by special guest George Coliln diCenzo
Microbes. These small but mighty organisms play many important roles in human society. They also have significant impacts on global nutrient cycles and plant and soil health. When it comes to growing plants, microbes are our friends, generally. But before getting into the role of microbes in supporting plant health through biological nitrogen fixation, let’s first discuss who microbes are, why we should care about microbes, and the role of nitrogen in plant nutrition.
Microbes – who are they and why should we care?
The term “microbe” is a shorthand way of saying “micro-organism”. A micro-organism is any organism too small to see without a microscope. There are many different types of microbes, including bacteria, archaea, fungi, and viruses, among others. A bacterium (plural: bacteria) is a specific type of microscopic organism that lacks a sub-cellular structure known as a nucleus, which is where DNA is stored in organisms like humans. A fungus (plural: fungi) is a specific type of microscopic organism that does have a nucleus. Bacteria (and archaea) are known as prokaryotes, whereas fungi (and plants and animals) are known as eukaryotes. Whereas all bacteria are microbes, not all fungi are microbes (e.g., mushrooms). So why should we care about these tiny organisms that we can’t see with a microscope? Well, because there are lots of them, they are everywhere, and they do a huge number of things!
While it would not be feasible to go around the world and count every microbe and weigh every living thing, we can use what we know to make informed guesses. In one study, scientists attempted to estimate the weight of all living things on Earth. Based on their analyses, they estimate that plants account for over 80% (by weight) of all living organisms on Earth. The next biggest group is bacteria, accounting for a bit over 12% of all living organisms! By comparisons, humans account for just 0.01%. The amount of microbes becomes even more impressive when you estimate how many there are, rather than how much they weight. Earth is home to about 8 billion (8,000,000,000) people and an estimated 5 nonillion (5,000,000,000,000,000,000,000,000,000,000) bacteria! They are also very diverse. Based on one estimate, bacteria may account for 75% of all the species on Earth. Consequently, as a group bacteria are able to do many different things and inhabit virtually every environment on Earth, including our skin, our digestive tracts, the Antarctic, deep sea hydrothermal vents, and so on.
Bacteria play many important roles in our environment and many have properties we find valuable. We can even thank bacteria for the lovely, earthy smell that appears after a rainstorm; that smell is caused by a compound called geosmin, which is produced by a soil bacterium called Streptomyces coelicolor. Streptomyces bacteria are also notable because they produce many of the antibiotics that we use in medicine. Microbes are also central to the production of many other pharmaceuticals and other bio-products. For example, most of the insulin used by people with diabetes is produced by certain bacteria and yeasts (a type of fungus) that were engineered to produce insulin. They are also crucial to the production of some of my favourite foods via fermentation. Bread, pizza dough, and related foods require Baker’s yeast. Cheese and yogurt, as well as cured meats like pepperoni, require lactic acid bacteria for their production. The olives we eat are first fermented with yeasts and lactic acid bacteria. Likewise, the production of chocolate depends on a complex fermentation process involving yeasts, lactic acid bacteria, and acetic acid bacteria. Many drinks – such as wine, beer, and kombucha – also require fermentation by microbes.
Last but certainly not least, many microbes are good friends with plants, and (sustainably) growing a healthy garden requires that the microbes not be ignored. Just like humans, plants are covered in microbes. In particular, the “rhizosphere”, the layer of soil around plant roots whose composition is directly influenced by plants, is a microbe rich environment. Microbes on plants and in the rhizosphere have several mechanisms by which they can promote the growth of plants. Some microbes can regulate production of plant hormones, thereby impacting plant growth rate. Similarly, some microbes have mechanisms by which they help protect plants from various abiotic stresses, helping plants survive stresses like drought and heat. Other microbes may outcompete, or directly inhibit, pathogenic bacteria in the rhizosphere, protecting plants from disease. Moreover, some microbes support the nutrition of plants. A notable example are the arbuscular mycorrhizal fungi (AMF). Aside from brassica plants (e.g., canola, kale, broccoli), nearly all land plants associate with AMF. Among other things, AMF help plants obtain phosphorus (the P of NPK fertilizers) and water from the soil. Biological nitrogen fixation is another key process through which microbes support plant nutrition by providing plants with a bioavailable source of nitrogen (the N of NPK fertilizers; the K is potassium).
What’s the deal with nitrogen?
All living organisms on Earth require nitrogen; without nitrogen, life on Earth would cease to exist. Fortunately, there is a lot of nitrogen; in fact, ~78% of Earth’s atmosphere is nitrogen in the form of nitrogen gas (N2). Unfortunately, that nitrogen gas is not bioavailable, meaning that very few organisms able to directly use nitrogen gas as a source of nitrogen. Instead, humans obtain nitrogen from the food that we eat, while plants obtain nitrogen from soils, primarily in the form of ammonia (NH3) or nitrate (NO3). However, most soils are not nitrogen-rich, and as a result, the growth and yield of plants are often limited by the availability of nitrogen. In natural ecosystems, bioavailable nitrogen (e.g., ammonia, nitrate) is added to the soil through mechanisms such as decomposition of dead organisms and conversion of nitrogen gas to ammonia via lightning strikes and biological nitrogen fixation.
To maximize the amount of food we produce, or to have dark green lawns, growers need to supplement their farms/gardens/lawns with nitrogen. There are many ways this can be done (e.g., compost, manure), but in conventional farms, nitrogen is primarily added via chemical fertilizers. Likewise, if you have ever added fertilizer to your lawn or house plants, nitrogen was likely the primary component of the fertilizer. Chemical nitrogen fertilizers are produced industrially via the Haber-Bosch process, which converts nitrogen gas to ammonia. The Haber-Bosch process was invented by the scientists Fritz Haber and Carl Bosch in the early 20th century, who were awarded Nobel Prizes for their invention in 1918 and 1931, respectively. Since its invention, the Haber-Bosch process has had major impacts (both positive and negative) on human society. On the positive side, the availability of chemical fertilizers led to large yield increases in conventional farms; by some estimates, half of the people alive today are alive because of yield increases supported by the Haber-Bosch process. However, this benefit comes with costs. It is extremely difficult to convert nitrogen gas to ammonia. To accomplish this difficult reaction, high temperatures (400˚C) and high pressures (200 atm) are used. It also requires a source of hydrogen, which comes from natural gas. As a result, the Haber-Bosch process is thought to use 1-2% of the global energy supply and is potentially the largest user of natural gas in agriculture. Nitrogen fertilizers are also estimated to be directly or indirectly responsible for about a third of the greenhouse gas emissions associated with agriculture. The impacts of nitrogen fertilizers are not limited to agriculture, as the same environmental effects are associated with nitrogen fertilizers applied to residential lawns. Considering this, where possible, we should reduce our use nitrogen fertilizers in favour of more sustainable solutions. One such solution is biological nitrogen fixation.
Biological nitrogen fixation as a source of bio-available nitrogen for plants
Unlike most organisms, there are a small number of bacteria (and archaea) that can use nitrogen gas by first converting it to ammonia in a process known as biological nitrogen fixation, which is dependent on an enzyme known as nitrogenase. Bacteria capable of fixing nitrogen are known as diazotrophs. Like the Haber-Bosch process, biological nitrogen fixation is energetically expensive to bacteria, and thus they only fix nitrogen when they need to. Biological nitrogen fixation is highly significant for global ecosystems. It is thought that biological nitrogen fixation accounts for 90% of non-anthropogenic, non-agricultural nitrogen fixation; the remaining 10% is via lightning strikes. Although soil bacteria performing biological nitrogen fixation are doing so to benefit themselves, it also introduces nitrogen into the local ecosystem, which can then be used by plants.
Plant-associated biological nitrogen fixation by bacteria can be generally divided into two categories: associative nitrogen fixation and symbiotic nitrogen fixation. Associative nitrogen fixation refers to when biological nitrogen fixation is performed by bacteria outside but in close association with plants, and it can occur in the soil or on plant surfaces. An example of a group of bacteria able to fix nitrogen are Azospirillum bacteria, while an example of a plant that associates with large populations of nitrogen-fixing bacteria is sugarcane. Another interesting example is a Mexican maize landrace that contains aerial roots coming off its shoot. After a rainstorm, the aerial roots become coated in a mucus that hosts nitrogen-fixing bacteria. Associative nitrogen fixation can partially support the nitrogen needs of plants. For example, one laboratory study showed that a specific Bacillus bacterium could provide maize with 30% of the nitrogen it needs. As such, cultivating healthy soils with an abundance of nitrogen-fixing bacteria can reduce the amount of nitrogen that a grower has to add to the field/garden. Indeed, some companies sell nitrogen fixing bacteria as bio-inoculants to support crop production, although the effectiveness of these products varies dramatically.
Unlike associative nitrogen fixation where the bacteria are outside the plant, in symbiotic nitrogen fixation, the bacteria are located within the plant and are found in specialized organs known as “root nodules” (often just called “nodules”). In fact, the bacteria are found inside the plant cells within the nodule. It is in part due to this intimate nature of the relationship that symbiotic nitrogen fixation can provide plants with more nitrogen than can associative nitrogen fixation. For some plant species, symbiotic nitrogen fixation can fulfill the entire nitrogen demands of the plant. This symbiosis is generally considered a “mutualism” as most people think that both of the interacting partners benefit; the plant gets nitrogen to support its growth, while the bacteria get carbon (e.g., sugars) and a safe place to replicate.
Not all plants are able to form root nodules, and thus symbiotic nitrogen fixation is largely limited to two classes of plants. One of these are the actinorhizal plants, whose nodules contain nitrogen fixing Frankia bacteria. Examples of actinorhizal plants are the alder tree and the goumi berry shrub. The second are legumes, whose nodules contain rhizobium (plural: rhizobia) bacteria. Examples of nitrogen-fixing legumes are lentils, soybean, common bean, peas, peanut, alfalfa, and clover. However, not all legumes fix nitrogen, for example, the Kentucky coffeetree that can be found in Kingston. In some cases, legumes can fulfill their entire nitrogen requirement via the symbiosis; in Canada, lentils and soybeans crops are inoculated with rhizobium inoculants and do not require the addition of chemical nitrogen fertilizer. As a result, legumes are a sustainable (and high protein) food source, and thus are a good food choice for consumers trying to reduce their carbon footprint. Legumes can also add some nitrogen back to the soil, enriching the soil for nearby plants or crops grown in a subsequent year. This is why farmers will generally include a legume in their crop rotations or as a cover crop. It is also why many people will recommend adding clover to your lawn; not only is the clover more drought resistant and able to stay green even during dry summer periods, but it is also a nitrogen-fixing plant and thus does not require chemical nitrogen fertilizers.
Indigenous communities have long recognized the benefit of symbiotic nitrogen fixation. A common form of agriculture practiced by Indigenous communities is the cultivation of Three Sisters gardens. In Three Sisters gardens, maize, squash, and beans are planted together. The squash shades the ground, keeping it moist and reducing weeds. The maize grows tall and can act as a support system for climbing beans. And in turn, the beans fix nitrogen, improving the quality of the soil for all three plant species.
How do you know if the legumes you planted are fixing nitrogen? Look at the roots! Nitrogen-fixing legumes will contain nodules located on their roots. Nodules vary in size depending on the plant species, but in all cases, they can be seen with the naked eye. Nodules comes in two general shapes. For some plants – like pea and clover – the nodules have an elongated cylinder-like shape. Other plants – like soybean and common bean – have nodules that are spherical. If your plant has root nodules, it means that there are rhizobia in the soil and presumably also in the nodule. However, not all rhizobia fix nitrogen equally. Some rhizobia are very effective at fixing nitrogen, others fix little to no nitrogen. At the same time, an inefficient nitrogen-fixing rhizobium might be highly competitive and take up all the space in the nodules. This is why growers will add commercial rhizobium inoculants to their crops, as it helps maximize the number of nodules containing rhizobia capable of fixing high amounts of nitrogen. However, even without adding a rhizobium inoculant, most soils will contain rhizobia able to fix nitrogen with legumes especially if the legume is native to the region, and as such, most legumes grown in home gardens should be nodulated.
Given that not all rhizobia fix nitrogen with a particular plant, the presence of a nodule in-and-of-itself is not enough to confirm nitrogen fixation is taking place. Nodules that are not fixing nitrogen will appear white. On the other hand, nodules actively fixing nitrogen will have a characteristic pink or red colour; for some nodules, you will only see this colour if they are cut open. This pink or red colour is caused by a protein known as leghemoglobin, which is similar to the hemoglobin that makes our blood red. Like the hemoglobin in our blood, leghemoglobin binds to oxygen. Nodules contain leghemoglobin to bind oxygen and ensure a low concentration of free oxygen in the nodule. This is important as oxygen inactivates the enzyme nitrogenase and thus stops nitrogen fixation from occurring. As an interesting aside, leghemoglobin is included as an ingredient in Impossible Burgers to give them a red blood-like colour reminiscent of meat.
Searching for more effective bean inoculants
One of the topics that my research group at Queen’s University studies is symbiotic nitrogen fixation. Recently, we have begun a new applied research project that aims to isolate new rhizobia from Ontario soils able to effectively fix nitrogen with common bean (Phaseolus vulgaris) or adzuki bean (Vigna angularis) plants. Commercial inoculants for these crops are not available, necessitating continued use of nitrogen fertilizers in conventional agriculture. We hope to isolate new rhizobia from Ontario soils that are adapted to the Canadian climate and that have high nitrogen-fixing potential with Canadian bean varieties. To do this, we are performing “nodule trapping” experiments. We plant beans in different soils in the lab and then collect the nodules, which will have rhizobia “trapped” within them. We then isolate the rhizobia from the nodules and grow them in the lab. Subsequently, we perform experiments to test if the individual rhizobia are effective at fixing nitrogen with different bean plants. In addition to doing this in the lab, we aim to supplement our rhizobium library by collecting nodules from bean plants grown in gardens or fields across Ontario.
In preliminary work last October, and in collaboration with the Kingston Area Seed Sanctuary Initiative (KASSI), we examined the roots of common bean plants grown in the Lakeside Community Garden and identified numerous nodules on several bean heritage varieties. We took a few of these nodules back to the lab and were able to successfully isolate rhizobia from three of the nodules, which we recently reported in a peer-reviewed report. Currently, we are trying to expand or rhizobium collection, after which we will begin testing their nitrogen-fixing abilities.
Take-away messages
Microbes are important members of all ecosystems on Earth, including garden and farm ecosystems. Microbes provide many functions that help plants grow, with one notable function being biological nitrogen fixation, i.e., the conversion of atmospheric nitrogen gas into ammonia. Biological nitrogen fixation supports plant nutrition and is a sustainable way of providing at least some of the nitrogen that plants need to grow. Incorporating nitrogen fixing plants into your garden, meadow, or lawn is an effective way of supporting the health of your soil. Not only that, but legumes like clover are nice looking additions to your lawn, while legumes like peas are tasty protein-rich foods.
If you have questions about biological nitrogen fixation, or if you grow common bean or adzuki bean and would be interested in helping us with our work, please reach out (george.dicenzo@queensu.ca or gd38@queensu.ca)! I would love to provide more information, get soils from your bean garden for nodule trapping experiments, and/or to come explore your bean plant root systems with you to look for and collect nodules. Happy gardening!