Lithotrophic Bacteria - Rock Eaters

So far in catabolism we have spent our time looking at microbes that use organic compounds as their source of food (energy and building blocks for making cells). Interest in these species is understandable since our metabolism is very similar, but this ignores a large group of microbes that are capable of using inorganic substances as their source of energy. They are termed lithotrophs, literally meaning rock eaters. General Concepts

§ Common habitats of lithotrophs include waste water, volcanoes, deep sea ocean vents, the atmosphere, mines, seawater, fresh water. Basically they can be found everywhere. § Energy is generated from reduced inorganic molecules. These molecules have high

potential electrons that can be used to drive ETS. Typical energy substrates include, H2,

+2 +2 CH4, CO, S, H2S, NH4, NO2, N2O, Fe and Mn . § Oxygen is often, though not always, the terminal electron accepter. Due to its willingness to accept electrons, oxygen gives the largest energy gain and is often employed by lithotrophs when available.

§ Cell carbon is often from CO2 frequently using the Calvin Cycle. There are also microbes that can grow heterotrophically. § Energy yields from lithotrophy are low per substrate oxidized and a large amount of substrate has to be metabolized per cell. § Their appetite for substrate makes lithotrophs important in the global cycling of elements that they attack. Lithotrophs have a major impact on the movement of nitrogen, sulfur and carbon though the biosphere.

To help the reader get a general idea of what these microbes are like, we look at two interesting lithotrophs. These are just two examples in a sea (pun intended) of many aquatic and terrestrial lithotrophs.

Nitrifying Bacteria.

Degradation of organic material typically results in the release of ammonia (NH3) into the environment. and nitrifying bacteria are more than willing to make a living oxidizing ammonia. They are ubiquitous in the environment and have even been discovered living in the sandstone of the Cologne Cathedral in Germany - The bacteria penetrate to depths of 15 cm in the sandstone blocks. These bacteria generate energy by oxidizing reduced forms of nitrogen to NO2 or NO3.

The concentration of nitrifiers depends upon the rate of NH3 production in the surrounding environment. The faster the rate, the higher the population of microbes. We will briefly look at the biochemistry of two nitrifiers, Nitrosomonas and Nitrobacter. Nitrosomonas oxidizes ammonia in the following reaction...

NH3 + 1 1/2 O2 HNO2 + H2O

Figure 1 - The reduction of ammonia by nitrifying bacteria Extracted electrons are donated directly to an ETS and no carrier is involved. The result of running the ETS is that a proton gradient is formed which can then synthesize ATP using ATP synthase.

Figure 2 - Energy generation in Nitrosomonas. Only two enzymes, ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) are involved in the oxidation of ammonia to nitrite.

Nitrite can be further acted on by another nitrifying bacteria, Nitrobacter. This microbe oxidizes nitrite to nitrate using oxygen as the terminal electron accepter. A proton gradient is established with resultant synthesis of ATP. Nitrobacter is often found in tandem with Nitrosomonas since the end product of Nitrosomonas metabolism is the energy substrate for Nitrobacter. This type of loose association is probably common in the environment and in this case benefits both organisms. Nitrobacter is provided with substrate and Nitrosomonas has its end product removed, which helps drive its metabolism.

Tube worms in the deep sea

The deep ocean was once thought to be a lifeless place. Lacking sunlight, which can only penetrate the first few 100 meters of water, the only living organisms were thought to be scavengers that feed off the organic material that trickled down from the ocean surface. In 1977 a geological deep sea expedition was begun to investigate areas of active volcanism deep beneath the ocean at depths of 1500 to 3700 meters. To the scientists great surprise a thriving community of clams, mussels, worms, shrimps and crabs was discovered surrounding these areas of active volcanism. This created a sensation in the scientific community. How did this community survive? What was the basis of the food chain? It couldn't be photosynthesis - everyone was intrigued.

The communities encircled areas where heated water was being ejected from "black smokers" so named because the liquid coming out of the vents was black due to rapid precipitation of black polymetal sulfides. These hydrothermal vents come about by sea water penetrating the ocean floor and passing over molten lava where it becomes superheated and loaded with reduced minerals. This hydrothermal fluid (350°C) then percolates up to the sea floor where it flows into the surrounding ocean (4°C) forming black smokers. (Remember that water at this depth is under great pressure and will not boil until it reaches temperatures of 450°C) The resulting temperature gradient and load of reduced minerals provides an ample source of energy for lithotrophs, mainly sulfur oxidizing bacteria, which are the primary producers in the deep sea food chain.

Some organisms in this biosphere feed on the microbial mats of bacteria that grow around the hydrothermal vents, but other organisms form symbiotic relationships with the primary producers.

One of the more interesting lithotrophs investigated is a bacterial species I will call the symbiont, reasons for which you will see in a minute (keep reading!).

This symbiont takes hydrogen sulfide, produced out of the black smokers and oxidizes it to sulfate using oxygen as the terminal electron accepter. The electrons extracted are used in an ETS to form a proton gradient which drives the synthesis of ATP. Cell carbon is obtained via the Calvin cycle. Its cell shape and coccoid morphology suggest it may be from the genus Thiovulum, but this association is tenuous at best. Analysis of the 16S ribosomal RNA will be required to correctly place it in the phylogenetic tree. The symbionts characteristics in itself are not unique, but where is makes its home is. It is contained in a tissue called the trophosome inside the tube worm Riftia pachytila.

A large tube worm is in a close symbiotic relationship with the symbiont. The worm provides a home for the microbe, giving it space to grow and providing both hydrogen sulfide and oxygen for the symbionts metabolism. These gases are delivered via a circulatory system that contains specialized hemoglobins that are capable of binding oxygen and hydrogen sulfide. The CO2 content of the worms blood is also high providing a carbon source for the assimilation into cell material. In return the symbiont provides a portion of its production from the Calvin cycle to the tube worm.

Figure 4 - The electron transport system in sulfide oxidizing bacteria. The endosymbiont of Riftia pachytila probably has a similar system for energy generation. flavoprotein (FP), cytochrome b (cyt b), cytochrome c (cyt c). It has not been possible to bring the endosymbiont into culture for in depth analysis and this has hindered research into its metabolism.

The existence of life on our planet that does not depend on the sun, but on terrestrial energy, has sparked the exciting possibility of life on other bodies in our solar system. Europa, a moon of Jupiter is a cold white sphere that may contain water several kilometers below its icy crust. If there is water, there may be life, at least microbial life. NASA's Galileo spacecraft has just completed its mission at Europa and soon scientist will have more information about the existence of liquid water on Europa. Carbon fixation:

Autotroph organisms have to reduce carbon dioxide to synthesize organic compounds, with often glucose as the starting point. Not only plants do it, but also photosynthetic bacteria (anoxygenic and oxygenic) as well as chemolithotrophs that oxidise inorganic substances. The “Calvin cycle” is not the only way to do this and thinking outside the box is a good idea if you want to have a good overview of what happens out there in nature.

Anaerobic Respiration

The last few sections have talked extensively about aerobic respiration. What defines it as aerobic is its use of oxygen as the terminal electron accepter. Since this is very similar to the type of respiration that humans use, our bias is obvious. Now let me fill you in on a little secret. Microbes are capable of using all sorts of other terminal electron accepters besides oxygen. Below we talk about a few examples of anaerobic respiration. The one thing that they all have in common is the use of an electron transport system in a membrane and the synthesis of ATP via ATP synthase. In both nitrate reduction and sulfate reduction there are two types of pathways, assimilatory and dissimilatory. Assimilatory pathways are methods for taking a nutrient in the soil, moving it into the cell and using it for biosynthesis of macromolecules. Dissimilatory pathways use the substrate as a place to dump electrons and generate energy. Here we examine dissimilatory pathways. Assimilatory pathways will be explained in the context of cell biosynthesis.

Nitrate reduction

Some microbes are capable of using nitrate as their terminal electron accepter. The ETS used is somewhat similar to aerobic respiration, but the terminal electron transport protein donates its electrons to nitrate instead of oxygen. Nitrate reduction in some species (the best studied being E. coli) is a two electron transfer where nitrate is reduced to nitrite. Electrons flow through the quinone pool and the cytochrome b/c1 complex and then nitrate reductase resulting in the transport of protons across the membrane as discussed earlier for aerobic respiration.

- - + - N03 + 2e + 2H N02 + H20

- - Figure 1 - The reaction for nitrate reduction. N03 , nitrate; N02 , nitrite

This reaction is not particularly efficient. Nitrate does not as willingly accept electrons when compared to oxygen and the potential energy gain from reducing nitrate is less. If microbes have a choice, they will use oxygen instead of nitrate, but in environments where oxygen is limiting and nitrate is plentiful, nitrate reduction takes place.

Denitrification

Nitrite, the product of nitrate reduction, is still a highly oxidized molecule and can accept up to six more electrons before being fully reduced to nitrogen gas. Microbes exist (Paracoccus species, Pseudomonas stutzeri, Pseudomonas aeruginosa, and Rhodobacter sphaeroides are a few examples) that are able to reduce nitrate all the way to nitrogen gas. The process is carefully regulated by the microbe since some of the products of the reduction of nitrate to nitrogen gas are toxic to metabolism. This may explain the large number of genes involved in the process and the limited number of bacteria that are capable of denitrification. Below is the chemical equation for the reduction of nitrate to N2.

- - N03 N02 NO N2O N2

Figure 2 - The reduction of nitrate to nitrogen gas. NO, nitric oxide; N2O, nitrous oxide; N2, nitrogen gas.

The advantage for the cell of carrying out a complete reduction of nitrate is two fold. The nitrate ETS serves as a place to oxidize NADH and free it to be used in catabolism of more substrate.

Denitrification take eight electrons from metabolism and adds them to nitrate to form N2 versus just two for nitrate reduction alone. Also, donation of electrons from NADH through the cytochrome b/c1 complex and eventually to nitrous oxide (N2O) reductase provides another opportunity to pump protons across the membrane. The figure below presents a schematic of the spacial arrangement of the denitrification enzymes in the membrane

Figure 3 - Denitrification in the membrane. NADH dehydrogenase complex (DH), nitrate reductase (NAR), nitrite reductase (NIR), NO reductase

(NOR), and N2O reductase (N2OR)

Nitrate reduction has been extensively studied in bacteria due to its significance in the global nitrogen cycle. Denitrification removes nitrate, an accessible nitrogen source for plants, from the soil and converts it to N2 a much less tractable source of nitrogen that most plants cannot use. This decreases soil fertility making farming more expensive. Intermediates of denitrification, nitrous oxide and nitric oxide, are gases and will sometimes escape the cell before being completely reduced. These compounds, when in the atmosphere, contribute to the greenhouse effect and exacerbate global warming. The use of high nitrate fertilizers in modern agriculture makes matters worse. For more information, there is an extensive review of denitrification available on line.

Sulfate reduction

The disimilatory reduction of sulfate seems to be a strictly anaerobic process as all the microbes

-2 capable of carrying it out only grow in environments devoid of oxygen. Sulfate (SO4 is reduced

-2 to sulfide (S ), typically in the form of hydrogen sulfide (H2S). Eight electrons are add to sulfate to make sulfide

-2 + acetate + SO4 + 3H + 2CO2 + H2S + 2H2O

Figure 4 - The reduction of sulfate to sulfide during growth on acetate.

The electron potential and energy yield for sulfate reduction is much lower than for nitrate or oxygen. However, there is still enough energy to allow the synthesis of ATP when the catabolic substrate used results in the formation of NADH or FADH. Substrates for sulfate reducers range from hydrogen gas to aromatic compounds such as benzoate. The most commonly utilized are acetate, lactate and other small organic acids (lactate, malate, pyruvate and ethanol are some examples). These compounds are prevalent in anaerobic environments where anaerobic catabolism of complex organic polymers such as cellulose and starch is taking place.

Biochemistry of sulfate reducers

Sulfate reducers take these growth substrates and metabolize them to acetate. The reducing power generated travels down an electron transport chain eventually reducing sulfate to hydrogen sulfide and generating energy using ATP synthase. Figure 5 - Pathway of sulfate reduction when grown on lactate. Lactate (in blue) is oxidized to acetate (in red) and the electrons remove eventually end up reducing sulfate (in blue) to sulfide (in red). Note that the energy gained in the process by SLP - converting acetyl phosphate to acetate - is used up to activate sulfate in the first step of sulfate reduction. Energy for metabolism is only generated via an electron transport chain.

Recent work in bioremediation of anaerobic sediments has resulted in the isolation of many novel sulfate reducing species capable of metabolizing environmentally intractable compounds including TNT, PCP and benzoate. It is becoming apparent that this group of bacteria are very important in recycling carbon to CO2 as part of the global carbon cycle in anaerobic environments. For a look at some recent research on sulfate reducing bacteria, check out the "Journal of Bacteriology"

Carbonate reduction

Several groups of microbes are capable of using carbonate (CO2) as a terminal electron accepter. Carbonate is a poor choice to leave your electrons with due to its low reduction potential and energy yields from CO2 reduction are low. However, carbonate is one of the most common anions in nature and its ready availability makes it a tempting target.

Several groups of microbes have evolved mechanisms to take advantage of carbonate. The most important group among these is the methanogens. Methanogens are Archaea and comprise one phylogenetic group that is very closely related. Methanogenesis seems to be highly conserved and have deeps roots in the phylogenetic tree of life. It must have evolve early on and practitioners of methanogenesis cannot mess with the genes too much, lest they die.

- + HC03 + 4H2 + H CH4 + 3H2O

Methanogens use compounds that contain very high energy electrons as their electron donors and in the process convert CO2 to methane (CH4). Above is shown the use of hydrogen as the source of electrons. They are the only group of microbes that produce a hydrocarbon as major end product of their metabolism.

Another group of carbonate reducing microbes are the homoacetogens. They utilize hydrogen as the electron source and use it to reduce CO2 to acetic acid.

- + - HC03 + 4H2 + H CH3-COO + 4H2O

Before we leave anaerobic respiration I want to emphasize several points § Anaerobic respiration is a common occurrence. Microbes that carry out this process are common in the environment and their activities greatly influence the global cycling of elements. § With the exception of methanogenesis these catabolic capabilities are not associated with any phylogenetic group, but are found scattered among the various groups of bacteria. § All of these processes are less efficient that oxidative phosphorylation. This may explain why aerobic organisms dominate the earth. That and the toxic effects of oxygen. § All types of respiration investigated so far involve a membrane system, the generation of a ion gradient and the formation of ATP via ATP synthase.

Metabolism - Fermentation

Fermentation is defined as an energy yielding process whereby organic molecules serve as both electron donors and electron accepters. The molecule being metabolized does not have all its potential energy extracted from it. In other words, it is not completely oxidized.

General Concepts

Microorganisms are capable of an amazing array of different types of fermentation. Most natural compounds are degraded by some type of microbe and even many man-made compounds can be attacked by bacteria. In environments devoid of oxygen (or other suitable inorganic electron accepter), this degradation involves fermentation.

Despite the many methods bacteria employ to ferment organic compounds, there are some unifying concepts that are true of all fermentations.

§ NAD+ is almost always reduced to NADH

Remember that metabolism involves the oxidation of the substrate. These electrons are removed from the organic molecule and most often given to NAD. (This is true both in fermentation and respiration). Below is shown an example of NAD reduction.

Figure 1 - oxidation of glyceraldehyde-3- phosphate to 1,3 bisphosphoglycerate. Electrons are removed from the carbon denoted in red and donated to NAD+. An inorganic phosphate is attached to the carbon shown

§ Fermentation results in a excess of NADH

Accumulation of NADH causes a problem for anaerobes. They have too much of it and it prevents further oxidation of substrate due to a lack of an NAD+ pool to accept electrons. In many fermentation pathways, the steps after energy generation are performed in part to get rid of the NADH.

§ Pyruvate is often an important intermediate

Many of the reactions that we will look at eventually end up making pyruvate. Pyruvate is a valuable intermediate because it can be used for cell synthesis and many different enzymes can act on it. It gives the microbe flexibility.

§ Energy is derived from Substrate-Level Phosphorylation (SLP)

The substrate is converted to a phosphorylated compound and in subsequent reactions the high energy phosphate is transferred to ATP.

Figure 2 - Phosphoenolpyruvate is converted to pyruvate with the formation of ATP. The phosphate hilited in blue is transferred from PEP to ATP.

§ Energy yields are low

SLP is an inefficient process and much of the energy of the electrons is lost. Typically energy yields are 1-4 ATP per substrate molecule fermented.

§ Oxygen is not involved

Fermentation can involve any molecule that can undergo oxidation. Typical substrates include sugars (such as glucose) and amino acids. Typical products depend upon the substrate but can include organic acids (lactic acid, acetic acid), alcohols (ethanol, methanol, butanol), ketones

(acetone) and gases (H2 and CO2)

Glycolysis - Embden-Meyerhoff-Parnas pathway (EMP)

EMP is the most commonly used series of reactions for oxidizing glucose to pyruvate and many bacteria, animals and plants employ this pathway in their catabolism. EMP is so ubiquitous that is worthwhile to use it as an example of a typical fermentation. It is an essential part of many organisms catabolism, even yours! However, it is not the only method for the fermentation of glucose. Remember that bacteria are remarkably creative and other pathways are present in different species. EMP can be divided into 3 stages, activation of glucose, hexose splitting and energy extraction

Activation of Glucose

Glucose is a relatively stable molecule and in order to degrade it, it must first be destabilized by adding high energy phosphates. In the first step a phosphate is donated from ATP (or phosphoenolpyruvate - the source of the phosphate depends on the species of microbe you look at) to glucose to form glucose-6-phosphate. The molecule is isomerized to fructose-6-phosphate (another sugar) and a second phosphate is added. Fructose-1,6-bisphosphate is easier to attack than glucose and is ready to be split.

Figure 3 - Activation of glucose by phosphorylation with ATP.

Hexose Splitting

Fructose bisphosphate aldolase then breaks the phosphate loaded fructose into two 3 carbon compounds, glyceraldehyde-3-phosphate (GAP) and dihydroxyacetonephosphate (DAP). This is the crucial step in the EMP pathway, converting the 6 carbon glucose molecule to two 3 carbon molecules that will eventually become pyruvate.

Figure 4 - Splitting of Fructose by aldolase.

Energy Extraction

In the next reaction, DAP is converted into GAP, which can be acted on by the rest of the EMP. The next step is a very important one. Inorganic phosphate is added to GAP to make 1,3-bisphosphoglycerate (BPG). No energy is required and in fact electrons are transferred from GAP to NAD+. This reaction is the payback for running the pathway and the phosphates added here are later transferred to ADP to make ATP. After several enzymatic rearrangements, the final product of the EMP pathway is pyruvate.

Figure 5 - Extraction of Energy. Note the two reactions hilighted in blue that yield energy. Remember that each glucose molecule that comes into glycolysis generates two GAP molecules that can then proceed down the latter half of the pathway.

The total reaction can be summarized as follows

+ 2 ATP + glucose + 4 ADP + 2 Pi + 2 NAD 2 ADP + 2 pyruvate + 4 ATP + 2 NADH

The blue highlight denotes energy put into the reaction. Subtracting this from the energy extracted, the net energy gain is 2 ATP per glucose. That is a lot of work for just 2 ATP. Fermentations do not yield large amounts of energy and this explains why fermenting microbes go through so much substrate without much growth.

Some of the NADH that is generated can be used for cell biosynthesis, but there is a large excess or reducing power. Fermenting bacteria must find a way to get rid of these extra electrons and they do it by adding them to pyruvate to form end products.

End Product Formation

One of the more familiar fermentations is conversion of glucose to ethanol to form alcoholic beverages. After the formation of pyruvate, ethanol is formed by two simple reactions. First, CO2 is removed from pyruvate to form acetaldehyde. Then acetaldehyde is reduced by, you guess it, NADH

Figure 6 - Oxidation of NADH. Acetaldehyde is reduced to ethanol (the active ingredient in alcoholic beverages). This is the final step in yeast fermentation of glucose to ethanol.

Another favorite microbial fermentation is the formation of lactic acid. This is performed by the lactic acid bacteria. Homofermentative lactic acid bacteria use the EMP pathway to make pyruvate and then reduce it to lactate using up their excess NADH in the process. Other bacteria use alternative pathways to generate lactate from glucose. Close examination of the heterofermentative pathway reveals that it does not use EMP at all. The take home message is, EMP is common, but there are many other ways of doing business.

Figure 7 - Formation of lactate by homofermentative bacteria. The pathway used is identical to glycolysis. The final end product is lactate which is excreted by the cells into their environment.

Figure 8 - Fermentation of glucose by heterofermentative bacteria. In this pathway the top part of the glycolytic pathway is not used. Note the recycling of NADH and the low yield. Only one ATP is generated per glucose fermented.

Microorganisms perform many more fermentations than what is covered here, but these examples give you a general idea. In the next section we look at some of the processes used to make fermented food products.

Fermentations of Importance to Humans

For millenia humans have taken advantage of the fermentations microbes perform. Over the years we have learned to control and optimize theses fermentations through trial and error. It was only in the last 100 years that the biochemistry behind fermentation has become clear.

In this section we take a little time off from the serious study of metabolism to look at the production of products we're all familiar with.

Several of the fermented products we consume depend upon yeast fermentation of glucose to ethanol as we just discussed in the section of fermentation.

Brewing Beer

Beer is a fermentation of barley and hops by yeast. The starch in barley is broken down into glucose and then fermented to ethanol by yeast. The finish product is aged and then packaged for distribution and consumption. It is an involved six stage process, beginning with the formation of malt from barley.

1. Malt - Barley is first soaked in water for 5 to 7 days. At this time the grain germinates and produces amylases (enzymes that degrade starch to glucose) and proteases (enzymes that break down proteins). These enzymes are essential to the brewing process. Amylase provides sugar for the yeast fermentation and the proteases solubilize compounds in the grain and hops important for the quality of the beer. The germinated malt is then dried and crushed. 2. Mash - Mashing solubilizes the starch and other flavors in the grain and extracts flavors and preservatives for the beer. The prepared malt is suspended in water mixed with boiled malt adjuncts (other grains, carbohydrates and sugars that provide a source of starch to be converted to sugar). This mash is then incubated at 65-70°C for a short time to allow the amylase to break down the starch to glucose. The temperature is raised to 75°C to inactive the enzymes and then allowed to settle. Insoluble matter sinks to the bottom and serves as a filter as the liquid (now called wort) is taken from the container. 3. Boiling with Hops - Hops and wort are combined and boiled for 2.5 hours. The liquid is removed and ready for fermentation. Boiling with hops serves several purposes - § Concentration § Sterilization, killing many microbes that might spoil the beer § Further inactivation of enzymes in the mash. § Solubilization of important compounds in the hops and mash. Some of these add to the flavor of the beer while others, especially from the hops, have antiseptic qualities and help preserve the beer. 4. Fermentation - Fermentation begins by adding the brewers yeast Saccharomyces carlsbergensis to the wort. The starter culture is usually obtained from a previous batch of beer and is added at a very high concentration (500 grams per 120 liters). Fermentation is at a low temperature between 3.3 and 14°C for 8 to 14 days. At this time the glucose in

wort is converted to ethanol and CO2. Other compounds in the wort are also fermented to add to the characteristic flavor of beer. 5. Aging - The fermented wort (green beer) is aged at 0 °C for a period of weeks or months depending on the brewer. At this time the yeast settle to the bottom of the vessel, bitter flavors are mellowed and other compounds are formed that enhance flavor. 6. Finishing - The beer is now prepped for packaging. This can involve filtering,

pasteurization, carbonation to 0.45 to 0.52% CO2, and clarification. All of these processes depend upon the beer being made and each brewery will specialize the fermentation, aging and finishing of their beer. This is often the inspiration for various advertising done by the brewery.

The beer is then put in containers and distributed to customers. Beer normally has a shelf life of about 6 months and after that starts to take on undesirable flavors.

Bread

Bread is a simple fermentation of sugar to CO2 and alcohol. The baker first combines flour, sugar, milk and other ingredients with a microorganism, usually a bread yeast such as Saccharomyces cerevisiae, but not always. The ingredients are mixed and then allowed to incubate at 27°C for a few hours. During this time the yeast convert the sugar present to ethanol and CO2. Most incubations are for less than 4 hours not leaving enough time for the yeast to increase in number. The CO2 produced causes the bread to rise (leaven) and become porous. The success of leavening is dependent upon the rate of gas production. This can be increased by adding more yeast, more sugar, or dough conditioners (various salts that the yeast need).

Tweaking a recipe by manipulating these factors can speed CO2 production, within reasonable limits. Adding too much of anything can either kill the yeast or cause the bread to rise too quickly. The temperature of incubation is another critical consideration. Saccharomyces grows best at 26 to 28°C and deviations from that temperature will usually result in slow or complete lack of leavening. Failure as a baker can normally be attributed to either not adding the exact amounts of ingredients or inappropriate incubation temperatures during leavening. Yogurt

Yogurt is a product of fermented milk. Lactic acid bacteria are the major microbes in many milk based fermented products. These bacteria are finicky having many growth requirements all of which can fortunately be satisfied by a milk mixture. Lactose in milk is fermented to lactic acid either via the homofermentative or heterofermentative pathway.

Production of yogurt starts by conditioning the milk. The water content of milk is first lowered 25% by vacuum evaporation and 5% milk solids are added. As a final conditioning step, the milk is heated to 86 to 93°C for 30-60 minutes. This causes some breakdown of proteins and other molecules and kills contaminating microbes that may compete with the starter culture. After cooling to 45°C a 1:1 mixture of Streptococcus thermophilus and Lactobacillus bulgaricus is added. Fermentation is at 45°C until the desired degree of acidity is reached. This usually occurs in 3-5 hours. The finished product may have other ingredients added (such as mold inhibitors or dye) and is packages with fruit. Yogurt is stored at 0-4°C until consumed to prevent spoilage.

There is some evidence that consumption of products containing active cultures of lactic acid bacteria can be beneficial. However, the health claims by proponents of this idea have yet to withstand serious scientific scrutiny. It is reasonable to assume that ingesting lactic acid bacteria may help deter other more sever pathogens such as E. coli or Salmonella typhimurium

Cheese

Cheese is also a milk fermentation, but its production is more complex. Different bacteria come into play and production periods are much longer than yogurt. Despite there being 20 classes and hundreds of varieties of cheeses the initial manufacturing process is surprisingly similar.

1. Curd Formation - Milk is first pasteurized and then fermented by a starter culture. This is usually a lactic acid bacteria with the specific species in use dependent upon the cheese being produced.

Rennet (a protease) is added to the fermentation and along with the lactic acid made by the added starter, causes the milk to form curds.

2. Curd Concentration - depending upon the cheese being made, the curds may be concentrated in some manner. The goal here is to remove the appropriate amount of whey (liquid left from curd formation). For fresh cheeses (cottage or mozzarella) no concentration takes place. For soft cheeses the curds are cut into large cubes and then ripened with a fungus or mold. Hard and semi-hard cheeses are cooked and then cut into small pieces to release more whey. 3. Ripening - prepared curd is then pressed into molds, salted and ripened for weeks to years. This process if different for each cheese.

The finished product is sold either as a complete mold (a wheel of cheese) or cut into smaller pieces. Most cheeses are stored at refrigerator temperatures.