Caldicellulosiruptor Saccharolyticus: an Ideal Hydrogen Producer?

Abstract

Caldicellulosiruptor saccharolyticus is an extremely thermophilic, strictly anaerobic, Gram-positive and cellulolytic microorganism

with a natural ability to produce hydrogen (H2) at nearly theoretical maximum yield, i.e. 4 mol/ mol of glucose. Due to its CO2-free

combustion and high energy density, among other desirable properties, H2is touted as a fuel of the future. Biological H2

production by thermophilic microorganisms utilizing waste biomass holds a huge potential as the most environment friendly

process for commercial H2production. For this reason, it is imperative to identify and develop a microorganism with the most

beneficial properties as an ideal H2producer may possess.

During this work, the physiology and metabolism of C. saccharolyticus was studied in detail, which revealed that – i) it can sustain

its growth in the absence of any means of removal of H2from the reactor, ii) it can utilize the sugars in wheat straw hydrolysate

effectively for its growth and H2production with 67% conversion efficiency, iii) Methane can replace N2as a sparging gas for

removal of H2from the culture, without affecting the growth and H2production by C. saccharolyticus, iv) by-products of its

fermentative metabolism can be converted to methane by anaerobic digestion, v) it is capable of assimilating sulphate as a primary

sulphur source, vi) can form biofilms when co-cultured with C. owensensis, which can be an effective means of retaining biomass

in the reactor, and vii) Up-flow anaerobic (UA) reactor with granular sludge offer better alternative that a continuously stirred-tank

reactor for improving its volumetric H2productivity (QH2).

Moreover, to improve the QH2further, the methods of evolutionary engineering were used to develop osmotolerant mutant strains

of C. saccharolyticus: i) C. saccharolyticusG10, capable of growing in a medium containing up to 100 g/L of glucose, and ii) C.

saccharolyticusAG6 capable of growing in a medium containing approx. 13.2 g/L of sodium aceate and 30 g/L of glucose. Indeed,

the cultivation of one of the osmotolerant strains in an chemically optimized medium improved the QH2.

Furthermore, experimente were performed to understand the barriers to genetic modification of C. saccharolyticus. The studies

revealed that the methylation of the foreign DNA by C5-cytosine-specific methyltransferase may help overcome the restriction modification system of C. saccharolyticus. In addition, uracil-auxorophic strains of C. saccharolyticus were developed, which can

be used as a host to perform genetic modifications.

In conclusion, the knowledge and newfound properties obtained in this study should be combined to create a strain of C.

saccharolyticus that will fulfil nearly all the requirements to make it into an ideal H2producer.

Abstract (Swedish)

Popular Abstract in English

What is common between the science of physics and global politics? They both define power in terms of energy. Indeed, the uneven distribution of petroleum reserves has been one of the main reasons of social, political and economic problems worldwide. Even more worrying is the rapid increase in the levels of greenhouse gases in the atmosphere. This is mainly caused by our heavy use of petroleum based fuels as a source of energy. This has led to global warming. What if we switch to using a fuel which does not cause any carbon dioxide emissions? Do we know any such fuel? Can we produce it in a way that is safe for the environment?

The answer you are looking for is – Hydrogen gas. It is in many ways an ideal fuel, termed by many experts as “the fuel of the future”. Indeed, it is a powerful and cleanest of the fuels producing nothing but water upon combustion. But, the current methods of producing hydrogen gas are harmful for the environment. These methods use petroleum based fuels for the production of hydrogen gas. Therefore, during this work, I studied a microorganism which can produce hydrogen gas using a method which is safer for the environment and does not depend on petroleum based fuels. The organism is - Caldicellulosiruptor saccharolyticus and the method is termed as thermophilic biohydrogen production.

During this work, I confirmed a few known features of Caldicellulosiruptor saccharolyticus and also discovered some new features. As a hydrogen producer, Caldicellulosiruptor saccharolyticus has many good properties. It has tools to break down almost any kind of biological polymer to individual sugars. And also is able to consume these sugars to produce hydrogen gas with utmost efficiency possible. It does not stop producing hydrogen gas even after its niche is saturated with the gas. It does not need any expensive nutrients for its growth, which helps in reduction of costs of hydrogen gas production.

But, in its natural state Caldicellulosiruptor saccharolyticus is not perfect. It cannot grow in an environment with very high amounts of sugars. This is a major drawback for its industrial application. Therefore, I exposed the organism to stressful conditions to study its response. The organism responded by adapting to changing conditions to give its variant - Caldicellulosiruptor saccharolyticus G10, which is able to grow in presence of high amounts of sugars. I also determined the suitable conditions which encourage the organism to form biofilm. Further studies showed that the biofilms of this organism can be helpful to increase its rate of producing hydrogen gas.

Also, genetic modification of Caldicellulosiruptor saccharolyticus has been a challenge for the researchers worldwide. I discovered that the problem lies in its defence mechanism. I also managed to create its variant - Caldicellulosiruptor saccharolyticus URA-, which can be an efficient host to study its possible genetic modifications.

Altogether, the knowledge obtained in this study can lead to create a modified variant of Caldicellulosiruptor saccharolyticus. This variant will have almost all the features of an ideal hydrogen producer needed for industrial production of hydrogen gas.



Popular Abstract in Swedish

Vad har den fysikaliska vetenskapen gemensamt med global politik? De definierar båda kraft i termer av energi. Och faktiskt, den ojämna distributionen av oljetillgångar har varit en av huvudanledningarna till sociala, politiska och ekonomiska problem i världen. Än mer oroväckande är den snabba ökningen av växthusgaser i atmosfären. Detta är främst orsakat av vår höga användning av petroleumbaserade produkter som energikälla, vilket har lett till global uppvärmning. Tänk om vi kunde växla om till ett bränsle som inte skapar någon koldioxidemission? Känner vi till något sådant bränsle? Kan vi producera det på ett sätt som är säkert för miljön?

Svaret du söker är – vätgas. Det är på många sätt ett idealiskt bränsle, beskrivet av många experter som ”framtidens bränsle”. Och ja, det är både kraftfullt och det renaste bränsle vi känner till då det endast bildar vattenånga vid förbränning. Men de nuvarande metoderna för vätgasframställning är skadliga för miljön eftersom de vid produktion använder sig av petroleumbaserade bränslen. Därför har jag i det här arbetet arbetat med studera en mikroorganism som kan producera vätgas med en metod som är både säkrare för miljön och inte är beroende av några petroleumbaserade bränslen. Organismen heter Caldicellulosiruptor saccharolyticus och metoden kallas termofilisk produktion av biovätgas.

I detta arbete bekräftade jag en del kända egenskaper hos Caldicellulosiruptor saccharolyticus och upptäckte även en del nya. Caldicellulosiruptor saccharolyticus har många goda egenskaper som vätgasproducent. Den har verktyg för att bryta ned nästan alla typer av biologiska polymerer till dess individuella sockerbeståndsdelar. Den är även ytterst effektiv på att konsumera dessa sockermolekyler med vätgas som produkt. Den slutar inte att producera vätgas även om dess omgivning är mättad av gasen. Den behöver inte några dyra näringsämnen för att kunna växa, vilket hjälper till att reducera kostnaden för vätgasproduktion.

Men Caldicellulosiruptor saccharolyticus är inte perfekt i sitt naturliga tillstånd. Den kan inte växa i en miljö med mycket höga halter av socker. Detta är en stor nackdel för dess industriella tillämpning. Därför utsatte jag organismen för stressande betingelser och studerade dess reaktion. Organismen svarade med att anpassa sig till de förändrade faktorerna vilket gav upphov till dess variant - Caldicellulosiruptor saccharolyticus G10, som är förmögen att växa i närvaro höga sockerhalter. Jag bestämde även de lämpliga förutsättningarna för att främja organismens bildning av biofilm. Vidare studier visade att organismens biofilmer kan vara gynnande för en ökad vätgasproduktion.

Genetisk modifikation av Caldicellulosiruptor saccharolyticus har dessutom varit en utmaning för forskare värden över. Jag fann att problemet ligger i dess försvarsmekanism. Jag lyckades även skapa dess variant, Caldicellulosiruptor saccharolyticus URA, som kan vara en effektiv värd vid studier av dess möjliga genetiska modifikationer.

Sammantaget kan den erhållna kunskapen i denna studie leda till en modifierad variant av Caldicellulosiruptor saccharolyticus. Denna variant kommer att inneha nästan alla de egenskaper som krävs av en idealisk vätgasproducent för industriell framställning av vätgas.

• author: Pawar, Sudhanshu
• supervisor: Ed van Niel
• opponent: Prof. Levin, David, University of Manitoba, Canada
• organization: Division of Chemical Engineering; Peter Rådström-lup-obsolete (research group)
• publishing date: 2014
• type: Thesis
• publication status: published
• subject: Engineering and Technology
• keywords: Caldicellulosiruptor owensensis, Caldicellulosiruptor saccharolyticus, hydrogen, volumetric hydrogen productivity, biofilm, osmotolerance, wheat straw hydrolysate, evolutionary engineering, CSTR, UA reactor and uracil auxotrophy
• pages: 206 pages
• defense location: Lecture hall C, Kemicentrum, Getingevägen 60, Lund University Faculty of Engineering, Lund
• defense date: 2014-10-24 10:00:00
• ISBN: 978-91-7422-371-2
• language: English
• LU publication?: yes
• id: b565a46b-babf-4f91-a5ee-2c3b8dfcc5a7 (old id 4690497)
• date added to LUP: 2016-04-04 12:51:55
• date last changed: 2018-11-21 21:11:08