Prior to this, the effect of different sonication parameters, specifically the time, on the enzyme induction was investigated, and it could be concluded that continuous sonication had a negative effect on enzyme induction. As such, sonication for 2, 8, and 20 hours were compared where 8 hours sonication were selected for further studies. The impact of three different frequencies, namely 40000 kHz and its resonance frequencies in audible sound and higher ultrasound frequencies, on the cellobiose induction of cellulase was studied by using 4W input power and 8-hour sonication. To acquire detailed information on the mechanism of sound influence on the microbial cell, enzyme activity results were used in combination with transcriptomics, metabolomics, proteomics, and secretomics and phosphoproteomics analyses. The OMICs analysis showed that based on the applied frequency, the cell responds differently by activating different metabolic pathways, resulting in the production of different proteins and phosphoproteins. The OMICs profiles of the audible sound and low frequency ultrasound treated samples had some similarities while showing significant differences with the high frequency ultrasound treated samples.}