MICROBIOLOGY 101/102 INTERNET TEXT

CHAPTER XIX: INDUSTRIAL MICROBIOLOGY

REVISION DATE: 12/07/99

GLOSSARIES

MICROBIOLOGY | GENETICS | MEDICAL

THE YEAR IN MICROBIOLOGY

Scientists build disease-fighting enzyme

TABLE OF CONTENTS

• Food Made by Microbial Activities
  • Cheese Manufacturing
  • Ethanol Fermentation
  • Bread Making
  • Other Fermented Foods
  • Silage
  • Microbes As Foods
• Non-Food Commercial Microbiology
  • Industrial Microbiology
  • General Industrial Processes
  • Pharmacological Industry
    • Products Produced by Molecular Biological Processes
  • Bioremediation

FOODS MADE BY MICROBES

As noted in Chapter 18 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. Insects are a staple of the diet of most non-Westerners. In various parts of the world one can experience the delights of fried grasshoppers, a variety of fat, juicy fried worms and crunchy crickets or water bugs. Some insects are even eaten alive as their flavor is considered the best in this state. Almost all peoples make and enjoy fermented beverages, some of which are produced in unusual ways such as having old women chew up and spit the raw material into containers to aid in the fermentation process. Certain countries consider dog and cat meat a special treat, and monkeys (including chimps--98.6% genetically human) and rats are a common food for many humans around the world. The expensive steaks you purchase in fine restaurants are juicy and tender because they have been hung in the cold room long enough to allow a thick layer of mold to cover the sides of beef; the mold releases proteases that tenderize the meat (you can purchase similar proteases in the store and sprinkle them on your steaks. 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 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

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 curdled, the liquid evaporated and microbes contaminating the milk grew. The 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 (REALLY SERIOUSLY HUNGRY) they gave it a try and found out that it didn't taste too bad. This experience probably happened numerous times given our ancestors propensity to carry milk & other liquids in the stomachs of dead animals. People quickly 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 born. The process of cheese manufacturing today follows these original steps:

• Milk from any mammal (e.g. cow, horse, camel, whale, guinea pig, human [Sold under the brand name of MOTHER'S-MILK CHEESE] etc. ) is mixed with RENNIN or a similar enzyme harvested commercially from a mold. The enzyme splits the milk protein (casein).
• A STARTER CULTURE of a known strain of bacteria that produces desired flavors is added and the lactose sugar is fermented by the bacteria to acids, and other flavoring products, which lower the pH to a point where the cleaved casein precipitates out as a CURD.
• The excess liquid, called WHEY, is drained away, sometimes under pressure and the semi-dried curd collected.
  • The whey, which use to be a waste product, is currently added to many foods because it is rich in proteins, vitamins and other cool nutrients.
• The curd is inoculated with SPECIAL BACTERIA (another "starter culture") and/or MOLDS or BOTH that are known to give a particular flavor to the cheese as they grow on the curd. An ARTIFICIAL inoculum of laboratory microbes is used today for many cheeses, but most cheeses around the world are NATURALLY inoculated by placing them in environments where the air is known to contain microbes that produce a desired cheese. Sometimes additional ingredients like salt and herbs are added to achieve a desired flavor in the product.
• The curd is RIPENED for various periods of time under specific conditions of temperature and humidity, often in caves where these environmental factors stay constant. During ripening the final flavors and consistencies of the cheeses are produced by the action of the various microbes growing in and on the cheese, as well as by abiotic chemical reactions.
• At the end of the incubation period the cheese is harvested packaged and sold.

FAQ: Why are there so many different flavors of cheese when the process is basically the same for all cheeses?

• ANSWER: The favor of a cheese is influenced by a range of subtle factors including:
  • The type (source) of milk used.
  • The diet the milk producer was eating when milked, which, in turn, is influenced by the local soil, plants and weather conditions prevailing in the area when the milk supply was obtained.
  • The particular genetic "STRAIN" of microbes the curd was inoculated with at the different stages. Just as humans that belong to the same species are extremely variable in the chemicals they produce (e.g. for making skin, hair and eye color for example) so are the thousands of strains of the cheese-making bacteria and molds. Each microbial strain contributes subtle different chemical flavors to its cheese.
    • Consider how wonderful is our ability to distinguish between all these 1,000s of different flavors.
  • Even the temperature and humidity during the ripening process influences the final flavor of a cheese. Thus two cheeses made from the same curd may produce recognizably different cheeses if the curd is split in half and each half ripened in a different cave. A good analogy might be two brothers raised by the same parents in the same community will likely be very different people which surprises no one.

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

• Some cheeses like cottage and cream cheese, are not ripened at all. Both of these cheeses are highly controlled as to the bacteria that are added to them.
• Cheeses are characterized by their water content as hard, semisoft and soft. The required level is achieved by squeezing the curd at various pressures to remove the desired amount of water.
• There are over 2000 different cheeses produced in the world.
• If cheese is made from contaminated milk it can spread FBD organisms to those that eat it; Listeria and Salmonella are the most common pathogens in cheese.
• The white covering on camembert cheese is a layer of penicillin mold. When you allow camembert cheeses to "ripen" just before eating, you're allowing the proteases produced by the mold to act on the milk protein of the cheese to soften it. How many of you eat the covering on the camembert? I do and I love it.

ALCOHOL FERMENTATION

Intentional 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 coinciding with man's use of containers to carry around liquids described above. 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 use; i.e., animal stomachs, bladders etc. as described above. After a successful raid on a hive our ancestors must have, like Winnie-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 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" (maybe the first "kegger" was really a "stomacher". So it is highly possible that the first alcoholic drink was mead (beer made from the sugar in honey).

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 were one of the early trade goods that humans bartered. Today the world produces >43 x 10⁹ gal of alcoholic drinks per year. So whatever your personal stand on "drinking" is, it is, I suspect, here to stay for as long into the future as anyone can see.

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 the fermentation wastes of sugar metabolism, so in order to continue using sugar anaerobically they have to excrete excess electrons which they do by making ethanol with them. 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. This is what you can buy in a store if you want to brew your own booze.

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. Following mashing, the liquid, or MALT WORT or WORT, containing the dissolved simple sugars is separated from the insoluble material.

6. Hops (a plant grown in the US mainly in Washington) are added to the wort, which is boiled to destroy extraneous enzymes, to extract the hop-flavors and to precipitate the proteins which could add unwanted flavors and cloudiness 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 (copper or stainless steel tanks today), 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 may be 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. However, most brewers consider that fresh beer is the better product as the flavor changes in beer as it reacts with even small quantities of oxygen are considered damaging to the flavor.

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 mutant yeast that imparts a unique flavor to 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 their product's flavor. Most ethanol for human consumption is produced by strains of two yeast species, Saccharomyces carlsbergensis and Saccaromyces cerevisiae, which are characterized as bottom and top yeast respectively, depending on where they settle in the fermenting container during the course of the fermentation process.

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 beer as a lab exercise you will see how this is done. In wine making the same basic process occurs only the source of the carbohydrates is grapes or some other carbohydrate-containing plant other than grain (e.g. blackberries, elderberries, dandelions etc.), malting is not required because grapes etc. contain only short sugar polymers that the yeast can ferment directly. The predominant flavor of the wine is the result of chemicals present in the grapes at the instant of harvest and that are produced during aging. Wine can be made from any fruit that has sufficient carbohydrates present to be converted into enough ethanol.

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 (if you dare, remind me to tell you an awful story illustrating this). 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 mold, to carry and to 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, plus the tough crust made it easy to handle, preserve and transport, so women experimented (the first "scientists"?) until they could reproduce this effect and over the years bread making developed into the process we know today.

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 (or added) 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 results 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 bread. The yeast also contributes important flavoring 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 ~1 million other Americans 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 health-food 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!!).

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 plant products are placed in pits or closed containers which maintain ANAEROBIC conditions, #microbes convert much of the complex carbohydrate, to lactic and other organic acids that are easily metabolized by the cattle and the microbes in the cow stomachs. These nutrients make them grow faster or produce more milk, so they can meet you on the hamburger bun or the "Got Milk" commercial.

MICROBES AS FOOD

This is one of these things that keeps popping up in, that I call "stupid science". The logic goes something like this:

"Hey! I've got a fantastic, sooo cool 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 oodles 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 City and the waste heat alone would melt the Polar ice caps. 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 "cool idea" isn't very workable.

However, there are a few microbe-foods that almost make using microbes for food look reasonable. These are mushrooms, molds in cheese, soy and tempeh, algae and yeasts. Some lakes in Africa naturally grow huge quantities of an eatable algae. There are tons of yeast produced as byproducts 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; you can find these algae in your local health-food store if you are interested (ask for Spirulina).

The take-home-lesson is that some 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. Some microbes are great in modifying many materials into byproducts that we enjoy eating, but most simply turn food into disgusting slop.

NON-FOOD INDUSTRIAL MICROBIOLOGY

Microbes have been used for about 100 years to produce industrial chemicals for human use. 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:

1. The classical industrial chemicals that can be manufactured either by microbes or organic chemical processes.

2. The biological chemicals that can not be synthesized by organic chemical processes, but only by living cells.

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 FERMENTERS. Fermenters are tanks that may hold 1,000 of gallons, or more, of a 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 monitoring the progress of the fermentation and for controlling the internal environment (e.g. the pH, temperature etc.).

All industrial microbial processes deal with similar problems:

• Finding the least expensive medium in which to grow the microbe so as to maximize yield and profits.

  • Often this is a waste product from another industrial process, such as corn steep liquor, sugar processing wastes or whey.

• Maintaining strain purity and developing better strains for improving the yield.

  • A single mutation may decrease the yield by a significant percentage or result in undesirable substances being produced. The industrial research laboratories constantly seek better strains for the production of their product.

• Preventing contamination by other microbes and by viruses (phage) that live on the microbe involved.

  • The media must be sterilized prior to being inoculated with the desired organism and purity must be maintained throughout the production process. A small quantity of a contaminant may produce an enzyme that can destroy the product in 1,000s of gallons of medium. For many microbes, viruses present a constant danger as a single virus can infect and destroy the desired microbe in an entire tank. The sterilization of large containers and huge quantities of media represent both an engineering and microbial challenge.

• Developing rapid and efficient methods for purification of the desired produce in a stable form that is safe to use.

  • The products of many fermentations are often unstable in the IMPURE FORM or subject to unwanted modifications if they are not purified quickly. The final growth mixture may contain dangerous substances from which the desired product must be separated. As every step in the purification results in a lose of the product, the search for more efficient purification procedures is never ending.

• Always striving to improve yield by modifying the strain, nutrients or environmental conditions.

  • As product yields are exquisitely sensitive to subtle modifications in the nutrient and the #environmental conditions, these are constantly monitored For example, the pH, oxygen content, nitrogen/phosphorous ratio etc. may be adjusted during the production process.

• Safe and inexpensive disposal of the massive quantities of waste products remaining after the product is formed.

  • The waste products of these large fermentations present major waste disposal problems as they are rich in organic matter that are highly polluting if released untreated into the environment. However, the cost of treatment cuts into the profit margin and increases the cost of the product.

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 (at this site go to "search", type in acetone & follow the steps until you reach the pathway), 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 Zionist, he asked that the British support the formation of a Jewish State in Palestine. Weismann subsequently made substantial contributions in the US to the production of synthetic rubber during the second world war which earned him the gratitude of the American government. 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 (read this short biography biography ), 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 inhibited the growth of that pathogen. He surmised that it might be used to fight bacterial infections and began to investigate it. Although he made little progress on it, and finally gave up, others began to investigate its possibilities and eventually a tiny amount of penicillin was isolated and given to a policeman suffering from a fulminating infection of S. aureus. He began to recover when the supply ran out and he died. The amounts of penicillin were so small in those early days that it was reisolated from the urine of patients and used again. However, clinical tests looked so promising that when the second WW came along the U.S. took over the investigation and the development of penicillin became, after the development of the atomic bomb, the second highest research priority in the war effort. From this followed the antibiotic era and the huge pharmacological industry that operates world wide to day.

The revolution in molecular biology offers the possibility of yielding a whole new range of pharmacologically active 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 (#cloning). 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 which can then be used for therapeutic purposes. For example, although clots are constantly forming in our bodies, they are 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, a cloned version of one of these clot-dissolving enzymes has been shown to be effective in saving lives and minimizing damage from strokes. However, their low concentration and difficulty of isolation have, until recently, made these clot-busting enzymes too rare and expensive to use widely. Now 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 manufactured by molecular biology technology is given below:

The following is a partial list of microbial produced commercial pharmacological and related biotech products:

1. Vitamins

2. Amino acids

3. Nucleic acids

4. Antibiotics

5. Alkaloids

6. Steroids

7. Non-Steroid Hormones/cell regulators (cytokines):

   1. Epidermal growth factor

   2. Proinsulin

   3. Insulin

   4. Human growth hormone

   5. Somatostatin

   6. Interferons

   7. Platelet-derived growth factor

   8. Fibroblast growth factor

   9. Tumor Necrosis Factor

   10. Other cytokines are coming on line all the time

8. Blood coagulating factor XIII

9. #Transgenic plants and animals

10. The #restriction enzymes

11. #Plasmids

12. Other enzymes (e.g. ligase, DNA polymerases etc.) used in molecular biological research

Biotech industries, which produce the biological materials listed above from genetically engineered microbes, plants or animals have developed rapidly in the last few years. Most of these industries, some of which are worth millions of dollars, did not exist 15 years ago and new biotech industries are appearing all the time as scientist find how to produce new biological products using genetically engineered microbes.

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. It is a very fluid situation and one should invest one's money in such enterprises carefully.

One direction that the biotech industry is taking 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. A cool novel, #Chromosome 6, you can read for extra credit is based on this idea.

BIOREMEDIATION

BIOREMEDIATION IS DEFINED AS THE USE OF MICROBES TO REMOVE POLLUTANTS FROM THE ENVIRONMENT.

Our industrial-based civilization has produced and contaminated the earth's surface with a huge number of dangerous pollutants, both natural and made-made. 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 wastes polluting our environment. The ones colored red are carcinogenic/toxic, the blue ones are just toxic (to things like the liver, brain and kidneys):

1. Benzene

2. Phenol

3. Chloroform

4. Carbon tetrachloride

5. Gasoline

6. Motor oils

7. Raw petroleum

8. Nitrate

9. Lead

10. DDT

In many cases the soil and ground water leaching from industrial and municipal toxic waste dumps contaminate vast quantities of ground water, making it dangerous for any subsequent use of that grand water. 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.

The basic principle of bioremediation is the same as that for #sewage treatment; That is, the use of microbial metabolism to "eat up" or metabolize pollutants so as to convert them into something harmless. The following general steps are utilized in bioremediation:

Define the pollution situation: What pollutants are present, how much of each are there, how dangerous are they, are they spreading and, if so, where and how fast.

Develop a microbial approach for dealing with the pollutants.

Isolate or stimulate a microbial population that will, by natural selection, "eat" or metabolize the pollutants.

Grow the POLLUTION-FIGHTING-MICROBES in large quantities or otherwise provide conditions that will stimulate their growth in the polluted environment.

Add the POLLUTION-FIGHTING-MICROBES to the polluted environment and provide the optimum nutrient and environmental conditions to allow the POLLUTION-FIGHTING-MICROBES to metabolize the pollutants.

The crucial step in this process is the isolation or enrichment of SUITABLE MICROBES that will effectively metabolize the desired pollutant. This is done using a technique developed by the early microbiologists called the ENRICHMENT CULTURE TECHNIQUE. The ENRICHMENT CULTURE TECHNIQUE works like this. A sample of a pollutant is added to a BASIC NUTRIENT MEDIUM in which the pollutant chemical (e.g. gasoline, phenol, turkey feathers etc.) is included as the MAJOR or only carbon and/or energy source. The medium is inoculated with soil which is likely to contain a diversity of microbes (e.g. rich garden soil, sewage etc.). The culture is incubated, usually under aerobic conditions, at a suitable temperature for a period of time and the concentration of the pollutant MONITORED. If a microbe happens to be present in the soil inoculum that CAN METABOLIZE THE POLLUTANT, it will grow and reproduce by following the #SURVIVAL OF THE FITTEST law of evolution. If the pollutant disappears, an inoculum is taken from the original flask and added to another and the process is REPEATED until you have a culture in which the POLLUTANT-DIGESTING ORGANISM predominates. This microbe(s) is isolated and studied to see if you can boost its pollutant-metabolizing abilities even more. Finally it is used as outlined above to treat the polluted material.

PROBLEMS WITH BIOREMEDIATION

• Often the concentration of a given pollutant is so low that it won't support good growth of microbes, yet the level is high enough to be dangerous. Under such conditions, additional nutrients, AT ADDED COST, have to be supplied.

• It is difficult to get the microbes into the polluted soil in a way that they can effectively remove the pollutant. One procedure involves digging up the contaminated soil, mixing it in large tanks with the microbes and nutrients until the pollutant is degraded and then returning the now POLLUTANT-FREE SOIL to its original place. Clearly, this is an expensive process when large areas of polluted land are involved.

• Many of the pollutants are recalcitrant or difficult for microbes to readily digest and thus the microbes take a long time to degrade them; further adding to the expense of the process.

• The limits of the pollution often are ill-defined. For example, seepage from a toxic land fill may have contaminated ground water in an area for years before its discovery and no one knows the extent of the contamination. In some circumstances, pollutants move only inches per year from the source, whereas in other cases it can travel for miles underground and turn up in well-water at a considerable distance from the pollution source. Defining the extent of an underground pollution problems takes years and millions of dollars.

  • Radioactive pollution of the Hanford nuclear works site is probably one of the most intensely investigated situations in the world, yet the extent of the problem is still unknown and its danger to the public is being intensely debated.

• The number of pollutants at a site may be unknown or poorly defined, so what works for one pollutant may not work on another pollutant.

Bioremediation has proven effective at treating pollution problems like aviation fuel spills in the ground on army bases, in the elimination of creosote from contaminated ground water and soil, in removing oil spills and in digesting a host of other organic pollutants. The process is COSTLY, but is proving to be more effective than such procedures as digging up contaminated ground and burying it somewhere else, incinerating the soil or treating the polluted water with expensive and dangerous chemicals to destroy the targeted pollutants.

Click here for a self assessment test of what you have learned.

Bioremediation Information:

http://biogroup.gzea.com/; Bioremediation discussion group.

http://www.clu-in.org/ ; EPA’s hazardous waste clean-up information site.

http://www.biotreat.state.pa.us/; Natural attenuation of chlorinated solvents workshop.

http://www.rtdf.org/; Remediation technology development forum.

http://www.westgov.org/itrc/; The interstate technology and regulatory working group.

http://www.nmsr.labmed.umn.edu/~lynda/index.html; University of Minnesota biocatalysis/biodegradation database.

http://web.utk.edu/~cebweb/cebfinal.html; Center for Environmental Biotechnology (CEB) at the University of Tennessee, Knoxville

http://www.mannlib.cornell.edu:10000/ccr2/Horror/Biof.tutorial.HTML ; Biofilms

Copyright © Dr. R. E. Hurlbert, 1999.
This material may be used for educational purposes only and may not be duplicated for commercial purposes.
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