Clostridium ljungdahlii

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Clostridium ljungdahlii
Systematics
Department : Firmicutes
Class : Clostridia
Order : Clostridiales
Family : Clostridiaceae
Genre : Clostridium
Type : Clostridium ljungdahlii
Scientific name
Clostridium ljungdahlii
Tanner et al . 1993

Clostridium ljungdahlii is a species of bacteria from the genus Clostridia . It lives in oxygen-free substrates and was first isolated from the waste of a chicken farm in 1988. Under unfavorable living conditions, it forms endospores after a few hours, which survive for several years as persistence stages and, under anaerobic conditions, become vegetative bacteria again.

All information about the bacterium comes from the examination of laboratory cultures, observations from the original substrates are not available. Due to its ability to fermentatively produce ethanol and acetic acid on the basis of synthesis gas ( synthesis gas fermentation ), it is of potential importance for industrial biotechnology , although production plants only exist on a pilot scale so far. In 2010, the genome of Clostridium ljungdahlii was sequenced and published in order to better understand the metabolism and to optimize the bacterium as a production organism .

features

Within the bacteria and especially within the clostridia, the species are distinguished primarily on the basis of their metabolic properties and molecular biological characteristics, while morphological characteristics can only be used to a limited extent for the determination of individual species.

morphology

Clostridium ljungdahlii is a rod-shaped, motile, gram-positive bacterium. The width of the bacterial cell is 0.6 μm with a length of 2 to 3 μm. The cell surface is surrounded by a cell envelope 0.1 to 0.2 μm thick. Like other clostridia, this one can also move actively with the flagella ( peritrich ) which are evenly arranged on the entire bacterial surface and do not form any cell colonies. The bacterium can form endospores at both cell poles (terminal and subterminal) .

metabolism

Clostridium ljungdahlii lives under anoxic ( anaerobic ) conditions, i.e. in an environment without oxygen. The pH optimum for the growth of the bacterium is in the slightly acidic range of 6.0 with a tolerance range between 4.0 and 7.0 in which growth occurs. The optimum temperature is around 37 ° C with a tolerance range of 30 to 40 ° C. Under these optimal conditions, the strain ATCC 49587 T ( T stands for type ) has a division rate of 0.26 divisions per hour on a fructose medium or a medium with hydrogen and carbon dioxide , i.e. the number of bacteria doubles approximately every four hours.

The reductive acetyl-CoA pathway in bacteria for carbon dioxide assimilation

Clostridium ljungdahlii is a homoacetogenic bacterium and is able to grow on different carbonaceous substrates. These include ethanol , pyruvate , arabinose , xylose , fructose and glucose as well as carbon monoxide or carbon dioxide together with hydrogen (gaseous in liquid medium). Methanol , ferulic acid , lactic acid , galactose , mannose , sucrose and starch, on the other hand, do not promote growth.

The uptake of carbon monoxide and carbon dioxide in the presence of hydrogen and their conversion to ethanol and acetic acid via acetyl-CoA takes place via the reductive acetyl-CoA route . This is also known as the "Wood-Ljungdahl-Weg" after its main discoverers Harland G. Wood and Lars G. Ljungdahl . In addition to carbon, the administration of vitamins and proteins (e.g. as yeast extract ) as a nitrogen source is necessary. The bacteria mainly produce acetic acid during their growth phase, while ethanol is mainly produced during the stationary phase. In addition, the main product depends on the pH value of the substrate: While the bacteria mainly produce acetic acid at higher pH values ​​between 5 and 7, they mainly produce ethanol at lower pH values ​​between 4 and 4.5.

The biochemical conversion of carbon monoxide and carbon dioxide takes place according to the following reaction equations:

For ethanol:
For acetic acid:

In the case of a hydrogen / carbon dioxide mixture, Clostridium ljungdahlii converts 4 mmol hydrogen and 2 mmol carbon dioxide into 1 mmol acetic acid, while a fructose medium converts 1 mmol fructose into an average of 2.44 mmol acetic acid. The conversion is similar to that of other acetogenic bacteria: Acetobacterium carbinolicum converts one molecule of fructose into 2.1 to 2.3 molecules of acetic acid, Acetobacterium woodi into 2.2 to 2.8; with Clostridium thermoaceticum one molecule of glucose is converted into an average of 2.55 and with Clostridium thermoautortrophicum into 2.5 molecules of acetic acid. Cultures for the production of ethanol from synthesis gas usually produce up to about 2 mmol of ethanol from 1 mmol of fructose. However, the growth of the bacteria and the production rate for ethanol can be controlled via the pH value - it has been shown that at a pH of 7.8 compared to pH 5.5 the cell density is about 1.5 times higher and at the same time the amount of ethanol contained in the medium increases by about 110%.

genetics

The genome of the bacterium was completely sequenced in 2010 as the second genome of an acetogenic bacterium according to Moorella thermoacetica . It has 4,630,065 bp and is one of the largest of the known genomes within the clostridia. 25.2% of the 4,184 identified genes have no known function. Notably, more than 3/4 of the coding sequences lie on one strand of the double helix. The genes identified include the genes for the biosynthesis of the flagella , which are set up in two clusters , as well as a neighboring gene cluster for the chemotaxis of the bacteria. Furthermore, the gene spo0A was detected, which encodes the regulatory protein Spo0A of the same name, which is necessary for spore formation . In addition, the genes of the sigma factors typical for sporulation could be detected, while the genes spo0F and spo0B as in all sequenced clostridia are missing.

The work of Köpke and co-workers focused primarily on the coding sequences that are of interest to the metabolic properties of the bacterium. It could be demonstrated that Clostridium ljungdahlii has a specific Rnf system. Compared to other known acetogenic bacteria, it has a modified metabolism that requires neither a cytochrome system nor sodium ions to provide energy .

ecology

Very little data are available on the ecology of Clostridium ljungdahlii , as the bacterium was examined exclusively under laboratory conditions after its isolation. It lives anaerobically , so it needs an oxygen-free substrate to form reproductive cells. Under aerobic conditions, it forms endospores after a few hours , which survive for several years as persistence stages even in oxygen-rich substrates and, under anaerobic conditions, become vegetative bacteria again.

The strain ATCC 49587 T , which was used for the first description, was isolated from the waste of a chicken farm.

Systematics

The first scientific description of Clostridium ljungdahlii was made in 1993 by the working group around Ralph S. Tanner from the University of Oklahoma . It is named after Lars G. Ljungdahl, who published central works on the elucidation of the metabolism of acetogenic bacteria and clostridia in general and, together with Harland G. Woods, is also the namesake of the Woods-Ljungdahl metabolic pathway.

Clostridium ljungdahlii is taxonomically assigned to the genus Clostridia, which is very species-rich with over 160 species described . The group of clostridia represents a very diverse group, whose phylogenetic classification and possible splitting into several genera is in constant discussion. The differentiation of the species within the genus is based primarily on the molecular biological analysis of the 23S rRNA , according to which the genus is divided into 12 homology groups. Clostridium ljungdahlii is the first known acetogenic bacterium in homology group I. It is closely related to Clostridium tyrobutyricum , a bacterium producing acetic and butyric acid, and Clostridium pasteurianum , which was detected by comparing the sequence 16S rRNA. Clostridium autoethanogenum, which is also described as a separate species, is probably a synonym for Clostridium ljungdahlii , as it cannot be distinguished from it.

Technical importance and research history

The bacterium was isolated by Suhakar Barik of the University of Arkansas for its physiological properties. He was specifically looking for bacteria that are able to use synthesis gas from coal gasification for the production of potentially interesting products. Together with JL Gaddy, also at the University of Arkansas, and other researchers, Barik was able to demonstrate the production of ethanol in 1988 by the bacterial culture that he isolated and assigned to the genus Clostridium . Ralph S. Tanner and D. Yang presented the new species scientifically for the first time in 1990 using Barik's culture as Clostridium ljungdahlii PETC T in a presentation and an abstract at the 90th annual meeting of the American Society for Microbiology in Washington DC. The official first description was by Tanner and staff until 1993 as Clostridium ljungdahlii strain ATCC 49587 T . Under this name, the culture was included in the American Type Culture Collection . In 1992, Gaddy patented the technical production of ethanol and acetic acid with bacterial culture. In the following years, further strains were isolated and cultivated with the strains DSM 13528 and PETC. In 2010 the genome of strain DSM 13528 was sequenced.

In particular, due to the potential use of hydrogen / carbon monoxide (synthesis gas fermentation) and hydrogen / carbon dioxide as a growth substrate, there is great economic interest in the bacterium for use in the field of industrial biotechnology . It is to be used for the production of ethanol, butanol and acetic acid, whereby a use for the production of butanol was only made possible by a gene transfer of the genes of Clostridium acetobutylicum which are important for the production of butanol . These genes are bcd for a butyryl-CoA dehydrogenase , hbd for a 3-hydroxybutyryl-CoA dehydrogenase , thlA for a thiolase , bdha for a butanol dehydrogenase and adhE for a butyraldehyde / butanol dehydrogenase. After installing the alien plasmid with the help of electroporation , butanol production on the basis of synthesis gas was determined in the newly developed strain, which is to be optimized in further steps.

In contrast to other acetogenic bacteria, Clostridium ljungdahlii is already used technically for the production of ethanol, whereby the synthesis gas fermentation is combined with biomass gasification . Systems from INEOS Bio in Great Britain, LanzaTech in New Zealand and China, and BriEnergy and Coskata in the United States are available on a pilot and demonstration scale.

supporting documents

  1. ↑ a b c d e Michael Köpke, Claudia Held, Sandra Hujer, Heiko Liesegang, Arnim Wiezer Antje Wollherr, Armin Ehrenreich, Wolfgang Liebl, Gerhard Gottschalk, Peter Dürre: Clostridium ljungdahlii represents a microbial production platform based on syngas. Proceedings of the National Academy of Sciences of the United States (PNAS) 107 (29), July 20, 2010. Full text , PMID 20616070 .
  2. ↑ a b c d e f g h i j k l R.S. Tanner, LM Miller, D. Yang: Clostridium ljungdahlii sp. nov., an acetogenic species in clostridial rRNA homology group I. International Journal of Systematic Bacteriology 43 (2), 1993: pp 232-236. ( Full text ( Memento of the original from April 18, 2010 in the Internet Archive ) Info: The archive link was inserted automatically and has not yet been checked. Please check the original and archive link according to the instructions and then remove this note. ), PMID 7684239 . @1@ 2Template: Webachiv / IABot / ijs.sgmjournals.org
  3. ↑ a b c J.L. Gaddy, EC Clausen: Clostridium ljungdahlii, an anaerobic ethanol and acetate producing micoorganism. US Patent 5173429, December 22, 1992. ( full text )
  4. ↑ JG Jungdahl: The Autotrophic Pathway of Acetate Synthesis in Acetogenic Bacteria. Annual Review of Microbiology 40, pp. 415-450. PMID 3096193 .
  5. ↑ Harland G. Wood, Steve W. Ragsdale, Ewa Pezacka: The acetyl-CoA pathway of autotrophic growth. FEMS Microbiology Letters 39 (4). doi : 10.1016 / 0378-1097 (86) 90022-4 .
  6. ↑ Asma Ahmed, Allyson White, Peng Hu, Randy Lewis, Raymond Huhnke: Ethanol from Syngas - Microbial Conversion. ( Memento of the original from June 26, 2010 in the Internet Archive ) Info: The archive link was inserted automatically and has not yet been checked. Please check the original and archive link according to the instructions and then remove this notice. SynGrant BioWeb, 2008.  @1@ 2Template: Webachiv / IABot / bioweb.sungrant.org
  7. ↑ Pradeep Chaminda Munasinghe, Samir Kumar Khanal: Biomass-derived syngas fermentation into biofuels: Opportunities and challenges. Bioresource Technology 101 (2010), pp. 5013-5022. PMID 20096574
  8. ↑ Jacqueline L. Cotter, Mari S. Chinn, Amy M. Grunden: Influence of process parameters on growth of Clostridium ljungdahlii and Clostridium autoethanogenum on synthesis gas. Enzyme and Microbial Technology 44 (2009), pp. 281-288. doi : 10.1016 / j.enzmictec.2008.11.002
  9. ↑ MD Collins, PA Lawson, A. Willems, JJ Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippie, JAE Farrow: The Phylogeny of the Genus Clostridium : Proposal of Five New Genera and Eleven New Species Combinations. International Journal of Systematic Bacteriology, October 1994; Pp. 812-826.
  10. ^ A b E. Stackebrandt, I. Kramer, J. Swiderski, H. Hippie: Phylogenetic basis for a taxonomic dissection of the Genus Clostridium . FEMS Immonology and Medical Microbiology 24 (1999); Pp. 253-258; PMID 10397308
  11. ^ S. Barik, S. Prieto, SB Harrison, EC Clausen, JL Gaddy: Biological production of alcohols from coal through indirect liquefaction. Applied Biochemistry and Biotechnology 18 (1), 1988; Pp. 363-378 doi: 10.1007 / BF02930840 .
  12. ↑ RS Tanner, D. Yang: Clostridium ljungdahlii PETC sp. nov., a new, acetogenic, gram-positive anaerobic bacterium, abstr. R-21. Abstr. 90th Annual Meeting on. Soc. Microbiol. 1990; S 249. American Society for Microbiology, Washington DC, 1990.
  13. ↑ UniProt .: " Clostridium ljungdahlii ", accessed on March 1, 2011.
  14. ↑ a b Michael Köpke: Genetic modification of Clostridium ljungdahlii for the production of 1-butanol from synthesis gas. Dissertation of the Faculty of Natural Sciences at Ulm University, 2009 ( full text ; PDF; 10.0 MB)
  15. ↑ a b c Michael Köpke, Sandra Hujer, Peter Dürre: Climate protection through innovative biotechnology. Laborwelt 6/2009.

literature

  • Michael Köpke, Claudia Held, Sandra Hujer, Heiko Liesegang, Arnim Wiezer Antje Wollherr, Armin Ehrenreich, Wolfgang Liebl, Gerhard Gottschalk, Peter Dürre: Clostridium ljungdahlii represents a microbial production platform based on syngas. Proceedings of the National Academy of Sciences of the United States (PNAS) 107 (29), July 20, 2010. Full text , PMID 20616070 .
  • RS Tanner, LM Miller, D. Yang: Clostridium ljungdahlii sp. nov., an acetogenic species in clostridial rRNA homology group I. International Journal of Systematic Bacteriology 43 (2), 1993: pp 232-236. ( Full text ), PMID 7684239 .
  • JL Gaddy, EC Clausen: Clostridium ljungdahlii, an anaerobic ethanol and acetate producing micoorganism. US Patent 5173429, December 22, 1992. ( full text )

Web links

This article was added to the list of articles worth reading on May 17, 2011 in this version .