MICROBIOLOGY 101 INTERNET TEXT

CHAPTER XX: INDUSTRIAL MICROBIOLOGY


 


REVISION DATE: 11/3/96 


 


TABLE OF CONTENTS


 

FOODS MADE BY MICROBES

The line between gourmet and rotten food is often a matter of perspective stemming from ones' upbringing and early gastronomic experiences. As discussed previously, one society will consider that slightly rotten pheasant is a taste experience of the highest order, while members of another group will gag at the very thought. We Americans use soy sauce in copious quantities on a variety of foods without realizing (or not caring) that it is a mixture of several rotted foods. Cheeses are simply a form of spoiled milk, many of which are covered with the very same molds that we throw out in disgust when we find them growing on our bread or tomatoes. The French and Germans consider snails such a gastronomical delight that they fight local snail-wars over the right to scour the woods for these slimy gastropods. Horse's milk and meat is eaten by more people than eat beef and cow's milk. Almost all peoples make and enjoy fermented beverages, some of which are produced in unusual ways such as having old women spit into the raw material. Certain countries consider dog meat a special treat, and monkeys and rats are a common food for many hominids around the world. As I discuss the industrial production of food, it is a good idea to consider the differences between people in a positive light and appreciate that other societies find our some of our dietary preferences as disgusting as we find theirs.

In the first part of this chapter the industrial production of several common foods will be described. In the second section other industrial uses of microbes will be presented. 


 

CHEESE PRODUCTION

Picture of cheese here

The discovery of the cheese-making process is very old and certainly was accidental. Early man learned to carry his water, beer and milk in natural containers like animal stomachs, bladders and lengths of intestines tied at the ends. These were tough, water-proof and light, and they could easily be tied around ones neck, shoulder or waist. The stomach of young cattle contains an enzyme, rennin, that cleaves the casein protein of milk making it easier to curdle when microbes convert the lactose sugar in milk to acid. This is the basis of cheese making. A likely scenario is that a calf's stomach, full of milk, was left in a cool corner of a cave or hut for several weeks during which time the milked cuddled, the liquid evaporated and microbes contaminating the milk grew. These molds and/or bacteria that grew on and in the curd as it continued to dry produced a unique flavor. When the owners finally returned they found a furry chunk of what once had been milk and being hungry (that is VERY HUNGRY) they gave it a try and found out that it didn't taste too bad. This experience probably happened numerous times given a propensity to carry milk in cow's stomachs. People surely realized that the semidried curd (the precipitated milk protein) was lighter to tote around than the milk and that it lasted a relatively long time before it spoiled so badly you couldn't stand to eat it. Finally someone came up with the idea of making it happen on purpose and the cheese industry was on its way. The process of cheese manufacturing today follows these same primitive steps:

How many of you like blue cheese (I'm mad for it)? Do you know what the crunchy blue things are in that cheese?

A FEW FACTS ABOUT CHEESE


 

ALCOHOL FERMENTATION

Intended alcohol production by humans is known to have been around for at least 10,000 years. It is not illogical to imagine that it is even older than that; probably corresponding with man's use of containers to carry around liquids. Like modern man, our ancestors relished honey and certainly raided wild bee hives for the sweet nectar they contained. Because of the liquid nature of honey early humans undoubtedly placed the honey in whatever containers they could conceive of; i.e., the animal stomachs, bladders etc. described above. After a successful raid on a hive our ancestors must have, like Winney-the-Poo, sat around the fire and enjoyed dipping their fingers in the "honey pot". Certainly they quickly discovered that by adding some water to the container they could make a sweet drink and even more certainly an occasional bag of honey-drink was left unattended for a period of time sufficient to allow fermentation to occur. Once the people returned and drank the now "modified" contents, the rest, as they say, "is history". So it is likely that the first alcoholic drink was mead.

Similar serendipitous discoveries that other natural materials could be fermented to produce the same taste as in mead probably followed in a relatively short period of time. Considering the fact that the wine trade is ancient, alcoholic products surely were one of the early trade goods that tribes of humans bartered for. Today the world produces >30 x 109 gal of alcoholic drinks per year. So whatever your personal stand on "drinking" is, it is here to stay for as long into the future as anyone can see I suspect.

ETHANOL PRODUCTION PROCESS

No matter how you cut it, ethanol is a waste product of the metabolism of certain microbes; that is it is the equivalent of "microbial pee". Microbes make ethanol because they don't have oxygen available to use it, so they must excrete it. The term "FERMENTATION" has numerous contextual meanings, but in relationship to ethanol production it refers to the metabolism of carbohydrates under anaerobic conditions. Any simple or complex carbohydrate can be fermented to produce ethanol. Usually this fermentation is carried out by certain ubiquitously distributed yeast, but a few bacteria are also able to produce ethanol in commercial quantities.

The commercial or industrial production of ethanol is produced as follows:

  1. A grain, usually barley, which is rich in complex glucose polymers (complex carbohydrates) is collected and wetted.
  2. As it is stored in a warm, dark place the seeds germinate (sprout) and release enzymes that break down or hydrolyze the polysaccharides to simple sugars which the yeast can metabolize. This process is called MALTING.
  3. The malt is dried and crushed to improve the extraction of the sugars. The dried material can be stored at this stage.
  4. In the next step, called MASHING, the sprouted malt is suspended in water where the enzymes continue to break down the polysaccharides to release simple sugars.
  5. The liquid, or MALT WORT or WORT, containing the dissolved simple sugars is separated from the insoluble material.
  6. Hops are added to the wort, which is boiled to destroy any extraneous enzymes, to extract the hop-flavors and to precipitate the proteins which could add unwanted flavors to the final product. The hop-flowers contain substances that inhibit spoilage microbes and aid in the final clarification of the product.
  7. Yeast are then added (PITCHED) and the mixture is placed in large closed containers, usually metal in large commercial operations, so the fermentation can proceed in the absence of air.
  8. The fermentation goes on for about 7 days at a cool temperature which is optimum for ethanol production.
  9. The brew (green beer) is usually aged (LAGERED) under a variety of conditions for various times as desired by individual brewers. During the aging, chemical changes occur spontaneously that subtly alter the flavor of the product.
  10. Finally the product is usually cleared (the yeast removed), bottled and pasteurized.

THE YEASTS AND OTHER DETAILS OF FERMENTATION

Each brewer has their own strain of yeast that is known to produce a particular flavor in their product. Companies go to great lengths to maintain the genetic purity of their yeast strain, as the wrong mutation can produce subtle changes in the product's flavor. Most ethanol for human consumption is produced by strains of two yeast species, Saccaromyces carlsbergensis and Saccaromyces cerevisiae, which are characterized as bottom and top yeast respectively, depending on where they settle in the fermenting container.

Light brews are generally made with yeast strains that convert more of the available sugars to ethanol, thus lowering the total caloric content of the beer. Since you have made wine as a lab exercise I will only note that in wine making the same basic process occurs only the source of the carbohydrates is grapes, malting is not required and the predominant flavor of the wine is the result of chemicals present in the grapes. Wine can be made from any fruit that has sufficient carbohydrates present to be converted into enough ethanol to make it a "wine".

Distilled ethanol products are made from the fermentation of other grains like corn and rye. At the end of the fermentation the material is boiled and the more volatile ethanol, which evaporates first, is collected in a concentrated form. The combination of the unique starting material, the yeast strains and the aging process all contribute to the unique flavors of distilled spirits. 


 

BREADS

Bread is another ancient product of microbial action that was certainly discovered by accident. Ancient man (in this case almost surely WOMEN) began to gather seeds for food, probably after seeing other animals eat them. However, dried seeds are hard to chew and if they're not broken open pass through the intestine without yielding any nutritional value. It was not difficult to recognize that breaking up the seeds with a stone yielded a more palatable food that digested easier and from there it was a small jump to mixing it with water to form the crushed material into a compact unit that was easy to carry and eat (remember they didn't have spoons to scoop up loose food). As these wet masses of crushed grain were placed near the fire to dry out, many of them baked. During the baking the grain developed a pleasing flavor and some of the wads of dough swelled up and became "bread" during the baking. The texture of the "bread" was clearly desirable so women experimented until they could reproduce this effect and over the years bread making developed into the process as we know it.

The rising of bread is due to a fortuitous combination of chemical characteristics. Wheat, and several related grains, make a group of proteins called glutens. These proteins have the characteristic of forming long molecular strings when they are "worked" or "kneaded" that bind the bread together in the sticky mass we call DOUGH. Gluten also contributes to the delightful flavor imparted to bread during baking. Bread rises due to the activity of contaminating yeast which metabolizes the sugar in the wheat and converts it into carbon dioxide. Because of the GLUTEN GLUE, the carbon dioxide is trapped within the bread which causes the bread to RISE from the pressure of the carbon dioxide buildup. This result in the formation of many small bubbles within the bread. When the bread is baked the protein is denatured and it and the starch harden into the food we know as bread. The yeast also contribute important favoring to the bread. Although, our knowledge of the biology of bread making is only a little more than 100 years old, people have known for several 1,000 years that in order to make bread you had to add a STARTER CULTURE of dough containing the yeast to each new batch of fresh bread dough.

This author and a high percentage of Northern Europeans suffer from a gluten induced inherited disease called CELIAC SPRUE. Those of us with this genetic condition become quite ill if we eat gluten containing foods, so we must avoid all wheat breads, pizzas, pastas, pies, cakes etc. So the next time you see someone carefully reading the label on a can in the supermarket, they may not be a healthfood enthusiast, but just one of us sprue-victims making sure there is no GLUTEN lurking in that container. Don't feel too sorry for us, as we have the perfect excuse for eating a LOT of Mexican food (OLE!!). 


 

OTHER COMMON FERMENTED FOODS

Since the eating of MICROBIAL ENHANCED (spoiled) food is quite common for humans it should not surprise the reader that there are a lot of such foods enjoyed by we humans, including the following:

SILAGE

A lot of the beef you eat and the milk you drink is produced from cows that are fed on a rich diet of fermented grass, chopped corn and other seed crops. When these are placed in pits or closed containers which maintain ANAEROBIC conditions, microbes convert much of the complex carbohydrate, to lactic and other acids that are easily metabolized by the cattle and which make them grow faster so they can meet you on the hamburger bum or the "Got Milk" commercial.

MICROBES AS FOOD

This is one of these things that keeps popping up in, what I call "pop-up science". The logic goes something like this: "Hay! I've got a fantastic, neat idea; here's these microbe things that grow like gangbusters on inexpensive waste products that we're just throwing away anyway; well why not grow these little micro "steaks" on garbage, press the little buggers into cakes or shape them into steaks, or whatever and sell it to people as the latest food fad and make boodles of money?".

Sorry, but it doesn't work out that way. First, most microbes taste so bad or are full of such toxic material that you wouldn't want them in the same county, much less in your lunch. Secondly, the "waste material" you're going to grow them on is either toxic or tastes terrible itself and besides you have to sterilize millions of tons of it before you can use it as microbial media, which takes buckets of money, an autoclave the size of New York and the waste heat alone would melt the ice cap. Thirdly, by the time you've treated them so they're palatable, you will have doubled the national debt. By now you've gotten the idea that the once "neat idea" isn't very workable.

There are a couple of microbe foods that almost make using microbes for food look reasonable. These are mushrooms and a some lakes in Africa that naturally grow some eatable algae. There are tons of yeast produced as by products of the ethanol industry, but eating yeast in anything but small, flavoring quantities is like doing the same with vanilla (dare you to try that). Sadly the people who eat the algae are so bad off nutritionally that........well the fact that they eat the algae says it all.

Now here is a story, the value of which you can judge for yourself: it seems that once upon-a-time a company was actually formed to use oil to grow oil-eating yeast as food to feed the ever-growing hungry horde of humans. Which is the equivalent of using new, fully operational BMWs as flowerpots. Guess what was the outcome of this enterprise?

The take-home-lesson is that microbes are great as supplements and flavor-enhancers in small doses as noted by the common usage of yeast as a flavoring agents in many foods, but they are duds as a main course.


 

NON-FOOD INDUSTRIAL MICROBIOLOGY

Microbes have been used for about fifty years to produce non-food chemicals for human use in industrial quantities. These microbial processes helped the allies WIN THE FIRST and SECOND WORLD WARS. In the former case microbial fermentation led to the formation of Israel. This section is divided into two parts, the classical industrial chemicals that can be manufactured either by microbes or organic chemical processes and the biological chemicals that can not be synthesized by organic chemical processes, but only by living cells themselves.

GENERAL DESCRIPTION OF HOW MICROBIAL PRODUCTS ARE MADE

All commercial FERMENTATIONS utilize similar techniques. The microbes are cultivated under rigorously controlled environmental conditions conducive to optimum production of the given product in rather humongous FERMENTORS. Fermentors are tanks that may hold 1,000 of gallons of culture. They must be made of materials , usually stainless steel, that can be heat sterilized and which will not react with the microbes or with the desired products. They must be able to be tightly sealed to prevent contamination and yet must contain numerous openings for monitors, air and media.

All industrial microbial processes deal with similar problems:


 

INDUSTRIAL CHEMICAL MICROBIOLOGY

Simple organic chemicals like ethanol, acetic acid (vinegar) acetone, butyric acid and lactic acid are readily made either by organic chemical synthesis or by microbial fermentation. The method of choice depends upon the price of the raw materials and on the availability of industrial facilities to carry out either process. That is, in some cases it is cheaper to manufacture ethanol by fermentation and in other cases by chemical conversion from petroleum or natural gas. Immediately proceeding the first world war the process of acetone-butanol fermentation by bacteria was discovered. When the war began England found itself cut off from a supply of acetone, a crucial ingredient in the making of gunpowder. Chaim Weismann, a Jewish biochemist was put in charge of developing the microbial process for the commercial production of acetone. His success made such an important contribution to the war effort that the British government offered him ANY REWARD he chose. Being an ardent Zioist, he asked that the British support the formation of a Jewish State in Palestine. Weismann also made substantial contributions in the US to the production of synthetic rubber during the second world war and earned the gratitude of the American government. Subsequently, when Israel, with the strong support of the US and British governments, became a nation after the second world war Wiesmann served as its first president.

Today acetone and butanol are more cheaply made from petroleum, but as these natural resources run low in the next century we may have to return to the microbiological technology. The following is a partial list of organic chemicals made commercially by microbes:

  1. 2,3, butainediol; buttery taste
  2. Enzymes
  3. Organic acids such as citric, lactic, ascorbic (vitamin C), acetic. These are utilized both as foods, and in industrial chemical processes.
  4. Polysaccharides
  5. Poly-beta hydroxybutyric acid
  6. Methane
  7. Hydrogen
  8. Biological pesticides

PHARMACOLOGICAL INDUSTRY

The second contribution of microbes to winning a war came through the serendipitous discovery of penicillin by the English microbiologist A. Fleming in 1929. Fleming, who was known as a bit of a character for painting pictures on petri dishes using different colored microbes, observed that a mold contaminating a plate of S. aureus was excreting something that was inhibiting the growth of that pathogen. He surmised that it might be a drug that could be used to fight bacterial infections and began to investigate it. Although he made little progress on it, others began to investigate its possibilities and eventually a tiny amount of penicillin was isolated and given to a policeman suffering from a terrible infection of S. aureus. He appeared to be recovering when the supply ran out and he died. The amounts of penicillin were so small in those early days that it was isolated from the urine of patients and used again. However, subsequent clinical tests looked so promising that when the second WW came along the US took over the investigation from the British and the development of penicillin became the second highest research priority in the war effort; second only to the development of the atomic bomb. From this followed the antibiotic era and the huge pharmacological industry that operates world wide to day.

The following is a list of microbial produced commercial pharmacological products:

  1. Vitamins
  2. Amino acids
  3. Nucleic acids
  4. Antibiotics
  5. Alkaloids
  6. Steroids

MOLECULAR INDUSTRIAL PRODUCTS

The revolution in molecular biology offers the possibility of yielding a whole new range of microbiological produces through the application of genetic engineering technology. As has been described in Chapter 10, it is now possible to move genes from one organism into a plasmid or into the genome of another organism. Under the proper conditions the cloned genes can be made to direct the synthesis of their protein product. In this way a substance that has a specific effect on another gene or gene product, but which is normally made in tiny amounts in a target organism can be made in commercially large quantities. These large quantities can then be used for therapeutic purposes. For example, clots are constantly forming in our bodies, but they are constantly being dissolved before they do serious damage by special "clot-dissolving enzymes". In the case of strokes or embolisms where life threatening clots form in the brain or lungs, these special enzymes have been shown to be effective in saving lives. However, their low concentration and difficulty of isolation have made these clot-busting enzymes too rare and expensive to use widely. However, these enzymes are now being made through genetic engineering technology in large enough quantities so as to become a standard treatment for stroke victims. A partial list of therapeutic agents is given below:
  1. Hormones: insulin, human growth hormone, somatostatin, interferons
  2. Epidermal growth factor
  3. Insulin
  4. Platelet-derived growth factor
  5. Proinsulin
  6. Fibroblast growth factor
  7. Blood coagulating factor XIII
  8. Transgenic plants and animals
In addition, there is a large industry in the production of genetic engineering materials which in turn are produced from genetically engineered microbes, plants or animals. These include most of the commercial restriction enzymes and plasmids as well as a number of other enzymes widely used in molecular biological research. Most of these industries, some of which are worth millions of dollars, did not exist 15 years ago. New biotech industries are appearing all the time.

Biotech industries are expected to be one of the fastest growing industries in the next century, particularly as the human population ages and as the information from the human genome project comes on line. However, a word of caution is advisable here. Many biotech industries fail because they could not make a commercially viable product due to some unexpected hitch developing along the way or they find that the market they thought was there really isn't or they were beaten out by a better or less expensive product. Many of the products that work under controlled laboratory conditions, fail to perform up to expectations in the real commercial world.

One direction that the biotech industry is going in is in the construction of transgenic plants and animals. Transgenic plants and animals are good vehicles for producing HUMAN GENE PRODUCTS. For example, pigs and goats have had human genes incorporated in them. One interesting system involves fusing the desired gene with the milk protein (casein) of a mammal, then when the animal lactates it produces large quantities of the human gene product which can then be cleaved from the milk protein and purified separately. 


 

BIOREMEDIATION

BIOREMEDIATION refers to the use of microbes to removed pollutants from the environment. In Chapter 19 the oldest form, sewage treatment, of bioremediation was described in great detail. However, our industrial-based civilization has produced and contaminated the earth's surface with a huge number of dangerous pollutants. Many of these substances are toxic and/or carcinogenic or harmful to the environment in other ways. Below is a small list of some prominent industrial pollutants polluting our environment. The ones colored red are carcinogenic/toxic, the blue ones are toxic:
  1. Benzene
  2. Phenol
  3. Chloroform
  4. Carbon tetrachloride
  5. Gasoline
  6. Motor oils
  7. Raw petroleum
  8. Nitrate
In many cases the soil and ground water leaching from the toxic and municipal waste dumps can contaminate vast quantities of ground water, making it dangerous for any use. The idea behind BIOREMEDIATION is to (1) isolate microbes that can DEGRADE or eat a particular pollutant and (2) to provide the conditions whereby it can do this most effectively, thereby eliminating that pollutant. The technology for doing this is still in the development stage, but companies have been formed which provide this service. The problems however are IMMENSE.