Methanosarcina barkeri is an extremophile

Versatility of Methanosarcina and problems with domestication and cultivation

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1 Versatility of Methanosarcina and Problems with Domestication and Cultivation Written by Vanja Michel Supervisor: Kurt Hanselmann Abstract: To study the cultivation of Methanosarcina spp. To simplify on a solid substrate, Metcalf et al. (2) developed an incubator which enables 119 or even 153 Petri dishes to be used at the same time. They also described a procedure that can be used to use this incubator within an anaerobic chamber, in which a gas mixture of N 2 / CO 2 / H 2 S (ratio 79.9: 10: 0.1) at the beginning and a mixture at the end of N 2 / CO 2 / H 2 (ratio 75: 20: 5) is used. The genome of M. acetivorans was completely sequenced and analyzed (by J.E. Galagan et al., 2002, 3). Another study analyzed the exceptional growth of M. acetivorans on carbon monoxide (4). The resulting information showed the versatile growth of Methanosarcina, which methane can form via the hydrogen path, the methylotrophic path and the acetate path. It found that using CO as a substrate resulted in the formation of acetate and fumarate and less methane was produced. This leads to the assumption that carbon monoxide acts as an inhibitor of methanogenesis. Introduction: In my case study I wanted to learn more about the methanogenic Archaea Methanosarcina. This group aroused my interest because it stands out among the archaea, which are still rather poorly studied, in that it is very diverse. So I asked myself what the difficulties in cultivating Methanosarcina are and how these problems can be solved. In doing so, I mainly focused on a possibility to cultivate these species on a large scale, which is of great interest and advantage for research. The Methanosarcina group is an interesting group among the methanogens. Fig. 1: Living culture of M. acetivorans: multicellular collections of archaea can be seen, as it has the greatest diversity of methane metabolisms. So I asked myself what different substrates methanosarcina can grow on. The species of the genus Methanosarcina belong to the family Methanosarcinaceae, the order Methanosarcinales, the class Methanomicrobia, the phylum of the Euryarchaeota and the domain of the Archaea. (1, a list of the species can be found under 1

2 Procedure: Since the Methanosarcina are strictly anaerobic, like all methanogenic organisms, a major problem in the cultivation of these species is to create a sufficiently oxygen-free environment in which they can survive. For a long time there was no known way of handling many Petri dishes at the same time. In 1997, however, W.W. Metcalf, J.K. Zhang and R.S. Wolfe developed an incubator that simplifies the cultivation of the Methanosarcina species. (2) In order to clarify the extraordinary properties of some species of the genus Methanosarcina, I have read various reports on this topic and I have particularly occupied myself with the various possibilities for the growth of this group. The sequencing of the genome of Methanosarcina acetivorans led to many findings and provides information about the diversity of substrates on which this species can grow, which is unusual for methanogens. (3, 4) I found the information to answer my questions mainly in reports which are freely accessible on the Internet by searching on the page "NCBI Literature Databases" (under PMC, PubMed Central, for interesting articles related to my topic Results: Incubator for the cultivation of Methanosarcina (2) To simplify the cultivation of Methanosarcina on agar plates, WW Metcalf, JK Zhang and RS Wolfe developed an incubator by modifying an airlock, which is intended for use in an anaerobic chamber A careful and precise procedure to prevent contact with oxygen is necessary, since the types of Methanosarcina, like the other methanogens, are strictly anaerobic and therefore extremely sensitive to oxygen. The airlock used by the authors of the study was rectangular and had two openings, Valves and a vacuum gauge (for the exact dimensions of all parts see Ori final report 2). They closed one of the two openings with an acrylic plate, for the other opening they developed a system to press the acrylic door against the rubber seal and thus prevent gas from escaping. To do this, they had to leave out some screws and replace them with brackets. Rotary locks have been attached to all sides of the door, which fit into the U-shaped brackets, making it possible to close the door airtight. A silicone pore sealer was used to seal the various openings. Fig. 2: Airlock that was used to build the incubator 2

3 To allow gases to enter, the airlock connection has been replaced by a three-way valve. They connected an opening of this valve to an external cylinder which contained 79.9% N 2, 10% CO 2 and 0.1% H 2 S by means of a copper pipe. The other port was connected to a cylinder with 75% N 2, 20% CO 2 and 5% H 2. A heated copper brush ensured that any oxygen was reduced. The vacuum pump was then connected with a PVC tube to the interior of the incubator and to a fume cupboard (in order to discharge the withdrawn gas). A turntable was installed on a ball bearing inside the incubator so that the treated Petri dishes can be worked well and they can be easily removed. This means that 119 Petri dishes on seven metal shelves can be treated in the incubator at the same time. If the turntable is left out, there is even enough space for 153 Petri dishes. To use the incubator, the Petri dishes are placed in it, the door is closed and the air is sucked out to a negative pressure of 0.5 atm (-50 kpa). The turn locks are then closed and the incubator is up to Fig. 3: A / B: Incubator with the door open or closed. Among other things, the following are shown: a) Rotary lock b) U-shaped brackets C: Incubator installed in an anaerobic chamber D: Placeholder filled with N 2 / CO 2 / H 2 S to approximately zero pressure. This process is repeated seven times. Last time a little negative pressure should be kept. If you want to remove the plates, the gas mixture is withdrawn again up to a pressure of -50 kpa and the chamber is filled with N 2 / CO 2 / H 2. This is repeated 13 times. When the vacuum is reached for the last time, the rotary locks are loosened and enough N 2 / CO 2 / H 2 is added until the door opens without any problems. In their experiments, these three scientists needed a gas mixture with H 2 S, since until now all methanogenic archaea seem to need H 2 S when cultivated on solid medium. They removed any remaining H 2 S using activated charcoal. If the incubator is not completely filled, placeholders can be used to save used gas. (see also Figure 2 D) 3

4 Versatile growth opportunities (3, 4) The formation of methane plays an essential role for all methanogenic organisms. However, several different types of methane formation do not occur in all groups of methanogens, and so in some cases they have a limited selection of substrates on which they can grow. The methanosarcina group is an exception here. This includes organisms that produce methane in all previously known ways and thus can also grow on the corresponding substrates. So far we have known organisms that grow using three groups of substances. Some use acetate, which they process into methane via acetyl-CoA. Others use CO 2 and H 2 as substrates for their growth (this is widespread among the methanogens). After many intermediate stages, they ultimately convert the CO 2 into CH 4 as well. A third possibility is the use of methylamines, methyl sulfides or methanol to produce methane via the intermediate product methyl-CoM. All of these substrates can be used by Methanosarcina species. Fig. 4: Different methanogenesis pathways: this figure shows which substances can be used to form methane. The so-called "acetoclastic pathway" is shown in blue, the "methylotrophic pathway" is green and the "hydrogen pathway" is red. The species Methanosarcina acetivorans is a particular example of this versatility. The size of the genome alone indicates that it has much broader growth opportunities than other archaeal representatives (M. acetivorans has a genome of 5,751,492 bp). This is the largest known genome among the archaea and the fourth largest genome of all sequenced prokaryotes. About 200 genes related to methanogenesis were found in this genome. M. acetivorans has two or three copies of the genes necessary for methanogenesis with acetate as well as those for the use of methanol, monomethylamine, dimethylamine and trimethylamine. However, due to the absence of some hydrogenases, this species is unable to use CO 2 and H 2 as a substrate. Further genes were found in the genome of M. acetivorans which indicate that further substrates are possible, but which are not yet known. Perhaps the most unusual type of growth in M. acetivorans is the use of carbon monoxide as a substrate. The conversion of CO is particularly important for humans, as this substance has a toxic effect even in low concentrations. Up until 2004, only 2 methanogenic species were known that could use CO as a substrate, namely Methanothermobacter thermoautotrophicus and Methanosarcina 4

5 barkeri. However, the occurrence of CO dehydrogenases in M. acetivorans indicated that this species can also use carbon monoxide. A study has shown that M. acetivorans can be cultivated in such a way that it grows with CO as a limiting substance. After getting used to this growth, the rate decreased as the CO partial pressure decreased. However, not as much methane was produced as one would actually have expected, which indicated other products. The scientists who carried out this study found that, along with methane, acetate and fumarate were the main products. The more CO there was, the less methane and the more fumarate and acetate were produced. Discussion: Research on archaea is much less advanced in some areas than research on vertebrates or bacteria, for example. Therefore, the development of the incubator for large-scale cultivation is of great importance, as it greatly simplifies the research work and thus paves the way for a better understanding of these interesting organisms. In addition, cultivation on a large scale can help to improve previous knowledge of methanogenesis. The sequencing of the genome of M. acetivorans is a first step towards a better understanding of the diverse group of Methanosarcina, which also includes thermophilic and halophilic species. Although the entire genome has been sequenced, there are still many unanswered questions for future researchers. For example, there are genes which indicate that M. acetivorans also grows in other ways that are not known to this day. The ability of M. acetivorans to grow on carbon monoxide could be useful in the treatment of toxic CO. This organism could be used to develop CO in reactions that forcibly produce CO into acetate, fumarate and methane. The problem with this task is oxygen sensitivity. There are probably simpler chemical methods, or organisms that are easier to work with, to break down carbon monoxide. The fact that less methane was formed with increasing CO concentration strongly suggests that carbon monoxide inhibits methanogenesis. In the presence of a sufficient concentration (or a sufficiently high partial pressure), other products are preferentially formed and the biosynthesis of methane decreases. List of sources 1. M.T. Madigan and J.M. Martinko, 2006, Brock, Biology of Microorganisms, Eleventh Edition, Appendix 2, pp. A-5 2. W.W. Metcalf, J.K. Zhang and R.S. Wolfe, An Anaerobic, Intrachamber Incubator for Growth of Methanosarcina spp. on Methanol-Containing Solid Media, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1998, p

6 3rd J.E. Galagan, C. Nusbaum et al., 2002, The Genome of M. acetivorans Reveals Extensive Metabolic and Physiological Diversity, Genome Res:, can be downloaded at 4. M. Rother and W.W. Metcalf, 2004, Anaerobic growth of Methanosarcina acetivorans C2A on carbon monoxide: An unusual way of life for a methanogenic archaeon, list of figures Fig. 1: E. Conway de Macario and A.J.L. Macario, found on Fig. 2: W.W. Metcalf, J.K. Zhang and R.S. Wolfe, 1998, An Anaerobic, Intrachamber Incubator for Growth of Methanosarcina spp. on Methanol-Containing Solid Media, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1998, p and all sources listed there Fig. 3: ibid. Fig. 4: J.E. Galagan, C. Nusbaum et al., 2002, The Genome of M. acetivo-rans Reveals Extensive Metabolic and Physiological Diversity, Genome Res: Appendix: Additional Material: The genus Methanosarcina is a very interesting and diverse group of the Archaea and on the Internet there is a multitude of exciting articles on a wide variety of topics. Here are a few examples: Growth form of Methanosarcina spp. with increased osmolarity: K.R. Sowers, J.E. Boone, and R.P. Gunsalus, 1993, Disaggregation of Methanosarcina spp. and Growth as Single Cells at Elevated Osmolarity, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1993, p Lipid structures from Methanosarcina spp .: G.D. Sprott et al., Hydroxydiether Lipid Structures in Methanosarcina spp. and Methanococcus voltae, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1993, p Halotoleranz: K.R. Sowers and R.P. Gunsalus, 1995, Halotolerance in Methanosarcina spp .: Role of N ε -acetyl-β-Lysine, α-glutamate, Glycine Betaine, and K + as Compatible Solutes for Osmotic Adaptation, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1995, p