Genomics and Transcriptomics of the Hydrogen Producing Extremely Thermophilic Bacterium Caldicellulosiruptor Saccharolyticus Ma
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Genomics and transcriptomics of the hydrogen producing extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus Marcel R. A. Verhaart Thesis committee Thesis supervisor Prof. Dr. Alfons J.M. Stams Personal Chair at the Laboratory of Microbiology Wageningen University Thesis co‐supervisor Dr. Servé W.M. Kengen Assistant Professor at the Laboratory of Microbiology Wageningen University Other members Prof. dr. ir. C.J.N. Buisman Wageningen University Prof. Dr. W.R Hagen Delft University of Technology Dr. Astrid E. Mars Wageningen UR Food & Biobased Research Dr. ir. E.W.J van Niel Lund University, Sweden This research was conducted under the auspices of the Graduate School WIMEK Genomics and transcriptomics of the hydrogen producing extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus Marcel R. A. Verhaart Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. dr. M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 19 November2010 at 4 p.m. in the Aula. Marcel R.A. Verhaart Genomics and transcriptomics of the hydrogen producing extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus, 178 pages. Thesis, Wageningen University, Wageningen, NL (2010) With references, with summaries in Dutch and English ISBN 978‐90‐8585‐796‐9 Abstract As fossil fuels are depleting, there is a clear need for alternative sustainable fuel sources. One of the interesting alternatives is hydrogen, which can be produced from biomass by bacteria and archaea. To make the application feasible, organisms are needed which have high hydrogen productivities as well as the capacity to handle a wide variety of substrates. Caldicellulosiruptor saccharolyticus has these abilities and this was the reason to select this organism to study hydrogen production at elevated temperatures. The aim of this study was to clarify the pathways involved in hydrogen production as well as studying the regulation of the hydrogen production on different levels. Especially the regulation of the disposal of reducing equivalents (NADH, reduced ferredoxin, NADPH), was an important aspect. The genome sequence was annotated and compared with transcriptome data. This revealed the presence of the Emden‐Meyerhof pathway, and the non‐oxidative part of the pentose phosphate pathway for the breakdown of glucose and xylose. The genomic data explained the high hydrolyzing capacity of this organism by the presence of a large number of carbohydrate active enzymes as well as a large variety of ABC transporters. With transcriptomics the substrate specificity of several of these transporters was predicted. The absence from the genome of some components involved in classical carbon catabolite repression explained the capacity of C. saccharolyticus to grow simultaneously on C5 and C6 sugars. When C. saccharolyticus was cultured on rhamnose, 1.2 propanediol production was observed. This observation could be explained by the presence of a rhamnose pathway, which could be unraveled using the genome sequence as well as the transcriptome data. As hydrogen pressure seems to be an important factor for hydrogen production by C. saccharolyticus, the effect of a high P(H2) was studied in a continuous culture. This showed a clear shift to lactate and ethanol production at higher P(H2). By comparing the transcriptome for the high and low P(H2) situation, this shift could be explained by the upregulation of the genes encoding an alcohol and a lactate dehydrogenase. This suggested that the electrons of NADH are most likely disposed of via lactate production, while the electrons of reduced ferredoxin might be shuttled via an oxidoreductase to produce ethanol.Overall this study revealed several new insights in the regulation of hydrogen production by thermophilic anaerobic organisms. Table of contents Chapter 1 Introduction and outline Page 7 Chapter 2 Hydrogen production by hyper‐ and extremely thermophilic Page 23 bacteria and archaea: Mechanisms for reductant disposal Chapter 3 Hydrogenomics of the extremely thermophilic bacterium Page 41 Caldicellulosiruptor saccharolyticus Chapter 4 Carbohydrate utilization patterns by the extremely thermophilic Page 77 bacterium Caldicellulosiruptor saccharolyticus reveal broad growth substrate preferences Chapter 5 Analysis of the rhamnose pathway in Caldicellulosiruptor Page 95 saccharolyticus Chapter 6 The effect of hydrogen pressure on the metabolism of the Page 109 thermophilic bacterium Caldicellulosiruptor saccharolyticus Chapter 7 General discussion Page 125 References Page 140 Summary Page 165 Samenvatting Page 168 List of publications Page 171 About the Author Page 172 Acknowledgements Page 173 Overview of completed training activities Page 176 6 Chapter 1 Introduction and outline Chapter 1 Introduction Fossil fuels To place the research described in this thesis in context, the fossil fuel problem should be addressed. These fuels are named “fossil” as these are the remains of thick layers of organic material of terrestrial or marine origin, which have been exposed for millions of years to heat and high pressure deep down in the earth. This resulted in coal, oil and natural gas, which are rich in hydrocarbons. Fossil fuel depletion Not only at the moment, these fossil fuels are the most important energy sources, but as predicted by the U.S. Energy Information, they still will be for the coming decades (200). It is also clear that the total energy demand is still increasing and will be twice as high in 2035 compared with 1990. As fossil fuels need millions of years to form, it is obvious that there will be a problem if energy demands increase. Although estimation of the depletion of the fossil fuel sources is difficult, all experts agree that there will be a need for alternatives in the future. At a certain point in time a situation will be reached where it is no longer economically feasible or physically possible to supply our energy demands with the fossil fuels. Environmental effects Another point of concern when using fossil fuels is the production of CO2 when combusted. For instance, in the US, 90% of all the CO2 produced is coming from fossil fuel combustion (201). CO2, together with some other gasses (e.g. water vapor, methane and ozone), is a so called greenhouse gas, which contributes to the greenhouse effect. This effect occurs when radiation from the sun, which is reflected by the earth’s surface, is absorbed by greenhouse gas in the atmosphere. Due to this absorption, the energy is trapped in our atmosphere and as a consequence makes the earth warm enough to live on. On the other hand if there is too much greenhouse gas present, the temperature will rise too much and can have very dramatic effects on the climate and biodiversity. Although it receives a lot of attention, the 8 Introduction and outline problems with these greenhouse gasses are far from solved. Predictions show that the CO2 production from fossil fuels only, will double in 2030 compared to 1990. Renewable energy sources As described above these negative effects of the use of fossil fuels, show the need to find alternative energy sources. Renewable energy is the term which is used for these alternatives, and it means that the energy is generated from naturally replenishable resources, such as wind, rain, tides, geothermal heat and sunlight. Sunlight in this case is not only the use of solar panels but also the generation of biomass which can be used as energy source. Renewable energy gained a lot of interest as shown by the investments in this area, which has increased fourfold from 2004 to 2008 to $120 billion. Also on the production side the interest is increasing as can be seen in table 1.1. Table 1.1 Increased interest in renewable energy shown by numbers; USD: US dollars; GW: gigawatt; GWth: gigawatts thermal; PV: photovoltaic. (161) SELECTED INDICATORS 2006 2007 2008 Investment in new renewable capacity 63 104 120 billion USD (annual) Renewables power capacity (existing, excl. 207 240 280 GW large hydro) Renewables power capacity (existing, incl. 1020 1070 1140 GW large hydro) Wind power capacity (existing) 74 94 121 GW Grid‐connected solar PV capacity (existing) 5.1 7.5 13 GW Solar PV production (annual) 2.5 3.7 6.9 GW Solar hot water capacity (existing) 105 126 145 GWth Ethanol production (annual) 39 50 67 billion liters Biodiesel production (annual) 6 9 12 billion liters Although these numbers show a clear increase, a lot more is needed to really replace fossil fuels, especially whenit is compared with the total power consumption, which was 18.8 9 Chapter 1 Terawatt in 2008 worldwide, and this will only increase in the future (200). If taken into account that the use of nuclear power has been quite stable in recent years and, because of political and environmental reasons, will most likely not increase in the near future, more investments and research on renewable resources are needed. Wind energy Although there is a lot of wind energy available, it is not always easy to harvest. This energy can be used in different ways, like for example, using wind turbines (electricity) or wind mills (mechanical power). In 2008 there was a existing wind power capacity of 121 GW (Table 1) which is a very small portion of the total renewable power capacity. Wind energy is sustainable and wind is widely available, but it also has some clear drawbacks. There is a visual impact of the large turbines and possible negative environmental effects, for example on birds and sealife. On the other hand there is also the problem that wind is not storable and the energy needs to be transformed to other energy carriers like hydropower. This will make wind energy a costly energy source as large investments need to be done if the proportions increase. So far wind energy seems to be more suited for local usage, when the installation costs are low and energy can be used immediately.