Clostridium ljungdahlii
Clostridium ljungdahlii is a species of bacteria of the genus Clostridium. She lives in oxygen-free substrates, and in 1988 first isolated from the waste of a chicken farm. Under unfavorable conditions of life it forms after a few hours endospores that survive for several years as resting stages and under anaerobic conditions back to vegetative bacteria.
All information about the bacteria come from the study of laboratory cultures, observations from the original substrates are not available. Because of its ability to produce by fermentation of ethanol and acetic acid on the basis of synthesis gas ( syngas fermentation ), it has a potential importance for industrial biotechnology, manufacturing, however, exist so far only on a pilot scale. In 2010, to better understand the metabolism and for the optimization of the bacterium sequenced and published the genome of Clostridium ljungdahlii as production organism.
Features
Within the bacteria, and especially within the Clostridia species are differentiated mainly due to their metabolic properties and molecular biological characteristics, while morphological features are available for the determination of individual species is limited.
Morphology
Clostridium ljungdahlii is a rod-shaped, motile, Gram- positive bacterium. The width of the bacterial cell is 0.6 microns at a length of 2 to 3 microns. The cell surface is surrounded by a 0.1 to 0.2 microns thick cell casing. Like other clostridia can actively move and does not form cell colonies also this with the bacteria on the entire surface evenly spaced flagella ( peritrichous ). At the two cell poles (terminal and subterminal ) can make the bacteria endospores.
Metabolism
Clostridium ljungdahlii lives under anoxic ( anaerobic ) conditions, ie in an environment without oxygen. The pH optimum for the growth of bacteria is in the slightly acidic range of 6.0 with a tolerance range between 4.0 and 7.0, takes place in the growth. The optimum temperature is about 37 ° C with a tolerance of 30 to 40 ° C. Under these optimal conditions, the strain ATCC 49587T has (T stands for type ), a division rate of 0.26 divisions per hour on a Fructosemedium or a medium with hydrogen and carbon dioxide, the number of bacteria that is doubling every four hours.
Clostridium ljungdahlii is a homoacetogenes bacterium and to grow in the position on various carbon-based substrates. These include ethanol, pyruvate, arabinose, xylose, fructose and glucose, as well as carbon monoxide or carbon dioxide, together with hydrogen (gas in the liquid medium). Methanol, ferulic acid, lactic acid, galactose, mannose, sucrose and starch, however, not lead to a growth promotion.
The inclusion of carbon monoxide and carbon dioxide in the presence of hydrogen and its conversion to ethanol and acetic acid via acetyl -CoA via the reductive acetyl-CoA pathway. This is according to his main discoverers Harland G. Wood and Lars G. Ljungdahl as " Wood - Ljungdahl pathway " known. In addition to carbon while the administration of vitamins, as well as proteins (e.g., as a yeast extract ) as a nitrogen source is needed. The bacteria produce during their growth phase, especially acetic acid, while ethanol is produced mainly during the stationary phase. In addition, the main product is dependent on the pH value of the substrate: While the bacteria form at higher pH values 5-7, especially acetic acid, producing at lower pH values between 4 and 4.5, especially ethanol.
The biochemical reaction of carbon monoxide and carbon dioxide takes place according to the following reaction equations:
In a hydrogen / carbon dioxide mixture 4 mmol hydrogen and 2 mmol of carbon dioxide are converted into 1 mmol of acetic acid by Clostridium ljungdahlii while on a fructose medium, a reaction of 1 mmol of fructose in an average of 2.44 mmol of acetic acid takes place. The implementation is similar to that of other acetogenic bacteria: Acetobacterium carbinolicum is a fructose molecule in 2.1 to 2.3 molecules of acetic acid in order Acetobacterium woodi in 2.2 to 2.8; in Clostridium thermoaceticum a reaction of one molecule of glucose in an average of 2.55 and in Clostridium thermoautortrophicum done in 2.5 molecules of acetic acid. Cultures for the production of ethanol from syngas typically produce up to about 2 mmol of ethanol from 1 mmol of fructose. Over the pH - value, however, can be the growth of bacteria and the production rate of the ethanol control - as could be shown that at a pH of 7.8 compared to pH 5.5, the cell density increases by about 1.5 times is and at the same time increases the amount of ethanol contained in the medium by about 110%.
Genetics
The genome of the bacterium was completely sequenced in 2010 as the second genome of an acetogenic bacterium to Moor Ella thermoacetica. It has 4,630,065 bp and is one of the largest in the known genomes within the clostridia. 25.2% of the 4,184 identified genes have no known function. Notably, there are more than three quarters of the coding sequences on one strand of the double helix. To the identified genes include the genes for the biosynthesis of the flagella, which are arranged in two clusters, as well as an adjacent gene cluster for the chemotaxis of the bacteria. Spo0A the gene was further demonstrated that encodes the same, required for sporulation Spo0A regulatory protein. In addition, the genes typical of the sporulation sigma factors could be detected, while the genes spo0F and spo0B missing, as in all sequenced clostridia.
The work of Koepke and staff focused mainly on the coding sequences that are interesting for the metabolic properties of the bacteria. It could be demonstrated that Clostridium ljungdahlii has a specific Rnf system. It thus has a comparison with other known acetogenic bacteria modified metabolism, which still required sodium ions for the energy supply neither a cytochrome system.
Ecology
About the ecology of Clostridium ljungdahlii are very few data, since the bacterium was investigated after its isolation solely under laboratory conditions. It lives obligate anaerobes, ie required for the formation of reproductive cells, an oxygen- free substrate. Under aerobic conditions, it is after a few hours endospores that survive for several years as resting stages in oxygen-rich substrates and under anaerobic conditions back to vegetative bacteria.
The strain ATCC 49587T, which was used for the original description, was isolated in the waste of a chicken farm.
System
The first scientific description of Clostridium ljungdahlii in 1993 by the research group led by Ralph S. Tanner from the University of Oklahoma. The name derives from Lars G. Ljungdahl, the central work on the elucidation of the metabolism of acetogenic bacteria and clostridia has published in general and in common with Harland G. Woods also the namesake of the Woods - Ljungdahl pathway.
Clostridium ljungdahlii is assigned taxonomically to the more than 160 described species very species-rich genus of clostridia. The group of clostridia represents a very diverse group, their phylogenetic classification and eventual splitting into two or more classes in constant discussion. The differentiation of species within the genus is based mainly on the molecular biological analysis of the 23S rRNA, after which the genus is divided into 12 homology groups. Clostridium ljungdahlii represents the first known acetogenic bacteria in the homology group I dar. There is a close relationship with Clostridium tyrobutyricum, an acetic and butyric acid -producing bacterium, Clostridium pasteurianum, and that was confirmed by comparison of the sequence of 16S rRNA. The Clostridium autoethanogenum also described as a separate species is probably a synonym of Clostridium ljungdahlii because it is indistinguishable from it.
Technical meaning and history of research
The bacterium was isolated from Suhakar Barik of the University of Arkansas, due to its physiological properties. He sought for specific bacteria that are able to use syngas 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 already in 1988 the ethanol production by the isolated of him and of the genus Clostridium associated bacterial culture. Ralph S. Tanner and D. Yang introduced the new type 1990 on the basis Bariks culture as Clostridium ljungdahlii PETCT in a presentation and an abstract at the 90th Annual Meeting of the American Society for Microbiology, Washington DC first scientifically ago. The official first description was by Tanner and co-workers in 1993 as Clostridium strain ATCC 49587T ljungdahlii. Under this designation, the culture was added to the American Type Culture Collection. Back in 1992, patented Gaddy technical ethanol and acetic acid production by the bacterial culture. In subsequent years, additional strains were with strains DSM 13528 and PETC isolated and cultured. 2010, the genome of the 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 a very large economic interest in the bacterium for use in the field of industrial biotechnology. It should be used for the production of ethanol, butanol and acetic acid, with a use for Butanolherstellung was only made possible by a gene important for the genes of Clostridium acetobutylicum Butanolherstellung. These are the genes 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 Butyraldehyd-/Butanol-Dehydrogenase. After installing the dissimilar plasmid using electroporation, a butanol production on the basis of synthesis gas could be found in the newly developed strain that is to be optimized in further steps.
Unlike other acetogenic bacteria Clostridium ljungdahlii is already used industrially for the production of ethanol, wherein the synthesis gas fermentation is connected to a biomass gasification. Of the undertakings INEOS Bio in the UK, LanzaTech New Zealand and China, and BriEnergy and Coskata in the United States are in pilot and demonstration scale available.