Microbiology

Microbiology is the study of microbes - microscopic living organisms. This group includes bacteria, fungi, and protozoa. This page looks at microbiology in the context of the rumen - part of the digestive system of  cows and other ruminants. Cows' guts and microbes Cows' guts (their digestive tracts, or intestines) are packed with microscopic organisms: bacteria, fungi, and single-celled animals calledprotozoa. They aren't alone in this - all animals carry a huge load of bacteria - but the microbes that live in one special gut compartment in cows (and other ruminants) are special: they digest cellulose. This makes a whole new energy source available to the cow. There's a lot of energy in cellulose, but most animals are simply unable to digest it because they don't have the necessary enzymes. That's where the microbes come in.  The ruminant digestive tract (University of Minnesota, 1996), showing the rumen,  where most cellulose digestion takes place.
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	When were rumen microbes first discovered? The single-celled animals (protozoa) living in the rumen were first discovered in 1843. Later that century, other researchers discovered that a range of bacteria also lived in this part of the gut. By the end of the 19th century, scientists were coming to realise that microorganisms were important in helping ruminant animals to gain nutrients from their food (Hungate, 1966). For example, when they killed the rumen bacteria in a sample of stomach contents (using an antiseptic) and then mixed the fluid with cellulose, the cellulose wasn’t digested.  But when rumen fluids and cellulose were mixed together without an antiseptic, various gases and acids were produced, which showed that the cellulose had been broken down. This was followed by research looking at what was actually produced during rumen fermentation. It turned out that one product, short-chain fatty acids, provided a large proportion of a cow’s energy needs (Annison and Bryden, 1998).
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	Why do cows need micro-organisms? Now we know that cows need rumen microorganisms to survive. Even though cows eat grass, they can’t digest it on their own. This is because cows can’t make the enzymes needed to break down some parts of plant cells. For example, cows can’t digest cellulose. To do this, they need an enzyme called cellulase. This is why the microorganisms are so important- they produce the cellulase and other enzymes necessary to break down the parts of plant cells that the cow can’t digest. Many different types of microorganisms live in cow guts, making different enzymes to break down different parts of plants.     Protein-structure model of cellulase The microorganisms digest the plant material and produce short-chain fatty acids, which the cow can then absorb through its gut wall and use for energy. As well as this, the cow also digests some of the microorganisms every day as they are washed out of the rumen into the abomasum. So you could say that the cow’s diet is actually made up of grass and microbes, not just the grass that it eats.     How can the microorganisms stay alive if some of them are always being digested by the cow? They keep their population numbers up by growing very quickly - they need to be able to reproduce before the food leaves the rumen. They can also attach themselves to solid lumps of food in the rumen to avoid being flushed out with rumen liquids.  Cows and their gut microbes are an example of symbiosis - two organisms living together. The relationship between cows and their gut microorganisms is mutualistic. This means that both organisms benefit from the relationship. The microbes get a suitable place to live and a supply of food delivered to their door. The cow gets energy from digesting the gut microbes and the short-chain fatty acids they produce.
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	How many microbes live in the rumen? The number of microbes in one drop of rumen fluid is more than 10 times bigger than the number of people on Earth! (If there are 1012microbes in 1ml of fluid, then 1 drop (1/20ml) contains 50,000,000,000. And 2008 data from the US Census Bureau tell us that there are around 6,602,000,000 people on Earth.) That’s a lot of microbes - about one thousand billion, or 1012, or 1,000,000,000,000 organisms per millilitre (Prescott, Harley & Klein, 2005). To give you an idea of how many there are in total, the rumen may hold up to 95 litres of food & fluid. There are all sorts of different types of microbes in the rumen, such as fungi, bacteria and single-celled eukaryotic organisms called protozoans. There are also some archaea, which are ancient microorgansisms that produce methane.
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	What types of bacteria are found in the rumen? Bacteria are the most important microbes involved in ruminant digestion, and there are many different types in the rumen (Prescott, Harley & Klein, 2005). Almost all of the rumen bacteria will die if they are exposed to oxygen- they are obligately anaerobic (Hungate, 1975). Here we'll look at some of the most important bacterial groups in the rumen - their relatives include some very useful microbes as well as some that make us sick. But first - something on how they're classified. Classification  Because there are so many organisms out there, we need a classification system to keep track of all of them. We classify organisms at different levels, grouping similar organisms together. For example, humans:               Domain              Eukaryota  Kingdom            Animalia  Phylum               Chordata                                Class                  Mammalia  Order                   Primates  Family                 Hominidae  Genus                 Homo species               sapiens So, back to the bacteria. Phylum Firmicutes Class Lactobacillales The Lactobacillales are usually harmless to humans- not all germs will make you sick!  “Lacto” means “milk,” and this gives us a clue about where these bacteria are found and what they're used for - making cheese and yoghurt. Others are used to make beer and wine. However, there are also harmful bacteria in this group, such asStaphylococcus aureus, which can cause infections, boils, abscesses and pneumonia in humans. You may have heard of “super bugs” infecting people in hospitals- these are often strains of S. aureus which have evolved to become resistant to antibiotics.    One species from this group, Streptococcus bovis, uses a wide diet of different food sources in the rumen, such as sugars and proteins, to makeshort-chain fatty acids.  Class ClostridiaThis class contains some important rumen bacteria that are able to digest a wide range of plant materials. Butyrivibrio fibrisolvens, a member of this class, is able to ferment a wide range of substrates, including cellulose, xylan (a structura polysaccharide similar to cellulose), proteins, sugars and fats. Close relatives of these rumen microorganisms are also found in the human gut (Fig. 1.). Other relatives of these bacteria are responsible for the diseases tetanus and botulism. Fig. 1. Clostridium difficile obtained from a human gut.  Source: Wiggs, 2007.  Phylum Bacteroidetes A large proportion of rumen bacteria belong to this group. These bacteria are rod-shaped and cannot survive in the presence of oxygen - they are obligate anaerobes (Fig. 2) (Prescott, Harley & Klein, 2005).  An important member of this group that is found in the rumen is Bacteroides succinogenes, a cellulose fermenter (Hungate, 1966). Fig. 2. Bacteroides biacutis. Source: Dowell, 2006.   Phylum Proteobacteria The phylum Proteobacteria is the largest and most diverse group of bacteria. They have many different shapes and ways of living. Some are free-living, some are parasitic, and others form mutualistic relationships with other organisms, such as those found in the rumen. Many Proteobacteria are responsible for diseases in animals and humans, such as Salmonella sp., which can cause typhoid. This order contains some bacteria found in the guts of other animals as well as ruminants. Just like cows, we need gut microorganisms to help digest our food. Escherichia coli (Fig. 3) is found in the guts of humans and animals. Fig. 3. A scanning electron micrograph of Escherichia coli.  Source: Rocky Mountain Laboratories, NIAID, NIH, 2005. Methane-producing bacteria (Phylum Euryarchaeota) These organisms belong to the domain Archaea, an ancient group separate from the eukaryotes and bacteria. The Archaea in the rumen use hydrogen gas and carbon dioxide to produce methane. This regulates fermentation in the rumen by lowering the amount of hydrogen gas, allowing bacteria which produce hydrogen to carry on metabolising. Some species that produce methane in the rumen include Methanobrevibacter ruminantium, Methanobacterium formicicum and Methanomicrobium mobile.   Note: Bacterial names Bacteria are often named for their shape. The name coccus comes from the Greek word meaning “granules” and is used to name spherical cells, such as those shown below (Fig. 4.). Rod-shaped bacteria are also very common- these are called bacilli. Spiral-shaped cells are called Spirilla,and if they are flexible, spiral-shaped bacteria are called spirochaetes. What about the bacteria Staphylococcus? Staphyle means “bunch of grapes” in Greek, and coccus means “granules”, so Staphylococcusmeans “bunches of granules”- which is just what they look like (Fig. 4). Fig. 4. Scanning electron micrograph of Staphylococcus aureus. Source: Ardurino and Carr, 2007.
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	What about protozoa and fungi? Protozoa Most of the protozoa found in the rumen are ciliates (phylum Ciliophora). Many ciliates use cilia (tail-like structures) to move around, and to move food particles into their mouths. Most ciliates do not live inside another organism, but some exist in the rumen, such as Entodinium. In the rumen, two types of ciliates are found; the holotrichs and the spirotrichs. The holotrichs convert soluble sugars into starch, while spirotrichs consume starch and cellulose (Hungate, 1975).  Fungi The fungi in the rumen are a bit different to the mushrooms in your local supermarket. They are very small (Fig. 5) - note that the scale bar on this diagram is 50µm, or 50 millionths of a metre. Unlike mushrooms, rumen fungi don’t need oxygen to survive: they are anaerobes. Rumen fungi have been shown to digest cellulose and xylans, which shows that they may play a role in helping the ruminant host to digest plant matter.  Fungi are very important to humans. They are excellent recyclers, breaking down animal and plant matter into molecules that can be re-used by other organisms.  Yeasts, a type of fungi, are used to make bread, wine and beer. Fungi also cause many diseases in plants and animals, such as the human diseases athlete’s foot and ringworm. Fig. 5. Candida albicans, the fungus that causes thrush in humans, magnified 200 times. Source: Y tambe, 2005
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	How do rumen microbes obtain energy? To obtain energy for growth and reproduction, organisms must break down large molecules into smaller ones. This process gives energy and is called catabolism. For growth to occur, larger molecules are made from smaller ones, using up energy. This process is called anabolism. Both processes are part of cellular metabolism. When plant material, such as grass, enters a cow's stomach, microorganisms immediately get to work to break it down. They do this with enzymes. Ruminants are unable to synthesise the enzymes needed to digest cellulose and other plant compounds. The microbes in the rumen, however, are able to make the various enzymes needed tohydrolyse cellulose, proteins, sugars and other materials. Rumen bacteria need carbon dioxide, nitrogen, sodium, and volatile fatty acids to grow (Hungate, Bryant & Mah, 1964). Some bacteria use only one type of food, but others use a range of different types (Table 1). Many bacteria are able to digest cellulose. Many can also digest xylan, a type of complex carbohydrate similar to cellulose. Others can digest proteins, or sugars such as amylose and lactose.  Glucose is released when cellulose is digested. Various microbes then ferment the glucose to produce short-chain fatty acids (SCFAs). The SCFAs are then taken up and used by the host animal. Carbon dioxide and methane gas are also produced by the microbes and are excreted by the host (e.g. in exhaled air & by belching). Table 1. Sources of carbon, energy and electrons and their definitions. (Modified from Prescott, Harley & Klein, 2005)  Carbon sources    -Autotrophs  Main carbon source is CO2  -Heterotrophs  Use carbon found in other organisms  Energy sources    -Phototrophs  Light  -Chemotrophs  Organic or inorganic compounds  Electron sources    Lithotrophs  Inorganic molecules  Organotrophs  Organic molecules
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	Fermentation Because there is very little oxygen present in the rumen, the microorganisms there must obtain energy from their food anaerobically(without oxygen). This is done through a process called fermentation. Fermentation involves the breakdown of glucose into alcohols or acids. Sound familiar? Fermentation is also used to make alcoholic drinks such as beer and wine. As we’ll see, it doesn’t yield as much energy as aerobic respiration.     Before fermentation can occur, larger molecules present in the plant material, such as lipids, proteins and polysaccharides, must first be broken down into their component parts. This is done by hydrolysis. Through hydrolysis, lipids are broken down into glycerol and fatty acids; proteins into amino acids, and polysaccharides into glucose and other simple sugars. Here, we’ll look at fermentation using glucose.     Whether there is oxygen present or not, the first step of respiration is glycolysis. In this process, glucose is broken down to form a compound called pyruvate. Glycolysis also generates 2 ATP molecules (the cell’s energy carriers) and 2 of NADH.     In anaerobic environments like the rumen, glycolysis may be followed by homolactic fermentation. This is a ‘recycling’ step that doesn’t generate any more ATP. Homolactic fermentation converts pyruvate into lactate and converts the NADH produced in glycolysis back into NAD+ so that it can be used again.     In our own cells (like those of all eukaryotes), where oxygen is present, further energy can be produced from pyruvate using the Krebs cycle and oxidative phosphorylation, producing a net total of 36 ATP per glucose molecule. However, this is ruled out in the anaerobic environment of the rumen.     The sparknotes site has a diagram showing how fermentation works.
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	Bibliography & useful websites Useful links http://www.textbookofbacteriology.net/ An online textbook written by an American university professor containing good general information about microbiology, also with some information on how microbes can affect human health Bibliography Annison, E. F. & W. L. Bryden.1998. Perspectives on ruminant nutrition and metabolism. Nutrition Research Reviews 11:173-198 Bauchop, T. 1977. Foregut fermentation. In: R. T. J. Clarke and T. Bauchop (Ed.). Microbial Ecology of the Gut (pp. 223-250). London: Academic Press. Clarke, R. T. J. 1977. Protozoa in the rumen ecosystem. In: R. T. J. Clarke and T. Bauchop (Ed.). Microbial Ecology of the Gut (pp. 251-275). London: Academic Press. Church, D. C. (Ed.) 1988. The Ruminant Animal: Digestive Physiology and Nutrition. Englewood Cliffs, New Jersey: Prentice Hall. Hespell, R. B. 1987. Biotechnology and modifications of the rumen microbial ecosystem. Proceedings of the Nutrition Society 46:407-413 Hungate, R. E. 1950. Mutualisms in Protozoa. Annual Review of Microbiology 4:53-66 Hungate, R. E. 1966. The Rumen and its Microbes. London: Academic Press. Hungate, R. E. 1975. The rumen microbial ecosystem. Annual Review of Ecology and Systematics 6: 39-66 Hungate, R. E., M. P. Bryant & R. A. Mah. 1964. The rumen bacteria and protozoa. Annual Review of Microbiology 18:131-166. Macy, J. M. & I. Probst. 1979. The biology of gastrointestinal bacteroides.Annual Review of Microbiology 33:561-594 Prescott, L. M., J. P. Harley & D. A. Klein. 2005. Microbiology. Sixth Edition. New York: McGraw-Hill. Tajima, K., R. I. Aminov, T. Nagamine, K. Ogata, M. Nakamura, H. Matsui & Y. Benno. 1999. Rumen bacterial diversity as determined by sequence analysis of 16S rDNA libraries. FEMS Microbiology Ecology 29(2):159-169 Thain, M. & M. Hickman. 2001. The Penguin Dictionary of Biology. Tenth Edition. London: Penguin Books.  U. S. Census Bureau. 2008. U.S. and World Population Clocks- POPClocks. Accessed on 16.04.08 fromhttp://www.census.gov/main/www/popclock.html Image credits Ardurino, M. J. and Carr, J. 2007. Electronic scanner image ofStaphylococcus aureus. Accessed on 06/04/08 fromhttp://en.wikipedia.org/wiki/Image:Staphylococcus_aureus_01.jpg Dowall, V. R. 2006. One of many en:commensal anaerobic Bacteroidesspp. in the gastrointestinal tract—cultured in blood agar medium for 48 hours. Accessed on 02/04/08 fromhttp://en.wikipedia.org/wiki/Image:Bacteroides_biacutis_01.jpg Rocky Mountain Laboratories, NIAID, NIH. 2005.  Escherichia coli: Scanning electron micrograph of Escherichia coli, grown in culture and adhered to a cover slip. Accessed on 02/04/08 fromhttp://en.wikipedia.org/wiki/Image:EscherichiaColi_NIAID.jpg Wiggs, L. S. 2007. Scanning electron micrograph of Clostridium difficilebacteria from a stool sample. Accessed on 02/04/08 fromhttp://upload.wikimedia.org/ Y tambe, 2005. Microscopic image (200-fold magnification) of Candida albicans ATCC 10231, grown on cornmeal agar medium with 1% Tween80. Accessed on 02/04/08 fromhttp://en.wikipedia.org/wiki/Image:Candida_albicans.jpg