AMITY UNIVERSITY RAJASTHAN

AMITY INSTITUTE OF BIOTECHNOLOGY MICROBIOLOGY ASSIGNMENT

ON

METHANOGENESIS, ACETOGENESIS & NITROGEN FIXATION

SUBMITTED BY:Deepak Kumar Tanwar

Btech. Bioinformatics

METHANOGENESIS

Methanogenesis is a microbial process, involving many complex, and differently interacting species, but most notably, the methane-producing bacteria. The biogas process is shown below in figure 1, and consists of three stages; hydrolysis, acidification and methane formation.

Figure 1: The process of methanogenesis). In the first stage of enzymatic hydrolysis, the extracellular enzymes of microbes, such as cellulase, protease, amylase and lipase externally enzymolize organic material. Bacteria decompose the complex carbohydrates, lipids and proteins in cellulosic biomass into more simple compounds. During the second stage, acid-producing bacteria convert the simplified compounds into acetic acid (CH3COOH), hydrogen (H2), and carbon dioxide (CO2). In the

process of acidification, the facultatively anaerobic bacteria utilise oxygen and carbon, thereby creating the necessary anaerobic conditions necessary for methanogenesis. In the final stage, the obligatory anaerobes that are involved in methane formation decompose compounds with a low molecular weight, (CH3COOH, H2, CO2), to form methane (CH4) and CO2 (Gate, 1999). The resulting biogas, sometimes referred to as 'gobar' gas, consists of methane and carbon dioxide, and perhaps some traces of other gases, notably hydrogen sulphide (H2S). Its exact composition will vary, according to the substrate used in the methanogenesis process, but as an approximate guide, when cattle dung is a major constituent of fermentation, the resulting gas will be between 55-66% CH4, 40-45% CO2, plus a negligible amount of H2S and H2 (KVIC, 1993). Biogas has the advantage of a potential thermal efficiency, given proper equipment and aeration, of 60%, compared to wood and dung that have a very low thermal efficiency of 17% and 11% respectively (KVIC, 1993). Methanogenesis or more particularly, the bacteria involved in the fermentation process are sensitive to a range of variables that ultimately determine gas production, and it is worth briefly outlining these factors. Temperature is perhaps the most critical consideration. Gasification is found to be maximised at about 35oC, and below this temperature, the digestion process is slowed, until little gas is produced at 15oC and under. Therefore in areas of temperature changes, such as mountainous regions, or winter conditions that may be more accentuated inland, mitigating factors need to be taken into account, such as increased insulation (Kalia, 1988), or the addition of solar heaters to maintain temperatures (Lichtman, 1983). Loading rate and retention period of material are also important considerations. In the KVIC model, retention ranges between 30-55 days, depending upon climatic conditions, and will decrease if loaded with more than its rated capacity (which may result in imperfectly digested slurry). KVIC state that maximum gas production occurs during the first four weeks, before tapering off, therefore a plant should be designed for a retention that exploits this feature. Retention period is found to reduce if temperatures are raised, or more nutrients are added to the digester. Human excreta, due to its high nutrient content, needs no more than 30 days retention in biogas plants (KVIC, 1983). Other factors likely to affect methanogenesis are pH; gas production is found to decrease with increasing acidity, and can result from over-loading the plant, which may stimulate the more fecund acidophiles, at the expense of the more tardy methane-producing microbes. Improved nutrient content, also, as mentioned above will increase the digestion process, and can be

manipulated by the addition of animal (and male human) urine, while toxic substances, such as heavy metals may inhibit gas production (KVIC, 1983). Understanding the process of methanogenesis allows manipulation, which can serve to maximise gas production in the field. Workers over the last twenty years have experimented with the digestion process, and have made strides in increasing gas yields, using techniques that can be similarly employed in a rural environment. Sharma et al (1988), have shown that biogas generation is increased when the particle size of organic material is small, in this case, less than 1mm. The workers recommend that a physical pre-treatment, such as grinding would improve a system's performance, and could also reduce the size of digester needed. A manual machine for physical pre-treatment of material would be a viable piece of equipment in a rural environment; indeed, there may be a similar piece of equipment already in use. Other workers have found that biogas production is accelerated by the presence of metal ions in biomass (Geeta et al, 1990). The species principally researched was water hyacinth (Eichornia crassipes Solms.), which flourishes in eutrophic water bodies. The plant characteristically grows at high densities, which often leads to clogging, and is therefore considered an environmental pest. Water hyacinth, however, also concentrates nickel from eutrophic environments, upto 0.27 kg h/day, which, when mixed with bovine excreta upto 25 parts per million (ppm) was found to enhance gas production by 40%. The use of E. crassipes in biogas systems can both increase gas production, and contribute to environmental management, by way of controlling a pest. Research in other areas has focused on the composition on the substrate, and its effect on gas production. Habig (1985), fermented a range of organic materials from marine macroalgae to vegetables and discerned that carbohydrate and protein are the principal components utilised during methanogenesis. Such work is invaluable in enabling a sound management and manipulation of methanogenesis, and can be of use to users in a rural environment.

NITROGEN FIXATION
Nitrogen fixation is the natural process, either biological or abiotic, by which nitrogen (N2) in the atmosphere is converted into ammonia. This process is essential for life because fixed

nitrogen is required to biosynthesize the basic building blocks of life, e.g. nucleotides for DNA and RNA and amino acids for proteins. Formally, nitrogen fixation also refers to other abiological conversions of nitrogen, such as its conversion to nitrogen dioxide. Nitrogen fixation is utilized by numerous prokaryotes, including bacteria, actinobacteria, and certain types of anaerobic bacteria. Microorganisms that fix nitrogen are called diazotrophs. Some higher plants, and some animals (termites), have formed associations (symbioses) with diazotrophs. Nitrogen fixation also occurs as a result of non-biological processes. These include lightning, industrially through the Haber-Bosch Process, and combustion. Biological nitrogen fixation was discovered by the Dutch microbiologist Martinus Beijerinck.

Biological nitrogen fixation

Biological nitrogen fixation (BNF) occurs when atmospheric nitrogen is converted to ammonia by an enzyme called nitrogenase. The formula for BNF is:

N2 + 6 H+ + 6 e 2 NH3 The process is coupled to the hydrolysis of 16 equivalents of ATP and is accompanied by the co-formation of one molecule of H2. In free-living diazotrophs, the nitrogenase-generated ammonium is assimilated into glutamate through the glutamine synthetase/glutamate synthase pathway. Enzymes responsible for nitrogenase action are very susceptible to destruction by oxygen. (In fact, many bacteria cease production of the enzyme in the presence of oxygen) Many nitrogen-fixing organisms exist only in anaerobic conditions, respiring to draw down oxygen levels, or binding the oxygen with a protein such as Leghemoglobin. Plants that contribute to nitrogen fixation include the legume family Fabaceae with taxa such as clover, soybeans, alfalfa, lupines, peanuts, and rooibos. They contain symbiotic bacteria called Rhizobia within nodules in their root systems, producing nitrogen compounds that help the plant to grow and compete with other plants. When the plant dies, the fixed

nitrogen is released, making it available to other plants and this helps to fertilize the soil. The great majority of legumes have this association, but a few genera (e.g., Styphnolobium) do not. In many traditional and organic farming practices, fields are rotated through various types of crops, which usually includes one consisting mainly or entirely of clover or buckwheat (family Polygonaceae), which were often referred to as "green manure."

Non-leguminous nitrogen-fixing plants

Although by far the majority of nitrogen-fixing plants are in the legume family Fabaceae, there are a few non-leguminous plants, such as alder, that can also fix nitrogen. These plants, referred to as "actinorhizal plants", consist of 24 genera of woody shrubs or trees distributed among in 8 plant families. The ability to fix nitrogen is not universally present in these families. For instance, of 122 genera in the Rosaceae, only 4 genera are capable of fixing nitrogen. All these families belong to the orders Cucurbitales, Fagales and Rosales, which together with the Fabales form a clade of eurosids. In this clade, Fabales were the first lineage to branch off; thus, the ability to fix nitrogen may be plesiomorphic and subsequently lost in most descendants of the original nitrogen-fixing plant; alternatively, it may be that the basic genetic and physiological requirements were present in an incipient state in the last common ancestors of all these plants, but only evolved to full function in some of them:

There are also several nitrogen-fixing symbiotic associations that involve cyanobacteria (such as Nostoc). These include some lichens such as Lobaria and Peltigera:

Mosquito fern (Azolla species) Cycads Gunnera

A sectioned alder tree root nodule.

A whole alder tree root nodule.

Microorganisms that fix nitrogen

Diazotrophs Cyanobacteria Azotobacteraceae

Rhizobia Frankia

Nitrogen fixation by cyanobacteria

Cyanobacteria inhabit nearly all illuminated environments on Earth and play key roles in the carbon and nitrogen cycle of the biosphere. Generally, cyanobacteria are able to utilize a variety of inorganic and organic sources of combined nitrogen, like nitrate, nitrite, ammonium, urea or some amino acids. Several cyanobacterial strains are also capable of diazotrophic growth. Genome sequencing has provided a large amount of information on the genetic basis of nitrogen metabolism and its control in different cyanobacteria. Comparative genomics, together with functional studies, has led to a significant advance in this field over the past years. 2-oxoglutarate has turned out to be the central signalling molecule reflecting the carbon/nitrogen balance of cyanobacteria. Central players of nitrogen control are the global transcriptional factor NtcA, which controls the expression of many genes involved in nitrogen metabolism, as well as the PII signalling protein, which fine-tunes cellular activities in response to changing C/N conditions. These two proteins are sensors of the cellular 2oxoglutarate level and have been conserved in all cyanobacteria. In contrast, the adaptation to nitrogen starvation involves heterogeneous responses in different strains.[4] Nitrogen fixation by cyanobacteria in coral reefs can fix twice the amount of nitrogen than on landaround 1.8 kg of nitrogen is fixed per hectare per day. .Symbiotic Nitrogen Fixation Symbiotic nitrogen fixation occurs in plants that harbor nitrogen-fixing bacteria within their tissues. The best-studied example is the association between legumes and bacteria in the genus Rhizobium. Each of these is able to survive independently (soil nitrates must then be available to the legume), but life together is clearly beneficial to both. Only together can nitrogen fixation take place. A symbiotic relationship in which both partners benefits is called mutualism.

Rhizobia
Rhizobia are Gram-negative bacilli that live freely in the soil (especially where legumes have been grown). However, they cannot fix atmospheric nitrogen until they have invaded the roots of the appropriate legume.

The Infection Thread

The interaction between a particular strain of rhizobia and the "appropriate" legume is mediated by:

a "Nod factor" secreted by the rhizobia and transmembrane receptors on the cells of the root hairs of the legume. Different strains of rhizobia produce different Nod factors, and different legumes produce receptors of different specificity.

If the combination is correct, the bacteria enter an epithelial cell of the root; then migrate into the cortex. Their path runs within an intracellular channel that grows through one cortex cell after another. This infection thread is constructed by the root cells, not the bacteria, and is formed only in response to the infection. When the infection thread reaches a cell deep in the cortex, it bursts and the bacteria are engulfed by endocytosis into endosomes. At this time the cell goes through several rounds of mitosis without cytokinesis so the cell becomes polyploid. The electron micrograph on the right (courtesy of Dr. D. C. Jordan) shows a rhizobia-filled infection thread growing into the cell (from the upper left to the lower right). Note how the wall of the infection thread is continuous with the wall of the cell. Once the thread ruptures, rhizobia are engulfed into membrane-bound symbiosomes within the cytoplasm (dark ovals). The cortex cells then begin to divide rapidly forming a nodule. This response is driven by the translocation of cytokinins from epidermal cells to the cells of the cortex. The photo on the left (courtesy of The Nitragin Co. Milwaukee, Wisconsin) shows nodules on the roots of the birdsfoot trefoil, a legume. The rhizobia also go through a period of rapid multiplication within the nodule cells. Then they begin to change shape and lose their motility. The bacteroids, as they are now called, may almost fill the cell. Only now does nitrogen fixation begin. The electron micrograph on the right (courtesy of R. R. Hebert) shows bacteroid-filled cells from a soybean nodule. The horizontal line marks the walls between two adjacent nodule cells. Root nodules are not simply structureless masses of cells. Each becomes connected by the xylem and phloem to the vascular system of the plant. The photo below on the left shows a developing lateral root on a pea root. On its right is a segment of a pea root showing a

developing nodule 12 days after the root was infected with rhizobia. Both structures are connected to the nutrient transport system of the plant (dark area extending through the center of the root). (Photomicrographs courtesy of the late John G. Torrey.) Thus the development of nodules, while dependent of rhizobia, is a well-coordinated developmental process of the plant. Although some soil bacteria (e.g., Azotobacter) can fix nitrogen by themselves, rhizobia cannot. Clearly rhizobia and legumes are mutually dependent. The benefit to the legume host is clear. The rhizobia make it independent of soil nitrogen. But why is the legume necessary? The legume is certainly helpful in that it supplies nutrients to the bacteroids with which they synthesize the large amounts of ATP needed to convert nitrogen (N2) into ammonia (NH3) In addition, the legume host supplies one critical component of nitrogenase the key enzyme for fixing nitrogen. The bacteroids need oxygen to make their ATP (by cellular respiration). However, nitrogenase is strongly inhibited by oxygen. Thus the bacteroids must walk a fine line between too much and too little oxygen. Their job is made easier by another contribution from their host: hemoglobin. Nodules are filled with hemoglobin. So much of it, in fact, that a freshly-cut nodule is red. The hemoglobin of the legume (called leghemoglobin), like the hemoglobin of vertebrates, probably supplies just the right concentration of oxygen to the bacteroids to satisfy their conflicting requirements. The metal molybdenum is a critical component of nitrogenase and so is absolutely essential for nitrogen fixation. But the amounts required are remarkably small. One ounce of molybdenum broadcast over an acre of cropland in Australia was found to be sufficient to restore fertility for over ten years. The photo at the right shows that the legume clover grows normally only where the supply of molybdenum is adequate. The soil shown here (in eastern Australia) is naturally deficient in molybdenum. Although the entire fenced-in plot was seeded to clover, the plant was able to flourish and fix nitrogen only where molybdenum fertilizer had been added (foreground).

Because of the specificity of the interaction between the Nod factor and the receptor on the legume, some strains of rhizobia will infect only peas, some only clover, some only alfalfa,

etc. The treating of legume seeds with the proper strain of rhizobia is a routine agricultural practice. (The Nitragin Company, that supplied one of the photos above specializes in producing rhizobial strains appropriate to each leguminous crop.) How did two such organisms ever work out such an intimate and complex living relationship? Assuming that the ancestors of the rhizobia could carry out the entire process by themselves as many other soil bacteria still do they must have gained some real advantage from evolving to share the duties with the legume. Perhaps the environment provided by their host, e.g., lots of food and just the right amount of oxygen, enabled the rhizobia to do the job more efficiently than before.

Nitrification
Ammonia can be taken up directly by plants usually through their roots. However, most of the ammonia produced by decay is converted into nitrates. This is accomplished in two steps:

Bacteria of the genus Nitrosomonas oxidize NH3 to nitrites (NO2). Bacteria of the genus Nitrobacter oxidize the nitrites to nitrates (NO3).

These two groups of autotrophic bacteria are called nitrifying bacteria. Through their activities (which supply them with all their energy needs), nitrogen is made available to the roots of plants. Both soil and the ocean contain archaeal microbes, assigned to the Crenarchaeota, that convert ammonia to nitrites. They are more abundant than the nitrifying bacteria and may turn out to play an important role in the nitrogen cycle. Many legumes, in addition to fixing atmospheric nitrogen, also perform nitrification converting some of their organic nitrogen to nitrites and nitrates. These reach the soil when they shed their leaves.

Denitrification
The three processes above remove nitrogen from the atmosphere and pass it through ecosystems. Denitrification reduces nitrates to nitrogen gas, thus replenishing the atmosphere. Once again, bacteria are the agents. They live deep in soil and in aquatic sediments where conditions are anaerobic. They use nitrates as an alternative to oxygen for the final electron acceptor in their respiration. Thus they close the nitrogen cycle.

ACETOGENESIS
Although some acetate (20%) and H2 (4%) are directly produced by acidogenic fermentation of sugars, and amino acids, both products are primarily derived from the acetogenesis and dehydrogenation of higher volatile fatty acids. Obligate H2-producing acetogenic bacteria are capable of producing acetate and H2 from higher fatty acids. Only Syntrophobacter wolinii, a propionate decomposer (3) and Sytrophomonos wolfei, a butyrate decomposer (4) have thus far been isolated due to technical difficulties involved in the isolation of pure strains, since H 2 produced, severely inhibits the growth of these strains. The use of co-culture techniques incorporating H2 consumers such as methanogens and sulfate-reducing bacteria may therefore facilitate elucidation of the biochemical breakdown of fatty acids. Overall breakdown reactions for long-chain fatty acids are presented in Tables 4-1 and 4-2. H2 production by acetogens is generally energetically unfavorable due to high free energy requirements ( Go,> 0; Table 4-1 and 4-2). However, with a combination of H 2-consuming bacteria (Table 4-2, 4-3), co-culture systems provide favorable conditions for the decomposition of fatty acids to acetate and CH4 or H2S ( Go,< 0). In addition to the decomposition of long-chain fatty acids, ethanol and lactate are also converted to acetate and H2 by an acetogen and Clostridium formicoaceticum, respectively. The effect of the partial pressure of H2 on the free energy associated with the conversion of ethanol, propionate, acetate, and H2/CO2 during methane fermentation is shown in Fig. 4-2. An extremely low partial pressure of H2 (10-5 atm) appears to be a significant factor in propionate degradation to CH4. Such a low partial pressure can be achieved in a co-culture with H2-consuming bacteria.