Physiological and genetic studies towards biofuel production in cyanobacteria

The main aim of this thesis was to contribute to the optimization of the cyanobacterial cell factory and to increase the production of cellulose as a biofuel (precursor) via a physiological and a transgenic approach. Chapter 1 provides an overview of the current state of cyanobacterial biofuel research and contains a detailed description of bacterial cellulose synthesis and regulation. Chapters 2, 3 and 4 focus on improving photosynthetic efficiency and describe our findings on how the photosynthetic machinery in the cyanobacteria Synechocystis sp. PCC 6803 responds to conditions of energy limitation and energy excess, we also describe how regulation may occur, specifically regarding state transitions. Additionally, in chapter 2 a new technique to directly measure the redox state of the PQ pool is introduced and we describe how different measuring techniques and growth conditions can affect results. Chapters 5 and 6 describe our efforts in producing cellulose as and extracellular biofuel via a physiological and a transgenic approach respectively. In chapter 5 we try to maximize cellulose production in the natural cellulose producing cyanobacteria Crinalium epipsammum and in chapter 6 we introduce cellulose synthase genes into Synechocystis sp. PCC 6803. In Chapter 7 all aspects of this thesis are discussed

Transition from exponential to linear photoautotrophic growth changes the physiology of <em>Synechocystis</em> sp. PCC 6803

Phototrophic microorganisms like cyanobacteria show growth curves in batch culture that differ from the corresponding growth curves of chemotrophic bacteria. Instead of the usual three phases, i.e., lag-, log-, and stationary phase, phototrophs display four distinct phases. The extra growth phase is a phase of linear, light-limited growth that follows the exponential phase and is often ignored as being different. Results of this study demonstrate marked growth phase-dependent alterations in the photophysiology of the cyanobacterium Synechocystis sp. PCC 6803 between cells harvested from the exponential and the linear growth phase. Notable differences are a gradual shift in the energy transfer of the light-harvesting phycobilisomes to the photosystems and a distinct change in the redox state of the plastoquinone pool. These differences will likely affect the result of physiological studies and the efficiency of product formation of Synechocystis in biotechnological applications. Our study also demonstrates that the length of the period of exponential growth can be extended by a gradually stronger incident light intensity that matches the increase of the cell density of the culture

The Redox Potential of the Plastoquinone Pool of the Cyanobacterium <i>Synechocystis</i> Species Strain PCC 6803 Is under Strict Homeostatic Control

A method is presented for rapid extraction of the total plastoquinone (PQ) pool from Synechocystis sp. strain PCC 6803 cells that preserves the in vivo plastoquinol (PQH(2)) to -PQ ratio. Cells were rapidly transferred into ice-cold organic solvent for instantaneous extraction of the cellular PQ plus PQH(2) content. After high-performance liquid chromatography fractionation of the organic phase extract, the PQH(2) content was quantitatively determined via its fluorescence emission at 330 nm. The in-cell PQH(2)-PQ ratio then followed from comparison of the PQH(2) signal in samples as collected and in an identical sample after complete reduction with sodium borohydride. Prior to PQH(2) extraction, cells from steady-state chemostat cultures were exposed to a wide range of physiological conditions, including high/low availability of inorganic carbon, and various actinic illumination conditions. Well-characterized electron-transfer inhibitors were used to generate a reduced or an oxidized PQ pool for reference. The in vivo redox state of the PQ pool was correlated with the results of pulse-amplitude modulation-based chlorophyll a fluorescence emission measurements, oxygen exchange rates, and 77 K fluorescence emission spectra. Our results show that the redox state of the PQ pool of Synechocystis sp. strain PCC 6803 is subject to strict homeostatic control (i.e. regulated between narrow limits), in contrast to the more dynamic chlorophyll a fluorescence signal

Normal- and iso-butyraldehyde production from proylene and synthesis gas using a water soluble rhodium catalyst

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