Methods Used in Environmental Biotechnology
In this article, we shall identify the various methods used in biotechnology in general and environmental biotechnology in particular.
Specifically, you will be acquainted with the trend of available methods and their improvement from the ancient traditional methods to the highly sophisticated methods that we have today.
Efforts will be made to show the techniques of each method, as well as their advantages and disadvantages over the traditional methods, if any.
Traditional Biotechnology Methods
The several methods used in biotechnology are generally divided into two major sectors, traditional and modern. The traditional methods comprise those methods that have been used since the ancient days and are generally based on the use of whole-organisms.
On the other hand, modern methods include those that use either parts of organisms or substances made from organisms. Typical examples of traditional methods are fermentation, nitrogen fixation and tissue culture, while the modern methods are gene and molecular based methods.
Microbial Fermentation
Fermentation involves the breaking down of complex organic substances into simpler ones by the activities of microorganisms. In the process energy is released. The energy is utilized by the microorganisms in the anaerobic environment or cells in the body of organisms as their source of energy.
Biochemically, fermentation is a process of oxidation of an organic compound by electron release. The electron is taken up by an electron acceptor which could also be an organic compound.
A typical example is the glycolysis (splitting of a sugar molecule and removing electrons from the molecule) of glucose to produce two molecules of pyruvate (a salt or ester of pyruvic acid).
The pyruvate is metabolized to various compounds, the nature of which determines the type of fermentation involved. In homolactic fermentation, lactic acid is produced from pyruvate; in alcoholic fermentation ethanol and carbon dioxide are produced and in heterolactic fermentation lactic acid as well as other acids and alcohols are produced.
Homolactic (LacticAcid) Fermentation
During lactic acid fermentation, the electrons released during glycolysis are passed to pyruvic acid to form two molecules of lactic acid.
Lactic acid fermentation is carried out by many bacteria, most notably by the lactic acid bacteria used in the production of yogurt, cheese, sauerkraut, and pickles. Some animal cells such as muscle cells can also use fermentation for a quick burst of energy.
Alcohol Fermentation
Alcohol fermentation also begins with glycolysis to produce two molecules of pyruvic acid, two molecules of ATP, and four electrons. Each pyruvic acid is modified to acetaldehyde and CO2. Two molecules
| Erythromycin | Antibiotic | Streptomyces (bacterium) | erythaeus |
| Invertase | Candy | Saccharomyces (fungi) | cerevisiae |
| Lactase | Digestive aid | Escherichia coli (bacterium) | |
| Neomycin | Antibiotic | Streptomyces (bacterium) | fradiae |
| Pectinase | Fruit juice | Aspergillus niger (fungus) | |
| Penicillin | Antibiotic | Penicillium notatum (fungus) | |
| Riboflavin | Vitamin | Ashbya gossypii (fungus) | |
| Streptomycin | Antibiotic | Streptomyces (bacterium) | griseus |
| Subtilisins | Laundry detergent | Bacillus subtilis (bacterium) | |
| Tetracycline | Antibiotic | Streptomyces (bacterium) | aureofaciens |
Table: Fermentations by Genetically- Engineered Organisms
| Product | Application | Organism |
| B. growth hormone | Milk production(cows) | Escherichia coli (E. coli) |
| Cellulase | Cellulose | E. coli |
| H. growth hormone | Growth deficiencies | E.coli |
| Human insulin | Diabetics | E. coli |
| Monoclonal antibodies | Therapeutics | Mammalian cell culture |
| Ice-minus | Prevents ice on plants | Pseudomonas syringae |
| Sno-max | Makes snow | Pseudomonas syringae |
| t-PA | Blood clots | Mammalian cell culture |
| Tumor necrosis factor | Dissolves tumor cells | E.coli |
B.=bovine H.=human
t-PA=Tissue plasminogen activator
Biological Nitrogen Fixation
Nitrogen is a critical limiting element for plant growth and production. It is a major component of chlorophyll, the most important pigment needed for photosynthesis, as well as amino acids, the key building blocks of proteins.
It is also found in other important biomolecules, such as ATP and nucleic acids. Even though it is one of the most abundant elements (predominately in the form of nitrogen gas (N2) in the Earth’s atmosphere), plants can only utilize reduced forms of this element. Plants acquire these forms of “combined” nitrogen by:
1) The addition of ammonia and/or nitrate fertilizer (from the Haber-Bosch process) or manure to soil,
2) The release of these compounds during organic matter decomposition,
3) The conversion of atmospheric nitrogen into the compounds by natural processes, such as lightning, and
4) Biological nitrogen fixation.
We will concentrate on biological nitrogen fixation.
Biological nitrogen fixation (BNF), discovered by Beijerinck in 1901 is carried out by a specialized group of prokaryotes. These organisms utilize the enzyme nitrogenase to catalyze the conversion of atmospheric nitrogen (N2) to ammonia (NH3).
These prokaryotes include aquatic organisms, such as cyanobacteria, free-living soil bacteria, such as Azotobacter, bacteria that form associative relationships with plants, such as Azospirillum, and most importantly, bacteria, such as Rhizobium and Bradyrhizobium, that form symbioses with legumes and other plants .
The process involves a reduction of atmospheric nitrogen in the presence of nitrogenase which catalyses the breaking of covalent bonds and the addition of three hydrogen atoms to each nitrogen atom.
Read Also : Basic Environmental Concepts and Theories
This process requires a large input of energy obtained by the oxidation of organic molecules. Non-photosynthetic free-living microorganisms obtain these molecules from other organisms, while photosynthetic microorganisms, such as cyanobacteria, use sugars produced by photosynthesis.
Associative and symbiotic nitrogen-fixing microorganisms obtain these compounds from their host plants’ rhizospheres. Nitrogen-fixing bacteria such as Azotobacter, Bacillus, Clostridium, and Klebsiella obtain their own source of energy, typically by oxidising organic molecules released by other organisms or from decomposition.
There are some free-living organisms that have chemolithotrophic capabilities and can thereby use inorganic compounds as a source of energy.
The principle of nitrogen fixation has been applied in the manufacture of synthetic fertilizer, which is used worldwide. Artificial fertilizer production is now the largest source of human-produced fixed nitrogen in the environment.
Ammonia is a required precursor to fertilizers, explosives and other products. The most common method is the Haber process. The Haber process requires high pressures (around 200 atm) and high temperatures (at least 400 °C), routine conditions for industrial catalysis.
This highly efficient process uses natural gas as a hydrogen source and air as a nitrogen source. However, the amount of energy involved is a concern and efforts are ongoing to reduce it to acceptable level or even replace the direct use of heat with a catalyst.
The rate of biological nitrogen fixation is complicated by edaphic, climatic and management factors. A legume-Rhizobium symbiosis might perform well in a loamy soil but not in a sandy soil, in the sub humid region but not in the Sahel, or under tillage but not in no-till plots. These factors affect the microsymbiont, the host-plant, or both.
Table: Well-studied Free-living Nitrogen-fixing Bacteria
| Species | Superfamily | Aerobic- AnaerobicAe-Ana | Characteristics |
| Clostridium pasteurianum | Firmibacteria(Low GC GRAM positive) | Ana | Isolated first (1893)• Acell free-extract. nitrogenase was first made (1962) |
| Klebsiella pneumoniae | Proteobacteria- gamma | Ana | Close to E.coli. Genetics was first studied |
| Klebsiella oxytoca | Proteobacteria- gamma | Ana | Isolated from rice root in Japan |
| Bacillus polymyxa | Firmibacteria (Low GC GRAM positive) | Ae | GRAM-positive bacteria |
| Azotobacter chroococcum | Proteobacteria- gamma | Ae | Azotobacter was isolated next.(1901). Widely used for research |
| Az. vinelandii | Proteobacteria- gamma | Ae | |
| Azospirilium brazilense | Proteobacteria- alfa | Ae | Isolated from rhizosphere of C4-plant. Widely studied as |
Plant Tissue Culture
Plant tissue culture is a method or technique to isolate parts of plants (protoplasm, cells, tissues, and organs) and grow them on artificial media in aseptic conditions in a controlled space so that parts of these plants can grow and develop into complete plants.
It is based on the theory of “totipotensi” (“total genetic potential”) propounded by Schleiden and Schwann in 1838. The theory states that ‘the plant cell as the smallest unit of a living organism can grow and thrive if kept in appropriate conditions’. The various tissue culture techniques now used widely in agriculture, disease control and bioremediation include but not limited to:
Mikropropagasi(micro propagation of plants)
This techniques is used to produce crops in large scale through mikropropagasi (micro propagation) or klonal (clone) propagation of various plants. Plant tissue in very small amounts can produce hundreds or thousands of plants continuously.
This technique has been used in industrial scale in various countries to commercially produce various types of plants such as ornamental plants (orchids, cut flowers, etc.). Fruit crops (bananas), crops and forestry industries (coffee, tea, etc.).
By using tissue culture methods, millions of plants with the same genetic characteristics can be obtained only from one eye buds. Therefore, this method becomes an alternative in the vegetative propagation of plants.
Improved crop
In crop improvement efforts through the glorification of the conventional methods, to obtain pure strains can take six to seven generations of self-pollination or crosses.
Through tissue culture techniques, homozygous plants can be obtained in a short time by producing haploid plants through pollen culture, anther or ovaries followed by chromosome doubling. Homozygous plants can be used as plant breeding material in order to improve the nature of the plant.
Production of disease-free plants (virus)
Tissue culture technology has contributed to making plant free from viruses. In plants that have been infected with the virus, the cells in the bud tip (meristem) is believed to be in an area that is not infected with the virus. In this way the meristem can be used to obtain virus-free plants.
Genetic transformation
Tissue culture techniques have become an important part in helping the success of plant genetic engineering (gene transfer). For example, bacterial gene transfer (such as cry genes from Bacillus thuringiensis) into the plant cells will be expressed after transgenic cell achieved plant regeneration.
The production of secondary metabolites, compounds
Plant cell culture can also be used to produce biochemical compounds (secondary metabolites) such as alkaloids, terpenoids, phenyl etc. propanoid. This technology is now available in industrial scale.
A typical example is the commercial production of “shikonin” a major component of zicao (purple gromwell, the dried root of Lithospermum erythrorhizon), a Chinese herbal medicine.
Modern Biotechnology Methods
Modern biotechnology is a term adopted by international convention to refer to biotechnological techniques for the manipulation of genetic material and the fusion of cells beyond normal breeding barriers.
The most obvious example is genetic engineering to create genetically modified/engineered organisms (GMOs/GEOs) through “transgenic technology” involving the insertion or deletion of genes.
In recent times, however, modern biotechnology is understood to also include manipulation of whole organisms or other parts of organisms, such as molecules, cells, tissues and organs.
Recent developments in biotechnology include genetically modified plants and animals, cell therapies and nanotechnology. Some of these techniques are not yet in everyday use but holds great promise for the future.
Genetic Engineering
Genetic engineering is the deliberate alteration of the characteristics of an organism by manipulating its genetic material using the DNA (deoxyribonucleic acid) technology. This involves the introduction of foreign DNA or synthetic genes into the organism of interest (microbe, plant or animal).
The introduction of new DNA does not require the use of classical genetic methods (gene segregation and linkage), however, traditional breeding (reproductive) methods are typically used for the propagation of recombinant organisms.
Genetic engineering is used to increase plant and animal food production; to diagnose disease, improve medical treatment, and produce vaccines and other useful drugs; and to help dispose of industrial wastes.
There are several methods for carrying out genetic engineering but these can be divided into three major categories, namely Plasmid Method, Vector Method and Biolistic Method.
The Plasmid Method
The plasmid method of genetic engineering is the most common and earliest technique. It is generally used for altering microorganisms such as bacteria. In the plasmid method, a small ring of DNA called a plasmid (a small circle of DNA that replicates itself independently of chromosomal DNA, especially in the cells of bacteria) is placed in a container with special restriction enzymes that cut the DNA at a certain recognizable sequence.
The same enzyme is then used to treat the DNA sequence to be engineered into the bacteria; this procedure creates “sticky ends” that will fuse together if given the opportunity.
Next, the two separate cut-up DNA sequences are introduced into the same container, where the sticky ends allow them to fuse, thus forming a ring of DNA with additional content. New enzymes are added to help cement the new linkages, and the culture is then separated by molecular weight.
Those molecules that weigh the most have successfully incorporated the new DNA, and they are to be preserved. The next step involves adding the newly formed plasmids to a culture of live bacteria with known genomes, some of which will take up the free-floating plasmids and begin to express them.
In general, the DNA introduced into the plasmid will include not only instructions for making a protein, but also antibiotic-resistance genes. These resistance genes can then be used to separate the bacteria which have taken up the plasmid from those that have not. The scientist simply adds the appropriate antibiotic, and the survivors are virtually guaranteed (barring spontaneous mutations) to possess the new genes.
Next, the scientist allows the successfully altered bacteria to grow and reproduce. They can now be used in experiments or put to work in industry. Furthermore, the bacteria can be allowed to evolve on their own, with a “selection pressure” provided by the scientist for producing more protein. Because of the power of natural selection, the bacteria produced after many generations will outperform the best of the early generations.
The VectorMethod
The second method of genetic engineering is called the vector method. It is similar to the plasmid method, but its products are inserted directly into the genome via a viral vector.
The preliminary steps are almost exactly the same: cut the viral DNA and the DNA to be inserted with the same enzyme, combine the two DNA sequences, and separate those that fuse successfully.
The only major difference is that portions of the viral DNA, such as those that cause its virulence, must first be removed or the organism to be re-engineered would become ill. This does yield an advantage – removal of large portions of the viral genome allows additional “space” in which to insert new genes.
Once the new viral genomes have been created, they are allowed to synthesize protein coats and then reproduce. Then the viruses are released into the target organism or a specific cellular subset (for example, they may be released into a bacterium via a bacteriophage, or into human lung cells as is hoped can be done for cystic fibrosis patients).
The virus infects the target cells, inserting its genome – with the newly engineered portion – into the genome of the target cell, which then begins to express the new sequence. With vectors as well, marker genes such as genes for antibiotic resistance are often used, giving scientists the ability to test for successful uptake and expression of the new genes. Once again, the engineered organisms can then be used in experiments or in industry.
The Biolistic Method
The biolistic method, also known as the gene-gun method, is a technique that is most commonly used in engineering plants – for example, when trying to add pesticide resistance to a crop. In this technique, pellets of metal (usually tungsten) coated with the desirable DNA are fired at plant cells.
Those cells that take up the DNA (again, this is confirmed with a marker gene) are then allowed to grow into new plants, and may also be cloned to produce more genetically identical crop. Though this technique has less finesse than the others, it has proven quite effective in plant engineering.
Examples of Genetically-engineered organisms
Animals
Transgenic animals are used as experimental models to perform phenotypic and for testing in biomedical research. Genetically- modified (genetically-engineered) animals are becoming more vital to the discovery and development of cures and treatments for many serious diseases.
By altering the DNA or transferring DNA to an animal, we can develop certain proteins that may be used in medical treatment. Stable expressions of human proteins have been developed in many animals, including sheep, pigs, and rats.
Some examples are: Human-alpha-1- antitrypsin, which has been developed in sheep and is used in treating humans with this deficiency and transgenic pigs with human-histo- compatibility have been studied in the hopes that the organs will be suitable for transplant with less chances of rejection.
Transgenic livestock have been used as bioreactors since the 1990s. Many medicines, including insulin and many immunizations are developed in transgenic animals. In March 2011, the bioactive recombinant Human Lysozyme was expressed in the milk of cloned transgenic cattle.
This field is growing rapidly and new pharming (production of human protein in animal milk) uses are being discovered and developed. The extent that trangenic animals will be useful in the medical field as well as other fields is very promising based on results thus far. Examples include:
Enviro-Pig
Enviro-Pig is genetically engineered to be able to break down phosphorus. It contains edited DNA from a pig and genetic material from mice. Normally, pigs are unable to metabolise phosphorus for which reason they excrete phosphorus in their feces.
This faeces then acts as fertilizer for crops but when they eventually run-off into streams and rivers, they lead to increase algal blooms and destroys habitats for fish. The genetically-engineered Enviro-Pig is made to rectify this environmental problem.
Cows(with human genes)
More recently in 2011 Chinese scientist have been breeding cows genetically engineered with genes from human beings to produce milk that would be the same as human breast milk.
Goats(that produce silkin their milk?)
A company called Biosteel has genetically engineered goats to produce milk with strong spider web like silk proteins in their milk. These particles are used by the company to make bulletproof vests and anti- ballistic missile systems for military contracts.
Pigs (that glow in the dark!)
In 2006 in Taiwan scientists used genetic material from a jelly fish and implanted it into pig embyros. The result? Pigs that glow bright green in the dark!
During the daylight hours these pigs have a tinge of green on their skin, snout and teeth but as soon as night comes they are light very fat fireflies trotting around their pigpen. The pig’s whole body including its internal organs and heart glow green.
Apes(with human genes)
Japanese scientists have implanted human genes into marmosets and are currently using the monkeys to work on a cure for Huntington’s disease and strokes in humans.
Again is it good to be putting human genetics into animals? I’m not sure, as said earlier there has to be a line somewhere, but where? It should also be noted that for a very long time scientists have been replacing the genes in mice (known as knockout mice) to perform these types of tests for cancer, Parkinson’s and other such diseases.
Figure: Glowing Pig
Plants
Transgenic plants have genes inserted into them that are derived from another species. The inserted genes can come from species within the same kingdom (plant to plant) or between kingdoms (bacteria to plant).
In many cases the inserted DNA has to be modified slightly in order to correctly and efficiently express in the host organism.
Transgenic plants are used to express proteins like the cry toxins from Bacillus thuringiensis, herbicides resistant genes and antigens for vaccination.
Transgenic carrots have been used to produce the drug Taliglucerase which is used to treat Gaucher’s disease. In the laboratory, transgenic plants have been modified to increase their photosynthesis (currently about 2% at most plants to the theoretic potential of 9-10%.
This is possible by changing the rubisco enzyme (i.e. changing C3 plants into C4 plants), by placing the rubisco in a carboxysome, by adding CO2 pumps in the cell wall, by changing the leaf form/size. Still other transgenic plants have been modified to fixate ambient nitrogen in the plant. Other genetically engineered plants are listed below:
Transgenic maize containing a gene from the bacteria Bacillus thuringiensis canola corn, including popcorn and sweet corn but not blue corn, cotton, flax, papaya, potatoes (Atlantic, Russett Burbank, Russet Norkatah, and Shepody), red-hearted chicory (radicchio), soybeans, squash (yellow crookneck), sugar beet, tomatoes, including cherry tomatoes
Cell Therapy
Cell therapy (also known as cellular therapy, cellular suspensions, glandular therapy, fresh cell therapy, siccacell therapy, embryonic cell therapy, stem cell therapy and organotherapy) describes the process of introducing new cells into a tissue in order to treat a disease.
Cell therapies often focus on the treatment of hereditary diseases, with or without the addition of gene therapy. Cell therapy is a sub-type of regenerative therapy. There are two major types of cell therapy the autologous cell therapy and the allogeneic cell therapy.
In autologous cell therapy, cells are harvested from a patient, treated or expanded and re-introduced back into the same patient. This is patient-specific and the fear of immunological incompatibility is low, compared with the allogeneic method.
The allogeneic method involves the harvesting of cells from one, or a few, universal donors followed by large scale expansion and banking of multiple doses before introduction to the recipient patient.
To reduce the rate of immune response incompatibility, the allogeneic approach utilises cell types that do not elicit immune responses, for which reason it has the potential to treat hundreds of patients from a single manufactured lot of cells.
The allogeneic methodology is used in the pharmaceutical industry for drug manufacturing because the product can be readily available for “off the shelf” distribution. The mesenchymal stem cells are the main cells used for this procedure due to their plasticity, established isolation procedures, and capacity for ex vivo expansion.
Nanotechnology
Nanotechnology involves creating and manipulating organic and inorganic matter at the nanoscale.
The techniques for nanotechnology involve atom-by-atom construction of objects that have potential applications in medicine, electronics, information technology, environmental monitoring and remediation, military equipment and weapons, and so forth.
It assumes that since the atom (cell) is the building block of all materials and molecules, the world’s needs could be met by utilising a limitless supply of atoms or cells to manufacture valuable materials/molecules.
Though not principally a branch of biotechnology, they share principles and involve technology at the lowest level of matter. It is generally believed therefore that experiences gained in one area will help in the other.
Bio-remediation
Bio-remediation is a biological method for restoring contaminated and polluted lands to their original state. The various types of bioremediation are either in situ or ex situ. The in situ techniques include bio-sparging, bio-venting bio-augmentation and phyto- remediation, while the ex situ methods are the various types of composting, i.e. windrow, land farming and bio piling.
Bio-sparging is an in situ treatment technique using natural microorganisms, like yeast or fungi, to decompose hazardous soil substances. Some microorganisms can ingest dangerous chemicals without harm. In turn, those pollutants are transformed into less toxic or nontoxic substances, usually in the form of carbon dioxide and water.
To be successful, bio-sparging requires active and healthy microorganisms. This is encouraged via increased bacterial growth in the soil, which creates optimal living conditions.
After the contaminants are regulated, the microorganisms reduce in number because their food source is gone. Bio-sparging can occur under aerobic and anaerobic conditions.
Bio-venting is another in situ remediation technology that uses microorganisms to biodegrade organic constituents in the soil. Bio- venting enhances the activity of indigenous bacteria and simulates the natural in situ biodegradation of hydrocarbons by inducing air or oxygen flow and if necessary, by adding nutrients.
During bio-venting, oxygen may be supplied through direct air injection into residual contamination in soil. Bio-venting primarily assists in the degradation of adsorbed fuel residuals, but also assists in the degradation of volatile organic compounds (VOCs) as vapors move slowly through biologically active soil.
Bio-augumentation is the introduction of a group of exotic natural microbial strains or a genetically engineered variant to complement or bio-stimulate indigenous microorganisms in contaminated soil or water.
Bio-augumentation is necessary only when it is certified that indigenous microbes are incapable of degrading the contaminants at desired rate but susceptible to bio-stimulation.
This process increases the reactive enzyme concentration within the bioremediation system and subsequently may increase contaminant degradation rates over the non- augmented rates, at least initially after inoculation.
Bio-augmentation is typically only applicable to bioremediation of chlorinated ethenes, although there are emerging cultures with the potential to biodegrade other compounds.
It is remediation tool of choice for sites where soil and groundwater are contaminated with chlorinated ethenes, such as tetracholroethylene and trichloroethylene. These compounds are completely degraded to ethylene and chlorine which are non-toxic.
Phyto-remediation, also called green remediation, botano-remediation, agro-remediation, or vegetative remediation, can be defined as an in situ remediation strategy that uses vegetation and associated microbiota, soil amendments, and agronomic techniques to remove, contain, or render environmental contaminants harmless.
Most of the plants used in phyto- remediation are metal accumulators. The idea of applying phyto- remediation to decontaminate contaminated lands was first introduced in 1983 although the concept had been used for over 300 years on wastewater management.
Composting, a major ex situ method, is a process during which biodegradable organic materials are degraded (or eaten) by microorganisms, resulting in the production of simpler organic and/or inorganic by-products (compost) and energy in the form of heat. Composting is classified further on the basis of the arrangement of the composting materials.
In windrow composting the organic matter or biodegradable waste, such as animal manure and crop residues is piled in long rows (windrows). This method is used to produce large volumes of compost.
These rows are generally turned to improve porosity and oxygen content, mix in or remove moisture, and redistribute cooler and hotter portions of the pile. The rate of composting in windrows depends on the initial ratios of carbon and nitrogen, the amount of bulking agent added to assure air porosity, the pile size, moisture content, and turning frequency.
Land farming is a bioremediation technique that is performed in the upper soil zone or in excavated stockpiled cells. Contaminated soils, sediments and sludges can be spread on the ground and periodically tilled to aerate and encourage bacterial growth.
Nutrients, minerals, and/or moisture may be added to speed degradation. Sometimes bacteria which have been selected for their success in breaking down hydrocarbons are added.
Contaminants are degraded, transformed, and immobilized by microbiological processes and by oxidation. Contaminated material is usually treated in lifts that are up to 50 cm thick. When the desired level of treatment is achieved, the lift is removed and a new lift is constructed.
It may be desirable to only remove the top of the remediated lift, and then construct the new lift by adding more contaminated media to the remaining material and mixing. This serves to inoculate the freshly added material with an actively degrading microbial culture, and can reduce treatment times.
In the bio-pile composting, also known as bio-cells, bio-heaps, bio- mounds, and compost piles organic is an important method for treating petroleum constituents in excavated soils.
This technology involves heaping contaminated soils into piles (or “cells”) and stimulating aerobic microbial activity within the soils through the aeration and/or addition of minerals, nutrients, and moisture.
The enhanced microbial activity results in degradation of adsorbed petroleum-product constituents through microbial respiration. Bio-piles are similar to land farms in that they are both above-ground, engineered systems that use oxygen, generally from air, to stimulate the growth and reproduction of aerobic bacteria which, in turn, degrade the petroleum constituents adsorbed to soil.
However, while land farms are aerated by tilling or plowing, bio-piles are aerated most often by forcing air to move by injection or extraction through slotted or perforated piping placed throughout the pile.
Bio-piles, like land farms, have been proven effective in reducing concentrations of nearly all the constituents of petroleum products typically found at underground storage tank (UST) sites.
Lighter (more volatile) petroleum products (e.g., gasoline) tend to be removed by evaporation during aeration processes (i.e., air injection, air extraction, or pile turning) and, to a lesser extent, degraded by microbial respiration.
The mid-range hydrocarbon products (e.g. diesel fuel, kerosene) contain lower percentages of lighter (more volatile) constituents than gasoline. Biodegradation of these petroleum products is more significant than evaporation. Heavier (non-volatile) petroleum products (e.g., heating oil, lubricating oils) do not evaporate during bio- pile aeration; the dominant mechanism that breaks down these petroleum products is biodegradation.
However, higher molecular weight petroleum constituents such as those found in heating and lubricating oils, and, to a lesser extent, in diesel fuel and kerosene, require a longer period of time to degrade than do the constituents in gasoline.
Efficiency of these techniques depends largely on soil characteristics, constituent characteristics and climate conditions.
In conclusion, various methods used in biotechnology and in particular environmental biotechnology can be divided into two broad categories, traditional and modern.
The traditional methods comprise those methods that have been used since the ancient days and are generally based on the use of whole-organisms.
On the other hand, modern methods include those that use either parts of organisms or substances made from organisms.
Typical examples of traditional methods are fermentation, nitrogen fixation and tissue culture, while the modern methods are gene and molecular based methods, including genetic engineering, cell therapy and nanotechnology.
Genetic engineering, which involves the transfer of genes or DNA, involves the use of three well-established methods used on the vehicle used for the transfer.
The methods are plasmid that uses the plasmid (a DNA ring that replicates independent of chromosomal DNA) as vehicle; the vector method that uses another microorganism and the biolistic method that uses metals and other markers as vehicle.
In this article, you have been acquainted with the various methods used in biotechnology in general and of environmental biotechnology in particular. You have learnt that the various methods used in biotechnology and in particular environmental biotechnology can be divided into two broad categories, traditional and modern.
Read Also : Land Environment and Biological Environment (Biosphere) Structure of the Environment
The traditional methods include fermentation, biological nitrogen fixation and plant culture while modern methods involved the methods used to manipulate the genetic composition of organisms for the achievement of specific goals.
The most important of these methods is the genetic engineering, which uses specialized techniques to alter the genetic composition of an organism. The techniques involve principally gene transfer from one organism known as the donor to another known as the target.
In the plasmid method, the transfer is made via a special DNA ring known as the plasmid while in the vector method, another organism, often a microbe is used to make the transfer. In a third method known as bio-listic, a metal usually tungsten or other suitable markers is used to make the transfer. You also learnt that biotechnology methods are specialized methods that have been used to change the outlook, composition and function of microbes, plants and animals for the achievement of specific goals.
