Dinitrogen Fixation In Azotobacter Chroococcum Biology Essay


There are many important factors that contribute usable nitrogen into Earth's atmosphere and biosphere and a large percentage of the nitrogen found on Earth is found as dinitrogen gas in the atmosphere, approximately 80%. Azotobacter chroococcum, a free-living nitrogen-fixing aerobic soil bacterium, is capable of fixing this dinitrogen gas into usable compounds necessary to support life. Using complex nitrogenase enzymes that are highly-oxygen sensitive, A. chroococcum is able to fix dinitrogen gas into ammonia, nitrates and other nitrogen-containing compounds into the soil that can be used by a wide variety of organisms for biochemical metabolic pathways. A. chroococcum is found widely dispersed throughout Texas and the world, and was chosen for use in this experiment because it is the most abundant of all the Azotobacter species (Azotobacter Chroococcum 2007). A chroococcum was also chosen for use, due to its usefulness in agricultural practices, its simple growing capacity, and its non-symbiotic nature. Due to higher alkalinity of Texas soils, with a pH of around 8.5, iron levels in the soil tend to be significantly reduced. This can be attributed to the large amounts of limestone (calcium carbonate) present. Azotobacter chroococcum, uses this free iron to help reduce superoxide compounds that form in the soils, and help the bacteria form protective cysts or slime layers, as well as aid in its extremely high respiration rate, among the highest of all organisms, that combined, protect a vital highly oxygen-sensitive dinitrogen fixing enzyme, nitrogenase. Because of this, an experiment was designed to test the effectiveness and efficiency of the nitrogenase enzyme in the free-living nitrogen-fixing aerobic bacterium, A. chroococcum. This experiment is testing whether the unique nitrogenase enzymes can be dependent upon higher levels of iron (II) compounds present in its environment, while being exposed to varied amounts (partial pressures) of molecular oxygen. It is hypothesized that under elevated levels of oxygen, A. chroococcum can use higher amounts of iron to help survive under super-oxidative stress, and while under anaerobic conditions, A. chroococcum will survive and multiply more quickly as well as utilize the excess iron to help fix dinitrogen gas at a more rapid rate, compared with atmospheric oxygen levels.


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Bacteria are largely responsible for continuing the nitrogen cycle when nitrogen is released into the atmosphere. Azotobacter chroococcum is an aerobic, free-living, dinitrogen fixing bacterium found in the soil that is commonly used for fertilizer due its nitrogen-fixing capabilities. A. chroococcum uses iron, molybdenum and other special minerals from the soil to help protect the highly oxygen-sensitive nitrogenase enzymes that fix the dinitrogen gas. This experiment was designed to test if A. chroococcum could survive and fix nitrogen under various oxygen levels, when supplied with specific amounts if iron compounds. A chroococcum was cultured in modified tryptic soy broth with added iron compounds and gently aerated at atmospheric, anaerobic and superoxidative conditions for 120 hours. The results showed that overall, there was a greater change in pH in the anaerobic flasks, indicating a greater amount of fixed nitrogen, in the form of ammonia, with positive results in the super-oxidative flasks, indicating growth and nitrogen fixation under higher oxygen levels is possible and one flask in particular (531mg) in the superoxidative group suggests that dinitrogen fixation occurs at a more rapid rate under higher oxygen levels with the presence of elevated iron levels, which may be due to increased cellular respiration and metabolic rates in response to higher oxygen levels. These data support the hypothesis that under superoxidative stress, dinitrogen fixation can occur with the presence of iron compounds, and supports further study in the superoxidative stress groups.

Background Research

In 1901, a Dutch microbiologist Martinus Beijerinck used an enrichment culture technique with a medium lacking in a combined nitrogen source, and discovered an aerobic microorganism that was capable of fixing molecular nitrogen, to which the name of Azotobacter chroococcum was given (Azotobacter Chroococcum 2007).

The genus Azotobacter comprises of large, gram-negative, facultative rods primarily found in neutral to alkaline soils that are capable of fixing dinitrogen gas non-symbiotically. Azotobacter is also of high interest because it has the highest respiratory rate of any living organism (Bulen, et al 1996). In addition to its ecological and physiological importance, Azotobacter is also of high interest due to its unusual ability to form a resting structure known as a cyst. Azotobacter cells are quite large for bacteria with diameter of about 2-4μm or larger. Pleomorphism is common and a variety of cell shapes and sizes have been described. On carbohydrate-containing media, extensive capsules or slime layers are produced and have been noted in literature. Azotobacter is able to grow on a wide variety of carbohydrates, alcohols and organic acids. The metabolism of carbon compounds is strictly oxidative and acids or other fermentation products are rarely ever produced (Linkerhägner & Oelze 1997).

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In regards to temperature, Azotobacter species are mesophilic organisms, meaning that they are found growing best at moderate temperatures. Many researchers regard 25-30 °C as the optimum temperature for growing and culturing Azotobacter (Saribay 2003). The minimum temperature of growth of

Azotobacter lies a little above 0 °C. Vegetative (consisting of little to no metabolism and found in an alginate or slime capsule) Azotobacter cells cannot tolerate high temperatures, and if kept between 45-48 °C, they degenerate and die (Saribay). This is curious since it shows no similarity to endospore activity, and is thought to protect the cell only from oxidative stress when nutrient limited. According to the study of Dhanasekar in 2003, Azotobacter, utilizing glucose and cultivated in a batch reactor, was reported to have optimum temperature at 30 °C; furthermore, even at 28 °C and 32 °C growth was decreased.

All members of the genus Azotobacter fix nitrogen and Azotobacter are able to develop on media with pH range from 4.5 to 8.5. Individual species of Azotobacter, and possibly even varying strains, differ in their sensitivity to an acid medium. It has been found, for example, that the minimum pH of the medium is about 5.5 for A. chroococcum. The optimum pH for Azotobacter lies within the pH range of 7.2 to 8.2 and observed at 7.5 (Linkerhägner & Oelze). Furthermore, growth is decreased at both acidic and alkaline pH range (Saribay), and growth also occurs on simple forms of combined nitrogen: ammonia, urea and nitrates (Tindale).

The oxygen-labile nitrogenase enzyme remains functional because of 'respiratory protection', where O2 consumption is uncoupled from ATP generation and the dissolved O2 concentration around the cell is reduced to very low levels (Saribay, Tindale). Iron is an essential nutrient for a similar species, A. vinelandii, and is required for respiratory protection, nitrogenase activity and protection against toxic oxygen products generated by active respiration both of the organism itself and surrounding organisms in the habitat. In its native soil environment, A. vinelandii is quite capable of extracting iron from the insoluble iron minerals that abound under aerobic, neutral-pH conditions (Zueng-Sang 2007). The bacterium releases siderophores for the solubilization, chelation and transport of iron into the cell. These include: catecholate, azotobactin, aminochelin, azotochelin, and protochelin (Tindale). These siderophores are produced under various iron-limited conditions, but are expressed in a unique sequential fashion. At limiting iron levels less than 10 µM, the catecholates are produced, followed by azotobactin at less than 3 µM iron (Tindale).

Respiration by iron-limited cells presents a considerable hazard: not only are superoxide radicals and hydrogen peroxide generated, but also iron-limited cells have very low superoxide dismutase (SOD) activity leaving the cell with little protection from these highly damaging compounds (Galindo, et al 2007). Vigorous aeration of iron-limited A. vinelandii cultures causes the upregulation of catecholate siderophore synthesis, but azotobactin is not similarly affected. It has been shown that the generation of hydroxyl radicals by the iron-catalysed Fenton reaction is limited when iron is chelated via azotochelin or protochelin (Tindale).

The expression of high-affinity, siderophore-mediated uptake systems for iron accumulation is controlled at the level of transcription by the dissociation of the ferric uptake regulator (Fur) from an iron-box operator sequence. In iron-limited medium, decreased levels of the corepressor, intracellular Fe 2+, allow Fur dimer dissociation from the promoter region and ensuing transcription of iron-regulated genes. This is a highly conserved control mechanism, found in most aerobes and facultative aerobes (Tindale). Thus, differential control of Azotobacter siderophore synthesis could be explained by differences in Fur affinity for catecholate or azotobactin Fur-binding operator sequences. Unfortunately, siderophore-specific operator sequences have not been identified in A. vinelandii and there has been no proof of the existence of a Fur homologue. The biological fixation of dinitrogen depends on the activity of the highly oxygen-sensitive nitrogenase enzyme complex (Galindo). Despite this sensitivity, species of the diazotrophic Azotobacter are able to grow under fully aerobic conditions (Bulen). For the survival of these bacteria under aerated conditions, one of the priorities of their entire metabolism is to protect the active nitrogenase from being damaged by oxygen. Protection of this enzyme from oxygen has been proposed to occur in Azotobacter mainly through two mechanisms: (i) high respiratory activity that removes oxygen already at the cell surface and (ii) reversible conversion of the enzyme into a protected inactivated state (Bulen, Saribay). The first mechanism is believed to explain the function of nitrogenase when cells grow diazotrophically in the presence of O2. The second mechanism is considered to be used to protect the reversibly inactivated enzyme from O2 damage when the respiratory protection becomes overburdened, such as with a sudden increase in the ambient O2 concentration (Bookrags 2005) or under conditions of phosphate limitation (Saribay). In the latter case, the respiration rate of cells is limited due to shortage of phosphate for oxidative phosphorylation. For growing cells which need an active nitrogenase system to provide their nitrogen requirement, the second protection mechanism can work only temporarily because it does not remove O2 and merely provides a barrier until a favorable habitat is encountered.

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Although the respiratory-protection hypothesis is generally accepted, some researchers have questioned it. Some authors found that at O2 concentrations ranging from 30% to 100% air saturation, A. vinelandii (a similar bacterium), showed almost constant respiration rates and negligible decreases in nitrogenase activity. These results are incompatible with the concept of respiratory protection. The observed a decrease in the cellular surface area per cell volume at elevated O2 levels and suggested that this decrease of cell surface may also provide some protection for the nitrogenase. In addition, it was hypothesized that the energy efficiency of cellular respiration is more important than the respiration rate as a protective mechanism.

Azotobacter are known to produce alginate under aerobic conditions. The formation of alginate is strongly affected by oxygen tension, especially in nitrogen-free medium and with limited phosphate (Saribay, Bulen). A possible link between alginate formation and protection of nitrogenase in this organism has not been examined so far in literature. Studies of the nitrogenase protection mechanisms of Azotobacter have mostly been based on either the respiration rates or acetylene reduction measurements as indications of nitrogenase activity (Galindo). In fact, the true biological function of alginate formation in bacteria is not fully understood. Alginate is important for cyst formation in A. vinelandii as a coating protective polysaccharide material (Galindo). This was evidenced by the fact that noncapsulate mutants of A. vinelandii were unable to form cysts. Such a coating protects the cells from desiccation and mechanical stresses. Under favorable growth conditions, the coat swells and the cyst germinates, divides, and releases a vegetative cell. However, the formation of a cyst in A. vinelandii does not explain the formation of alginate by vegetative cells under conditions not favoring cyst formation (Saribay, Bulen, Galindo).

For the protection of nitrogenase in nitrogen-fixing microorganisms, a low intracellular oxygen concentration is essential (Tindale). For A. vinelandii the increase of viscosity of the culture broth during the course of cultivation as a result of increasing biomass and alginate concentrations can reduce the oxygen transfer rate from the gas phase to the aqueous phase and from the bulk liquid to the cell surface. It is hypothesized that A. chroococcum act in a similar manner. To avoid a high oxygen transfer rate into the cell, an effective oxygen barrier on the cell surface can be even more crucial. The present communication provides evidence for the importance of alginate capsule formation on the cell surface for the survival of diazotrophically growing A. vinelandii under aerobic conditions. Variations in the quantity and quality of the alginate produced are being studied under different culture conditions, and based on the experimental results, new protection mechanisms for nitrogenase against oxygen and oxygen byproducts are being proposed.



The obligate aerobic bacterium Azotobacter chroococcum was cultured in modified tryptic soy broth (TSB) under varied levels of oxygen with varied amounts of iron (in the form of ferrous sulfate) mixed with the broth. The broth was modified and the iron content of the broth was changed by adding ferrous sulfate to standard TSB. To better represent a vitamin-enriched soil environment, a multivitamin was added as a fine powder along with the ferrous sulfate. Once all materials were gathered and necessary changes were made to the TSB, the flasks were plugged with 3 cotton balls (5 cotton balls for the 2000ml flasks) wrapped in cheesecloth and aluminum foil was placed over the cotton balls in cheesecloth and the opening of the flasks to ensure that no further dust particles were allowed in, yet air was still free to flow. With the modified TSB in the appropriate flasks (culturing flasks were left in 125ml flasks, while the excess broth was placed in a 2000ml flask), the flasks were steam autoclaved at 121°C for 15-20 minutes. The flasks were allowed to cool overnight in the closed autoclave or allowed to refrigerate overnight. The cultures were then inoculated with a specified number of colonies (Controls- 20 colonies, all others- 4 colonies). The cultures were immediately grown and incubated in 125ml flasks and were aerated on a platform shaker at about 25-30 strokes per minute between 28-30 °C for 120 hours. In the anaerobic cultures and super-oxidative cultures, the flasks were not wrapped as previously mentioned. These flasks were carefully washed out with a foam (64% ethyl alcohol by volume) and hot water. The flasks were then filled with its appropriate modified Tryptic Soy Broth. The stoppers with holes (8 in total) were allowed to sit in the antiseptic foam (64% ethyl alcohol by volume) for 20 minutes and quickly rinsed with distilled water and placed snuggly in the appropriate flask opening. The flasks and stoppers were then allowed to boil on a hot plate in a fume hood for 15 minutes to ensure sterility. The anaerobic chambers consisted of a specialized long plastic bag containing a smaller bag with 5.0g of calcium carbonate, iron powder, citric acid, and inert extender. The flasks were then allowed to cool and then inoculated with 4 cultures from the same Petri dish as the controls. The flasks were each subsequently placed in their respective appropriate section in the specialized plastic bags and the liquid reagent was poured down the specialized canals and allowed to mix with the bags of 5.0g of iron powder, calcium carbonate, citric acid, and inert extender. The ends of the bags were then folded over and, in their respective containers; the anaerobic bags were placed in plastic containers with a sealable twisting lid and immediately allowed to culture in a non-moving incubator at 30°C. After 4 hours, all 4 of the chambers were observed to have their respective indicators change from a medium light blue to complete white in color to ensure that the reactions were working properly. Separately, the necessary oxygen to be used was made by electrolyzing water, with added sodium sulfate electrolyte to stimulate the reaction, into hydrogen and oxygen gas. Each gas was collected in a separate piece of glassware with the open ends of the glassware submerged in the water-electrolyte solution. A 12.5V DC power supply was used as the power supplier for this reaction as well as five 9.0V batteries setup in series with alligator clips attached to the ends of the batteries. The positive ends of the battery in series and the positive end of the power supply were attached to specialized metal rods and placed in the opening of the upside down, submerged flask filled with water-electrolyte solution in a tub full of water-electrolyte solution. The same process was done for the negative ends of the batteries and the negative end of the power supply in an opening of a different flask submerged in the same tub of the same water-electrolyte solution. The series connection was fully attached and the power supply was turned on when electrolyzing the water-electrolyte solution. Upon completion of the electrolysis, each gas was tested using the gas syringe to capture (separately) the specified gas and the gas was placed in an upside down test tube. A glowing wooden splint was then placed through the opening of the test tube into the gas collected at the top. These tests went as expected, the suspected oxygen gas allowed the splint to glow brighter for a longer period of time than the hydrogen. The hydrogen gas test used a glowing splint and when tested, made the glowing splint quickly die out with the noted "pop". Once the separate gases were tested for accuracy, the oxygen gas was collected using a gas syringe with closable end valve and ordinary aquarium tubing open to the atmosphere as well as the oxygen gas trapped in the glassware. Oxygen was then captured in the syringe 20cc, or cubic centimeters, at a time and the valve was then closed to prevent leakage or mixing with atmospheric gas. Then, the end of the syringe was attached to aquarium tubing with an attached one-way valve to prevent any atmospheric oxygen from entering and to keep any pressure in the flask from escaping back out through the tubing, and was connected to the rubber stopper placed in the flask. The syringe was then let open and the oxygen was then pressed through the tubing and valves into the flask. This process was repeated until 40cc of oxygen was pushed through into each of the flasks, and at the completion of this procedure, the tubing was bent and pinched permanently with a clip to prevent leakage into the atmosphere. The elevated levels of oxygen were immediately incubated in the platform shaker with the previously specified settings. As a control, the experiment used the atmospheric oxygen percentage (about 18%) which is what is naturally found in the air with the broth being made iron-sufficient (about the level of an iron-sufficient soil) with the addition of about 375mg of ferrous sulfate added as well as the multivitamin to better represent the presence of normal soil minerals, including the necessary molybdenum for the Fur). As a control, for each of the 3 oxygen tests (anaerobic, atmospheric, and super-oxidative), there is a flask with 177mg of ferrous sulfate, a flask with 354mg of ferrous sulfate, a flask with 531mg of ferrous sulfate, and a flask with 887mg of ferrous sulfate. This allows for the controls and complete data for comparison amongst the different levels of oxygen and varied levels of iron with only one variable being tested per flask per test. At the conclusion of the culturing and experimentation, samples were analyzed by titrating the broths with a known concentration of acetic acid (6M) and used in coordination with a calibrated Vernier pH probe in combination with a stirring plate placed under the flasks with a stirring magnet submerged and spinning in the broths, in order to determine the dinitrogen-fixing capabilities of each of the cultures as well as how much dinitrogen gas was fixed into ammonia. Via titration, it was assumed that the more acetic acid that was used to reach the equivalence point, the more ammonia that was created. It was noted, that the initial pH readings of the broth were 8.2. At the conclusion of the culturing and titrating, the results of the titration curves were analyzed and determined based upon the amount and concentration of acetic acid used to titrate the ammonia over a given time period and the equivalence points were found. It is observed that for this experiment, 18 drops from the burette is equivalent to 1.0 ml. Furthermore, all observations from start to finish of this experiment were recorded in a log book and several pictures were taken to help explain experimental design and growth patterns.


Anaerobic Flasks- Titration Results

Ferrous Sulfate Used In Flask

177 mg

354 mg

531 mg

887 mg

Acetic Acid (6M) Used At Equivalence Point




1.63 ml

Starting pH (Before Titration)





Ending pH (After Titration)





Change in pH





% Change In pH





Superoxidative- Titration Results

Ferrous Sulfate Used In Flask

177 mg

354 mg

531 mg

887 mg

Acetic Acid (6M) Used At Equivalence Point





Starting pH (Before Titration)





Ending pH (After Titration)





Change in pH





% Change In pH





Control (Atmospheric) Flasks- Titration Results

Ferrous Sulfate Used In Flask

177 mg

354 mg

531 mg

887 mg

Acetic Acid (6M) Used At Equivalence Point





Starting pH (Before Titration)





Ending pH (After Titration)





Change in pH





% Change In pH






These data support the hypothesis that A. chroococcum can survive and fix dinitrogen gas into ammonia under anaerobic and super-oxidative conditions. Certain patterns were noticed with varying groups of data:

With the anaerobic flasks, these data suggest that as iron levels increase while under anaerobic conditions, the less dinitrogen gas that will be fixed. In the 177mg anaerobic flask, .000034mole: mg was used, whereas at 354mg, 531mg, and 887mg, .000018mole: mg, .000013mole: mg, .000011mole: mg were used respectively. This pattern shows a trend in which less nitrogen is fixed as the iron content is increased, in the anaerobic chambers.

The controlled atmospheric flasks suggest that 531mg is the optimal iron level under normal atmospheric conditions. These data suggest that 354mg is not enough iron content to support dinitrogen fixation, as is seen with only .00000037mole: mg being used. The flask with 531mg, however, is the best to use under atmospheric conditions.

The superoxidative group's data suggest that 177mg and 354mg is enough to support growth and dinitrogen fixation. However, the data strongly suggest that 531mg is significantly better for supporting life and nitrogen fixation while under increased oxygen levels. These data support my hypothesis, and even go on to suggest that more dinitrogen can be fixed while under super-oxidative stress with the presence of increased levels if iron. This is thought to be due to the increased cellular respiration rates along with increased metabolism rates due to the higher oxygen levels in these flasks.

The expected results, based on common knowledge and theory, were that the anaerobic flasks would fix the most dinitrogen gas, that the super-oxidative flasks would fix the least dinitrogen gas, but would still survive and fix nitrogen, and that the controlled flasks would be somewhere in between the anaerobic and super-oxidative results.

These data overall support the hypothesis in suggesting that A. chroococcum will grow under super-oxidative stress. However, these data did reject the hypothesis by suggesting that the anaerobic did not, overall, fix the most dinitrogen gas. Instead, these data suggest that the super-oxidative flasks fixed the most dinitrogen gas overall. As suggested by the results in the tables and graphs, the most dinitrogen fixed came from the 531mg flask in the superoxidative group followed by the 177mg flask in the anaerobic group. The smallest results came from the 354mg in the controlled group.

In all groups and in all flasks, only 1 iron content and 1 oxygen level were compared at a time, to prevent skewed results due to more than variable in each flask, individually. All flasks were titrated; however, there is no data for the 534mg flask in the super-oxidative group due to loss of data after data collection was taken. Possible sources of error in these experiments include, but are not limited to: minute differences in the mass of iron content added, differences in oxygen added, different results due to each flasks being cultured in a slightly different area in the incubator and receiving more or less heat and/or light, the electrolysis reaction not being as pure as thought, the anaerobic chambers not working identically, and instrumental error measurement while doing titrations. All of these factors could have contributed to varying results and any inaccurate data.

As for future research, more trials should be done to check for any inconsistencies that may have occurred with the first trials. Also, oxygen content could possibly be even higher and more specific in the super-oxidative groups. Subsequently, different ranges and amounts of iron could be used while culturing, or even using iron in a different form or compound could be done to have more data to compare and analyze. Ferrous iron versus ferric iron should be explored since both are important for chelation and utilizing the Fur sequence. For obtaining results, chromatography or spectrometry could have been used to determine the nitrogen content of the flasks and cultures.


Overall, these data support and reject parts of the hypothesis. It supported the hypothesis that under super-oxidative stresses, A. chroococcum will survive and fix dinitrogen. In fact, the data goes further to suggest that under super-oxidative stress, more dinitrogen can be fixed, which is thought to be due to increased cellular respiration and metabolic rates. These trials strongly support the hypothesis.

However, these data also reject parts of the hypothesis. By fixing more dinitrogen gas under super-oxidative stress, the results rejected the hypothesis that under anaerobic conditions, the most dinitrogen gas will be fixed into ammonia and nitrates due to the lack of stress on the bacteria. However, according to the data, this hasn't been the case. Although, the anaerobic tests do suggest that more dinitrogen was fixed than in the controlled atmospheric flasks. This, also somewhat supports the hypothesis that the most dinitrogen will be fixed in the anaerobic flasks, as the data suggest that more dinitrogen was fixed than in the controlled flasks.

Source of error in this experiment could have come from small differences in the mass of iron content added or different mixtures of ferrous sulfate being used, which could have altered the data by being different from what is expected. This may be avoided by using a liquid to determine exact amount that is added. Also, small differences in oxygen that was added may have altered the results for the anaerobic and super-oxidative flask groups. This may be better controlled by using a more reliable and more efficient reaction in both cases. Different results due to each flask being cultured in a slightly different area in the incubator is something that cannot really be controlled due to a lack of space, or the use of more than 1 incubator. The electrolysis reaction not being as pure as thought, may have altered the data by giving an unspecified amount of oxygen to different flasks. This may be avoided by using a better reaction at a more rapid pace; however, this too, is hard to control. Incorrect measurements while doing titrations could have severely altered the data and given inaccurate data regarding the amount of nitrogen fixed. This may be controlled by doing the titrations more slowly and under higher stirring speeds or using a different method of measuring the ammonia.

Overall, 12 flasks were used, 4 for anaerobic conditions, 4 more atmospheric, and 4 more super-oxidative. All cultures were grown between 29-30 degrees Celsius. After 120 hours, the flasks were titrated with a known concentration of acetic acid (usually 6 Molar) and were graphed using a Vernier pH probe, and analyzed to compare all data. The results somewhat reject and support the hypothesis by showing that, overall, the super-oxidative flasks fixed the most dinitrogen gas.