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Physiology of Caldicellulosiruptor saccharolyticus: a hydrogen cell factory

Willquist, Karin LU (2010)
Abstract (Swedish)
Popular Abstract in Swedish

Biovätgas är en mycket energirik och förnyelsebar energikälla för bilar och elproduktion som genererar mycket lite av växthusgasen koldioxid. Vätgasen kan framställas av billiga råvaror såsom hushållsavfall, industriellt avfall och av lignocellulosa. Lignocellulossa är en komplex polymer som finns bland annat i olika träslag och gräs. Detta leder till att råvaropriset minskar och att produktionen av biovätgas inte konkurrerar med livsmedelsproduktionen. Den största utmaningen för biovätgas är att vätgasutbytet, dvs. den mängd vätgas per förbrukad råvara, är mycket lågt.



Detta arbete är unikt i sitt slag eftersom det fokuserar på en bakterie, Caldicellulosiruptor... (More)
Popular Abstract in Swedish

Biovätgas är en mycket energirik och förnyelsebar energikälla för bilar och elproduktion som genererar mycket lite av växthusgasen koldioxid. Vätgasen kan framställas av billiga råvaror såsom hushållsavfall, industriellt avfall och av lignocellulosa. Lignocellulossa är en komplex polymer som finns bland annat i olika träslag och gräs. Detta leder till att råvaropriset minskar och att produktionen av biovätgas inte konkurrerar med livsmedelsproduktionen. Den största utmaningen för biovätgas är att vätgasutbytet, dvs. den mängd vätgas per förbrukad råvara, är mycket lågt.



Detta arbete är unikt i sitt slag eftersom det fokuserar på en bakterie, Caldicellulosiruptor saccharolyticus, som kan omvandla ett brett spektrum av råmaterial till vätgas, med ett maximalt vätgasutbyte, vilket aldrig har avhandlats förut. Bakterien isolerades från en träbit i en varmkälla i New Zeeland. Bakterien förökar sig optimalt vid 70ºC och endast i syrefria miljöer. Jag har studerat hur olika processparametrar, såsom högt eller lågt vätgas- eller koldioxidtryck, påverkar dess tillväxt, vätgasutbyte och metabolism. Bland annat har jag sett att vid ett högt koldioxidtryck ökar saltkoncentrationen i tillväxtmediet vilket i sin tur inhiberar bakteriens tillväxt och leder till minskad vätgasproduktivitet. Ett högt vätgastryck kan också leda till ett reducerat vätgasutbyte eftersom det inhiberar själva vätgasproduktionen vilket resulterar i ökad mjölksyraproduktion. På samma sätt som våra muskler bildar mjölksyra som en slaggprodukt vid ofullständig förbränning vid syrebrist, kan bakterien bilda mjölksyra för att upprätthålla förbränningen när det finns för mycket vätgas i dess omgivning. I mitt arbete har jag identifierat vilka parametrar och signalmolekyler, både i tillväxtmediet och inuti cellen, som orsakar minskad vätgasproduktion och ökad produktion av mjölksyra och etanol, en annan oönskad metabol biprodukt.



Jag har även studerat varför Caldicellulosiruptor saccharolyticus har bättre förutsättningar att producera mer vätgas än andra närbesläktade bakterier. Den höga tillväxttemperaturen spelar en avgörande roll, eftersom den påverkar termodynamiken i systemet och gör att vätgasreaktionen blir mer fördelaktig. En annan viktig drivkraft är energimetabolismen. Precis som hos oss människor måste Caldicellulosiruptor saccharolyticus få i sig kolhydrater som sedan kan omvandlas till energi. Eftersom denna bakterie har anpassats till en miljö med låga kolhydratskoncentrationer, har den utvecklat effektiva transportsystem för kolhydrater. Förutom att bakterien kan tillgodogöra sig låga halter av kolhydrater utvinns så mycket energi som möjligt vid vätgasproduktionen, vilket förklarar varför Caldicellulosiruptor saccharolyticus är mer benägen att producera vätgas än andra närbesläktade bakterier.



Generellt kan man säga att Caldicellulosiruptor saccharolyticus besitter många egenskaper som är användbara i en framtida biovätgasproduktion. Vikten av att studera bakteriers metabolism, för att optimera processen och framställa höga kvantiteter av vätgas, framhävs i denna avhandling. Dessutom rekommenderas att isolera bakterier med liknande fysiologiska egenskaper som Caldicellulosiruptor saccharolyticus. (Less)
Abstract
A high substrate conversion efficiency is a prerequisite for an economically feasible biohydrogen production. Caldicellulosiruptor saccharolyticus is a strictly anaerobic extreme thermophilic bacterium that is able to convert the theoretical maximum of 4 mol/mol glucose to H2. It can grow and produce H2 on a broad spectrum of sugars ranging from monomers (hexoses and pentoses) to more complex sugars such as lignocellulosics, thereby rendering it industrially interesting. Moreover, it is capable of maintaining a growth and production of H2 at elevated partial H2 pressures (PH2), which significantly reduces the cost of gas upgrading. These qualities, which make C. saccharolyticus a superior H2 cell factory, may be attributed to its... (More)
A high substrate conversion efficiency is a prerequisite for an economically feasible biohydrogen production. Caldicellulosiruptor saccharolyticus is a strictly anaerobic extreme thermophilic bacterium that is able to convert the theoretical maximum of 4 mol/mol glucose to H2. It can grow and produce H2 on a broad spectrum of sugars ranging from monomers (hexoses and pentoses) to more complex sugars such as lignocellulosics, thereby rendering it industrially interesting. Moreover, it is capable of maintaining a growth and production of H2 at elevated partial H2 pressures (PH2), which significantly reduces the cost of gas upgrading. These qualities, which make C. saccharolyticus a superior H2 cell factory, may be attributed to its adaptation to a monosaccharide-poor environment. Characteristics that it has developed to survive in this harsh environment include: i) the generation of cellulolytic and (hemi)-cellulolytic enzymes which can degrade complex polymers, ii) a possession of a high number of high affinity ATP binding cassette (ABC) transport systems to translocate a large spectrum of monomers and dimers, and iii) a lack of glucose-repression enabling co-metabolization of several types of sugars. To fuel this high affinity transport system, they are forced to oxidize glucose to acetate to generate adequate amounts of ATP. H2 is an electron sink, formed to reoxidize NADH and reduce ferredoxin in this process.



In addition, C. saccharolyticus can conserve energy by using pyrophosphate (PPi) as an additional energy carrier. It lacks cytosolic PPase activity, but is instead able to utilize the energy in the PPi bond by i) a membrane-bound proton-translocating PPase that generates a proton motive force, ii) PPi-phosphofructokinase (PPi-PFK) that uses PPi instead of ATP, and/or iii) pyruvate phosphate dikinase (PPDK) that uses PPi and PEP to generate pyruvate and ATP. Moreover, the ATP/PPi and NADH/NAD ratios increase when the growth rate decreases in the transition to the stationary phase.



To ensure a high acetate flux, the enzymes around the pyruvate node are strongly controlled in C. saccharolyticus. Lactate dehydrogenase (LDH) is constitutively expressed but strongly regulated at the enzyme level by both the ATP/PPi and the NADH/NAD ratios. These experimental data were used to derive a kinetic model over LDH rendering it possible to simulate lactate formation during batch growth in C. saccharolyticus. Such lactate formation occurs in the transition to the stationary phase as a result of the combination of an increased osmotic pressure and a raise in PH2. This is attributed to the fact that, in conditions promoting high growth rates, LDH is inactive, leading to a low lactate formation. However, LDH is activated when the ATP or NADH levels increase and the PPi levels decrease, leading to a partial metabolic redistribution to lactate. Lactate is a vital extra electron sink for maintaining a high glycolytic flux since this flux is inhibited by increased NADH levels.



However, C. saccharolyticus also possesses several shortcomings which need to be addressed. These include i) elevated dissolved H2 concentrations and osmotic pressures triggering lactate formation although C. saccharolyticus is more tolerant to PH2 than many other microorgansims, ii) sparging with CO2 which inhibits growth and H2 productivities, iii) a sensitivity to increased osmotic pressures resulting in cell lysis and decreased H2 productivities, iv) a low cell number compared to mesophilic co-cultures resulting in a low volumetric H2 productivity, and v) the addition of CO2 or acetate addition as a requirement to initiate growth on xylose and arabinose. The latter does not constitute a problem provided that a sugar mixture with both pentose and hexose sugars is used.



Despite these shortcomings, C. saccharolyticus can be considered a superior H2 cell factory. The aspects of its many positive qualities for a biohydrogen process and possible origins for these qualities are discussed in this thesis. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Professor SOUCAILLE, PHILIPPE, Département de Génie Biochimique et Alimentaire, Toulouse, France
organization
publishing date
type
Thesis
publication status
published
subject
keywords
growth activation, hydrogen tolerance, hydrogen yields CO2 inhibition, physiology, enzyme kinetics, Caldicellulosiruptor saccharolyticus, biohydrogen, energy metabolism
pages
196 pages
publisher
Applied Microbiology (LTH)
defense location
Sal B Kemicentrum, Getingevägen 60, Lund university, Faculty of Engineering
defense date
2010-04-16 09:00
ISBN
978-91-7422-239-5
language
English
LU publication?
yes
id
cc93124a-65ce-4d66-bc09-7696d9110744 (old id 1567666)
date added to LUP
2010-03-26 12:33:23
date last changed
2016-09-19 08:45:15
@phdthesis{cc93124a-65ce-4d66-bc09-7696d9110744,
  abstract     = {A high substrate conversion efficiency is a prerequisite for an economically feasible biohydrogen production. Caldicellulosiruptor saccharolyticus is a strictly anaerobic extreme thermophilic bacterium that is able to convert the theoretical maximum of 4 mol/mol glucose to H2. It can grow and produce H2 on a broad spectrum of sugars ranging from monomers (hexoses and pentoses) to more complex sugars such as lignocellulosics, thereby rendering it industrially interesting. Moreover, it is capable of maintaining a growth and production of H2 at elevated partial H2 pressures (PH2), which significantly reduces the cost of gas upgrading. These qualities, which make C. saccharolyticus a superior H2 cell factory, may be attributed to its adaptation to a monosaccharide-poor environment. Characteristics that it has developed to survive in this harsh environment include: i) the generation of cellulolytic and (hemi)-cellulolytic enzymes which can degrade complex polymers, ii) a possession of a high number of high affinity ATP binding cassette (ABC) transport systems to translocate a large spectrum of monomers and dimers, and iii) a lack of glucose-repression enabling co-metabolization of several types of sugars. To fuel this high affinity transport system, they are forced to oxidize glucose to acetate to generate adequate amounts of ATP. H2 is an electron sink, formed to reoxidize NADH and reduce ferredoxin in this process. <br/><br>
<br/><br>
In addition, C. saccharolyticus can conserve energy by using pyrophosphate (PPi) as an additional energy carrier. It lacks cytosolic PPase activity, but is instead able to utilize the energy in the PPi bond by i) a membrane-bound proton-translocating PPase that generates a proton motive force, ii) PPi-phosphofructokinase (PPi-PFK) that uses PPi instead of ATP, and/or iii) pyruvate phosphate dikinase (PPDK) that uses PPi and PEP to generate pyruvate and ATP. Moreover, the ATP/PPi and NADH/NAD ratios increase when the growth rate decreases in the transition to the stationary phase. <br/><br>
<br/><br>
To ensure a high acetate flux, the enzymes around the pyruvate node are strongly controlled in C. saccharolyticus. Lactate dehydrogenase (LDH) is constitutively expressed but strongly regulated at the enzyme level by both the ATP/PPi and the NADH/NAD ratios. These experimental data were used to derive a kinetic model over LDH rendering it possible to simulate lactate formation during batch growth in C. saccharolyticus. Such lactate formation occurs in the transition to the stationary phase as a result of the combination of an increased osmotic pressure and a raise in PH2. This is attributed to the fact that, in conditions promoting high growth rates, LDH is inactive, leading to a low lactate formation. However, LDH is activated when the ATP or NADH levels increase and the PPi levels decrease, leading to a partial metabolic redistribution to lactate. Lactate is a vital extra electron sink for maintaining a high glycolytic flux since this flux is inhibited by increased NADH levels. <br/><br>
<br/><br>
However, C. saccharolyticus also possesses several shortcomings which need to be addressed. These include i) elevated dissolved H2 concentrations and osmotic pressures triggering lactate formation although C. saccharolyticus is more tolerant to PH2 than many other microorgansims, ii) sparging with CO2 which inhibits growth and H2 productivities, iii) a sensitivity to increased osmotic pressures resulting in cell lysis and decreased H2 productivities, iv) a low cell number compared to mesophilic co-cultures resulting in a low volumetric H2 productivity, and v) the addition of CO2 or acetate addition as a requirement to initiate growth on xylose and arabinose. The latter does not constitute a problem provided that a sugar mixture with both pentose and hexose sugars is used.<br/><br>
<br/><br>
Despite these shortcomings, C. saccharolyticus can be considered a superior H2 cell factory. The aspects of its many positive qualities for a biohydrogen process and possible origins for these qualities are discussed in this thesis.},
  author       = {Willquist, Karin},
  isbn         = {978-91-7422-239-5},
  keyword      = {growth activation,hydrogen tolerance,hydrogen yields CO2 inhibition,physiology,enzyme kinetics,Caldicellulosiruptor saccharolyticus,biohydrogen,energy metabolism},
  language     = {eng},
  pages        = {196},
  publisher    = {Applied Microbiology (LTH)},
  school       = {Lund University},
  title        = {Physiology of Caldicellulosiruptor saccharolyticus: a hydrogen cell factory},
  year         = {2010},
}