SOUnd-DRIven BIOtechnology

• 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. 

Ämnesord

TEKNIK OCH TEKNOLOGIER  -- Industriell bioteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Industrial Biotechnology (hsv//eng)

TEKNIK OCH TEKNOLOGIER  -- Industriell bioteknik -- Bioprocessteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Industrial Biotechnology -- Bioprocess Technology (hsv//eng)

Nyckelord

Biokemisk processteknik

Biochemical Process Engineering

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