Laboratory Methods in Environmental Science

Introduction

Laboratory methods in environmental science are used in the analyses of air, soil, and water samples recovered from a variety of environmental sites. The analyses can be geared toward the detection of living material such as bacteria in, for example, a water body that is suspected of being contaminated. Analyses can also detect nonliving material such as heavy metals, phosphate, nitrogen, antibiotics, and pharmaceuticals.

The variety of compounds that can be detected as part of environmental analyses are echoed by the laboratory methods that can be used. Some methods have existed for over a century. A well-known example is the use of nutrients incorporated into a semi-solid support to grow microorganisms that may be present in a sample. As well, techniques of molecular biology, which target regions of the genetic material of organisms, have been very useful in detecting biological material in environmental samples. Bacteria can now be detected and even identified to the species level without having to actually grow the bacteria. This enables the detection of organisms that may be difficult to grow under laboratory conditions.

Detection of organisms and compounds has become very sensitive, with detection in the parts per billion range (equivalent to a drop of water in an Olympic-sized swimming pool) routinely obtainable.

Laboratory methods enable the monitoring of the progress of an environmental clean up by detection of the disappearance of the pollutant, and they are also important in ensuring that environmental standards imposed on a particular industry and land use are being maintained. In the event that regulations are being breached, the laboratory data can be used in legal proceedings.

Historical Background and Scientific Foundations

Some laboratory methods used in environmental science have been in use for hundreds of years. The best example is the detection of bacteria in soil and water samples. Although more sophisticated genetically based detection techniques were developed beginning in the 1970s, a mainstay in the environmental science laboratory are growth-based techniques that utilize agar and agarose.

Agar and agarose solidify nutrients that would otherwise remain in solution. Both can liquefy when heated and become a semi-solid when they cool.

Agar is an uncharged network of strands of gelactose, which is extracted from two species of seaweed; Gelidium comeum and Gracilaria verrucosa.

Agarose is a purified version of agar that is comprised of repeating molecules of galactopyranose. The side groups that stick out from the galactopyranose backbone are arranged such that two adjacent chains can link together into a helix. This linkage is so compact that water can be trapped inside. Helices can cross-link with one another to form a three-dimensional network of water-containing agarose chains.

Solid media are prepared by heating up the agar or agarose and the nutrients to form a solution. The solution is then sterilized, typically in a steam-heat apparatus known as an autoclave. The sterile medium is then poured into one half of sterile plastic or glass growth chambers (Petri plates) and the lid is placed over the still hot solution to keep impurities from settling onto the surface. As the solution cools, the nutrient-containing agar or agarose becomes a semi-solid. When bacteria contact the surface of the medium, they are able to extract the nutrients from the medium. Repeated cycles of bacterial growth and division yields mounds of bacteria that can be seen with the unaided eye. Each mound (or colony) is produced from a single bacterium, there-

WORDS TO KNOW

BIOREMEDIATION: The use of living organisms to help repair damage such as that caused by oil spills.

DEGRADATION: The microbial breakdown of a complex compound into simpler compounds with the release of energy.

ENZYME: A protein that catalyzes a chemical reaction, usually by lowering the energy at which the reaction can occur, without itself being changed by the reaction.

GENOME: The total content of genetic material in organisms.

MICROCOSM: A miniature representation of a system that is used to model the system and study interactions.

SUPERFUND: Legislation that authorizes funds to clean up abandoned, contaminated sites.

fore counting the number of colonies can be used to accurately determine the number of living bacteria in a certain volume of the sample.

The history of agar and agarose goes back centuries, purportedly to mid-sixteenth century China. Soon after, a flourishing agar manufacturing industry was established in Japan and continued until the countries’s industrial structure was destroyed in World War II (1939–1945). In the United States, seaweed beds found along the southern California coast have made the San Diego area an important center of agar manufacture.

In 1882, German microbiologist Robert Koch (1843–1910) reported on the use of agar as a means for growing microorganisms. This discovery fueled the establishment of microbiology as a research discipline. Hundreds of different types of agar-based growth media have since been designed. Some are nonspecific and support the growth of a variety of bacteria. Other so-called selective media allow the growth of only a few or one type of bacteria.

Likewise, the use of agarose has proved tremendously useful in electrophoretic techniques. The agarose matrix can be constructed to have differently sized tunnels through the agarose strands, allowing the agarose to act as a sieve to separate molecules on the basis of the size. Since the agarose has no charge, an electrical current can be passed through it, which can drive the movement of charged samples such as DNA through a slab of agaros, with larger pieces of DNA moving more slowly than smaller pieces.

Some laboratory methods used to examine environmental samples are based on biochemical analysis. This approach enables the analysis of samples for compounds that are found in living organisms and/or for detection of chemical reactions that occur in living organisms. An example is an assay known as biochemical oxygen demand, which measures the use of oxygen as an indication of the presence and the approximate quantity of life in water samples.

To detect a target biomolecule, the compound must be obtained in pure form from the thousands of other compounds that may be present in the sample. Once isolated, the target compound can be studied, for example, to determine its concentration in the environmental sample and to indicate if it poses a health threat. Assays can be qualitative (i.e., is a compound toxic?) or quantitative (i.e., how toxic is the compound?).

Some of the laboratory methods that examine bio-molecules such as amino acids, proteins, and carbohydrates have existed since the nineteenth century. Other analyses such as the determination of the sequences of genetic material and protein are more recent, having been established beginning in the 1970s.

Most biomolecules occur in very small amounts in the cell, and their detection and analyses ranges from precipitation, centrifugation, and gel electrophoresis to sophisticated techniques of chromatography.

Centrifugation involves the rapid spinning of a suspension or solution. The force generated forces molecules to separate. Depending on the centrifugation speed and composition of the sample, molecules as small as deoxyribonucleic acid (DNA; the genetic material of most organisms) can be obtained. Electrophoresis techniques exploit the size and charge of biomolecules to separate various molecules based on their differing movements toward positively (anode) or negatively (cathode) charged poles of an electric field.

Chromatography consists of allowing a liquid or gas sample to flow through a tube or column packed with a solid material that may be coated with molecules that are recognized by regions of the surface of the target compound. The different components of the mixture separate because they travel through the tube at different rates, depending on the interactions with the stationary material and on the size of the openings between the packed material of the column. If the stationary material is charged, the chromatography column will allow separation according to charge, which is termed ion exchange chromatography. The biological activity of biomolecules has been exploited to design a form of chromatography known as affinity chromatography. This simply means that the system is tailored so that the column material has affinity for the target molecules in the sample. As examples, a target enzyme will recognize a particular compound that it can act on, and hormones will recognize and bind to their receptors. The specific interactions will retain the target compounds in the column, while other compounds will pass through the column.

In both biochemical and molecular biology analyses, sophisticated equipment and software and the use of automated systems that allow thousands of samples to be screened in a day is making it possible to analyze environmental samples in a shorter time, with higher accuracy, and less expensively than was possible when analyses were done individually and by a person.

Increase in public environmental awareness and the need to better control and reduce pollution has spurred research to discover or genetically tailor organisms to use toxic compounds as a nutrient source. The subsequent breakdown (degradation) of the compounds can generate by products that are less harmful. In the laboratory, test-tube experiments can be done to see if environmental samples are capable of causing degradation of a certain pollutant. If successful, the actual organism can be isolated. Such laboratory studies have led to the discovery of bacteria that are capable of degrading pollutants such as heavy metals and toxic chemicals in soil and water, explosive residues, or spilled petroleum. Crude oil is naturally biodegradable, however, in nature the degradation can take years or decades. Laboratory studies continue to search for ways to genetically modify these natural organisms to speed up the degradation process, which would be useful in dealing with oil spills.

In a similar strategy, transgenic plants (plants that are engineered to contain and express non-plant genes, particularly genes from bacteria that code for the production of degradative proteins) are being explored for the removal of heavy metals and explosive compounds from soils.

Molecular techniques of environmental analyses generate huge amounts of data. Making sense of the information is a challenge. Bioinformatics (also called computational biology) was established beginning in the 1980s in response to the need to better detect target regions of genetic material and improve the storage of the information. Finding genes and determining the structure and function of the proteins produced are part of bioinformatics.

Bioinformatics utilizes statistical techniques and computer programs to search through existing databases of DNA or protein sequences to find matches for the environmental samples being analyzed. Obtaining information is a multi-step process. Databases are examined, or browsed, to see if there is a match in DNA or protein sequences to the sequence that is being compared. If a match is obtained, it can provide a valuable clue as to the nature of the target compound and even its function. Examples of databases are the European Molecular Biology DNA Sequence Database (EMBL), GenBank, SwissProt, and the Protein Identification Resource (PIR).

In this way, compounds and even living organisms can be identified in a sample without the need to culture the samples to grow the organisms or to use other tech-

niques to isolate the compounds. As an example, a 2008 initiative of various branches of the U.S. government, including the National Institutes of Health, has begun to explore the use of bioinformatics to identify potentially harmful compounds in air, water, and soil samples.

The area of bioinformatics concerned with protein sequences and the function of proteins is known as proteomics. Using proteomics, three-dimensional structures of the protein molecules can be generated using computer graphics and by comparison with similar proteins obtained as crystals.

Laboratory examination of environmental samples for genetic material has benefited from a technique that uses DNA chip technology. A gene chip is waferlike in appearance, and resembles a microtransistor chip. However, instead of transistors, a DNA chip contains an orderly and densely packed array of single strands of DNA. The array is made by spotting DNA samples on the surface of the chip in a predetermined pattern, either by hand or using robotic automation. The latter can produce very small spots and so has been called a microarray.

Since DNA is normally double-stranded, this array of bits of DNA can bind the complementary strands of genetic material. Thus, hundreds of different bits of genetic material can be detected using a single chip.

Typically, the pieces of DNA attached to the chip are labeled with a fluorescent dye—a compound that fluoresces a certain color when illuminated with light of a certain wavelength. The DNA in the environmental sample is separated into its two single strands by gentle heating. Another fluorescent dye that fluoresces a different color under the same wavelength of light is added to the sample. When the sample is added to the chip, the binding of sample DNA to the DNA tethered on the chip is evident by the appearance of both colors at the spot where binding has occurred.

Since the sequence of the tethered pieces of DNA and their pattern on the chip are known, the binding pattern of the sample DNA reveals the sequence of the sample pieces.

Vast amounts of information are obtained from a single experiment. Up to 260,000 genes can be probed on a single chip.

Impacts and Issues

Advancements in laboratory analyses have made detection of a variety of compounds in a variety of samples extremely sensitive. As a result, laboratory methods are a crucial part of environmental science.

Detection of various pollutants is crucial in determining their origins and so in correcting the problems. For example, if bioremediation of gasoline-polluted soil is performed, analyses of the sample aids in monitoring the success of the degradation of the pollutant.

The use of laboratory-generated genetically engineered bacteria is being done more often in controlled conditions when the polluted soil has been removed and taken to another location for treatment. As of 2008, bioremediation has been used to help clean tens of thousands of sites involving contaminated soil and water around the globe. In the United States these include the sites whose cleanup has been done under a program popularly known as Superfund. In the Superfund program, heavily contaminated sites in the United States that have long since been abandoned, or whose clean up costs cannot be assumed by those identified as being responsible for the contamination, are cleaned up with funding provided by the Environmental Protection Agency (EPA). Since the program’s inception in 1980, almost 1,300 sites have been designated for remediation.

Laboratory research is always underway to identify bacteria that have the genetic capability to be exploited in bioremediation. The research focuses on bacteria that already live in the presence of pollutants, since they must have developed ways of coping with the normally toxic levels of the particular pollutant.

Research is also being done to use genetically engineered plants to remove cadium and other heavy metals from contaminated soil. This approach is known as phytoremediation. Experiments conducted at the U.S. Agricultural Research Service have shown, for example, that the alpine pennycress is able to accumulate high levels of cadmium in its leaves. Subsequently, the plants can be harvested and a new crop planted to absorb even more cadmium from the soil.

Such laboratory studies are very promising in the remediation of polluted soils. Furthermore, at a cost of $250—$1,000 per acre, the approach is very economical compared to the cost of conventional clean up, which can reach $1 million per acre.

Laboratory analyses are also an important facet of waste management. For example, analyses of runoff from landfills can detect leakage of potentially toxic compounds. The sensitivity of the detection techniques can enable a leakage to be detected before it begins to substantially affect the environment surrounding the landfill, including wildlife and people.

As well, laboratory analyses of environmental samples help determine if an industry or other land user is complying with emission rules that have been established for the operation or the particular land use.

See Also Biodegradation; Biofuels; Bioremediation; Clean Energy; Environmental Assessments; Hybrid Vehicles; Mathematical Modeling and Simulation; Real-Time Monitoring and Reporting; Surveying; Wastewater Treatment Technologies

BIBLIOGRAPHY

Books

Atlas, Ronald M., and Jim Philip. Bioremediation: Applied Microbial Solutions for Real-World Environment Cleanup. Washington, DC: ASM Press, 2005.

Livingstone, James. Agriculture and Soil Pollution: New Research. Hauppauge: Nova Science Publishers, 2006.

Wang, Zhendi, and Scott Stout. Oil Spill Environmental Forensics: Fingerprinting and Source Identification. New York: Academic, 2006.

Brian D. Hoyle

IN CONTEXT: BASIC RESEARCH HELPS EXPLAIN ENVIRONMENTAL PROCESSES, PROBLEMS, AND SOLUTIONS

The 2007 Nobel Prize in chemistry went to German chemist Gerhard Ertl (1936–) for his groundbreaking studies in the fundamental chemical processes that take place when gas phase molecules collide with the surface of a solid. Such surface chemistry studies have broad application in industry and the environment. Applying techniques developed during early semiconductor studies, Ertl used ultrahigh-vacuum environments to record the behavior of gas molecules on extremely pure surfaces of metals. Ertl’s work provided insights into the mechanism of the Haber-Bosh process, an important industrial process in which nitrogen and hydrogen are converted to ammonia using an iron surface as a catalyst. The ammonia produced is largely used for agricultural applications. Surface chemistry explains a range of reactions including the process used to clean exhaust emissions from modern automobiles (a chemical reaction in which carbon monoxide is converted to carbon dioxide on a platinum surface), the destruction of atmospheric ozone on the surfaces of ice crystals in the upper atmosphere, and is critical to the development of fuel cells that will use environmentally friendly hydrogen instead of petroleum-based fossil fuels.