Appendix D: Microbiology FAQs

Microorganisms are organisms that are too small to be seen with the naked eye. Most microorganisms are smaller than 0.2 mm in length and may be no more than 1 or 2 micrometers (µm) or even smaller. Living microorganisms include bacteria and archaea, fungi, and some protozoa. Viruses are the smallest microorganisms but are not living organisms. Only some microorganisms (bacteria, archaea, and fungi) contribute significantly to processes that remove contaminants from the environment. These FAQs focus primarily on bacteria and only mention other microorganisms as relevant.

Bacteria are single-celled organisms that come in many shapes and sizes. The structure of a typical bacterium is shown in Figure D-1. The outermost layer of the bacterium is the capsule. This layer protects the bacterium from the environment. Inside the capsule is the cell wall, which maintains the shape and structural integrity of the microorganism. Inside the cell wall, a cell membrane acts as a selective barrier between the outside aqueous environment and the inside of the cell. The cell membrane surrounds the gel-like cytoplasm, where most of the biochemical reactions occur within the bacterium. The cytoplasm also contains many small organic and inorganic chemicals. Inside the cytoplasm are two key structures: the chromosome and the ribosomeA multi-component biological molecule which is part of the protein-synthesizing machinery of the cell.. Some bacteria also have proteinLarge organic compounds made of amino acids arranged in a linear chain and joined together by peptide bonds (US Navy 2009).-based appendages (called flagella or pili), which help bacteria move in their environment.

Figure D-1. The structure of a typical bacterium.

Like all living organisms, bacteria are made of four major classes of biomoleculesClasses of compounds produced by or inherent to living cells including phospholipids, nucleic acids (e.g., DNA, RNA), and proteins.: lipidsA diverse range of organic compounds that are defined as being insoluble in water but soluble in non-aqueous solvents. Lipids include oils, waxes, and sterols., polysaccharides, proteins, and nucleic acids. All of these major biomolecules consist of six key elements: carbon (C), hydrogen (H), oxygen (O), nitrogen (N), phosphorus (P), and sulfur (S). These elements are collectively referred to as CHONPS.

Lipids are a large and diverse group of organic compounds that are insoluble in water but soluble in nonpolar solvents such as chloroform. In bacteria the most important lipids are called phospholipids. Phospholipids consist of glycerol backbone with two attached fatty acid chains and a phosphate group linked to an organic molecule such as choline. Phospholipids are important because they align themselves with their hydrophobic (water-hating) fatty acid tails away from the water, and their hydrophilic (water-loving) phosphate heads towards the water. This alignment, as shown in Figure D-2, results in the formation of a membrane. The cells of all living organisms have a membrane that acts as a semi-permeable barrier that isolates and protects the processes that occur within the cell from the outside environment.

Figure D-2. Phospholipid.

The analysis of the fatty acid components of phospholipids (phospolipid fatty acids, PLFAs) can aid in understanding the microbial processes occurring at a contaminated site. For example, different microorganisms produce specific types of phospholipids. The variety and relative abundance of PLFAs can identify the types of microorganism present in a sample (see Section 5). The PLFAPhospholipid fatty acids derived from the two hydrocarbon tails of phospholipids. composition of bacteria can also change in response to environmental factors. These changes can determine whether bacteria in a sample are under stress. The analysis of PLFAs can also identify groups of microorganisms that are metabolically active in an environment. See Section 7).

Sugars, also known as carbohydrates, are simple molecules composed of carbon, oxygen, and hydrogen. Sugars either exist as single molecules (monosaccharides) or as polymers (polysaccharides). Monosaccharides are often used by bacteria as energy sources and are components of other important biomolecules. Polysaccharides are components of the bacterial cell wall and the capsule. Capsules help bacteria attach to surfaces, form biofilms and provide protection from adverse environmental conditions. The sugars present in microorganisms typically do not provide useful information about the types or numbers of contaminant-degrading bacteria in an environment. None of the current EMDs described in this document characterize sugars derived from bacteria and other microorganisms. 

Proteins are polymers consisting of linear chains of amino acids. These chains can fold over on themselves to form complex molecular structures. Depending on their three-dimensional structure and amino acid sequence, proteins can serve a wide variety of biological functions. In simple organisms like bacteria, many proteins serve as catalysts and are responsible for the biochemical reactions that are required for a bacterium to live, grow, and reproduce. Despite the importance of proteins in bacterial processes and contaminant-degrading activities, the diversity of these biomolecules currently prevents standardized methods for their extraction, analysis, and identification. None of the current EMDs described in this document characterizes microbial proteins at the molecular (amino acid sequence) level. Figure D-3 illustrates protein structure.

Figure D-3. Example of a protein.

Source: Myoglobin 3D structure. Aza Toth at en.wikipedia.org. 05:39, 27 February 2008. http://en.wikipedia.org/wiki/File:Myoglobin.png 

Enzymes are specialized proteins that catalyze biochemical reactions. Catalysts increase the rate of a chemical reaction by lowering the activation energy (the energy required to initiate the reaction - see Figure D-4). As a catalyst, enzymesAny of numerous proteins or conjugated proteins produced by living organisms and facilitating biochemical reactions (based on USEPA 2004a). are not destroyed in this process and can facilitate the same reaction many times. Enzyme-catalyzed reactions convert one or more starting compounds (substrates) into one or more products. Enzymes are often specific for the type of reaction that they catalyze and typically only catalyze reactions with a limited range of substrates. One type of EMD, enzyme activity probes (EAPs)Transformation of surrogate compounds (probes) resembling contaminants produces a fluorescent (or other distinct) signal in cells which is then detected using a microscope. which are described in Section 8, detects the presence of specific enzymes in bacteria by using alternative substrates for enzymes that normally catalyze key reactions involved in contaminant biodegradationA process by which microorganisms transform or alter (through metabolic or enzymatic action) the structure of chemicals introduced into the environment (USEPA 2011)..

Figure D-4. Schematic of an enzyme lowering the activation energy of a reaction.

Source: Fvasconcellos at en.wikipedia.org. 28 May 2008. http://en.wikipedia.org/wiki/File:Carbonic_anhydrase_reaction_in_tissue.svg

 

Nucleic acids occur in all living organisms in two forms known as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA is the key information-containing molecule in all living cells. The information in DNA enables a bacterium to produce all of the proteins needed for the organism to live and grow.

Nucleic acids are polymers of nucleotides. Nucleotides consist of a sugar, a phosphate group, and one of several nitrogen-containing, ring-shaped components called bases. Nucleotide acids are joined together in a linear chain by bonds formed between the sugar of one nucleotide and the phosphate group of another. This bonding produces a sugar-phosphate backbone common to all nucleic acids. The information in DNA and RNA is present in the bases attached to this backbone. In DNA these bases are either adenine (A), guanine (G), cytosine (C), or thymine (T). In RNA the bases are the same except thymine (T) is always replaced by uracil (U).

DNA and RNA have small but important differences. In RNA, the sugar in the nucleotide monomers is a called ribose. In DNA this sugar is missing an oxygen atom and is therefore called deoxyribose. RNA is also usually a single-stranded molecule, whereas DNA is usually a double-stranded molecule consisting of two anti-parallel strands bound together through bonding (base pairing) between the bases on each strand (A:T and G:C). Consequently, the sequence of bases on one strand of DNA has the complementary sequence of bases running in the opposite direction. For example, if the sequence of bases in one strand of DNA is ATCG, the complementary sequence on the opposite strand would be TAGC. Figure D-5 illustrates this relationship.

Figure D-5. Nucleic acids, DNA, and RNA.

The chromosome is the organized structure of DNA found in a bacterial cell. In bacteria, often only one circular chromosome exists and may consist of as little as 600,000 bases pairs (600 kilobase pairs [kbp]) of DNA to over 9,000,000 base pairs (9 megabase pairs [Mbp]). Also in bacteria, smaller amounts of non-essential DNA are often present in the cytoplasm, which are separate from the chromosome. These smaller pieces of DNA are called plasmids and may range in size from ~10 kbp to almost 2 Mbp.

The DNA in chromosomes and plasmids is organized into smaller functional sections called genes. Individual genes typically contain all the information required for a cell to make a single specific protein. The information in genes is encoded in the sequence of bases (A, T, C, and G) in the DNA. A typical bacterium such as Escherichia coli (E. coli) has approximately 4,500 protein-encoding genes in its approximately 4.5 Mbp chromosome, suggesting that the average geneA segment of DNA containing the code for a protein, transfer RNA, or ribosomal RNA molecule (based on Madigan et al. 2010). is approximately 1kbp in size. The totality of genetic information of a bacterium (chromosome plus plasmids) is called the genome of that bacterium.

Many of the EMDs discussed in this document focus on detecting and quantifying genes. Some genes are particularly important because they encode the enzymes that catalyze the reactions involved in contaminant biodegradation. The presence of a specific bacterial gene in a sample can often indicate that microorganisms in that sample can catalyze a specific reaction. Furthermore, individual bacteria typically only have a limited number of copies of specific genes in their genomes. Measurement of the number of copies of a specific gene in an environmental sample can therefore indicate the abundance of organisms with a specific activityRefers to when a microorganism performs a specific function (e.g., sulfate reduction, metabolism of benzene).

Ribosomes are responsible for producing the proteins a bacterium needs. Ribosomes consist of both protein and RNA. Bacterial ribosomes use three major forms of RNA to make protein: messenger (mRNA), ribosomal (rRNA), and transfer (tRNA). mRNA contains the information the ribosome uses to determine which protein to make. This form of RNA is generated when a gene in the chromosome is “read” (transcribed), by an enzyme called RNA polymerase. The transcriptionThe first step in activation of a biochemical pathway where a complementary RNA copy is synthesized from a DNA sequence. process uses the sequence of DNA bases in a gene as a template to make a copy in the form of mRNA. The sequence of bases in the mRNA is then used to by the ribosome to join the correct sequence of amino acids together as a linear chain. The process of converting the information from the mRNA into protein is called translationThe second step of gene expression where messenger RNA (mRNA) produced by transcription is decoded by the cell to produce an active protein. and is shown in Figure D-6.

 

Figure D-6. Transcription and translation.

Source: Dhorspool at en.wikipedia.org. 28 November 2008. http://en.wikipedia.org/wiki/File:Central_Dogma_of_Molecular_Biochemistry_with_Enzymes.jpg  

Unlike most organisms, in which growth indicates an increase in size and mass, most bacteria grow by dividing one cell into two. This process is called binary fission. The two cells generated by binary fission are identical to the original cell, contain the same biomolecules, and have identical genetic information.

All microorganisms need five things to grow: an electron donorA chemical compound that donates electrons to another compound (based on USEPA 2011)., an electron acceptorA chemical compound that accepts electrons transferred to it from another compound (based on USEPA 2011)., a carbon source, other nutrients (such as HNOPS and trace metals), and water. Bacteria obtain all of these materials from the outside the cell. 

Electron donors are energy sources for bacteria. Energy is extracted from electron donors by removing electrons from the compound and transferring them to electron acceptors. Many of the bacteria relevant to understanding contaminant biodegradation in soils, sediments, and groundwater use organic compounds as electron donors. Because organic chemicals contain carbon, they can act as both the electron donor and the carbon source for bacteria. Bacteria that use organic compounds as both their electron donor and carbon source are called chemoheterotrophsBacteria that use organic compounds as both their electron donor and carbon source.. In contrast some bacteria, called lithoautotrophsBacteria that grow only using inorganic chemicals such as ammonia, iron, or hydrogen as their electron donor., grow only using inorganic chemicals such as ammonia, iron, or hydrogen as their electron donor. Since there is no carbon in these electron donors, these organisms must use use carbon dioxide (CO₂) as their carbon source.

Once the energy has been extracted from an electron donor by a bacterium, the electrons are finally transferred to another chemical called the terminal electron acceptor. By definition, aerobic organisms use oxygen (O₂) as their terminal electron acceptor. Conversely, anaerobic microorganisms use chemicals other than oxygen. Common terminal electron acceptorsCompounds used by microorganisms to support respiration. In aerobic organisms the terminal electron acceptor is oxygen (O₂). Anaerobic organisms use compounds other than O₂. These include common naturally–occurring compounds such as nitrate (NO₃⁻) or sulfate (SO₄²⁻) or anthropogenic contaminants such as chlorinated ethenes (e.g. perchloroethylene). Atoms from electron acceptors are typically not incorporated into biomolecules made by organisms that reduce these compounds during respiration. that anaerobic bacteria use include, but are not limited to, nitrate (NO₃^-), iron (Fe³⁺), manganese (Mn⁴⁺), and sulfate (SO₄^2-). Some bacteria and archaea can also use carbon dioxide (CO₂) as an electron acceptor.

The process of removing electrons from an electron donorA chemical compound that donates electrons to another compound (based on USEPA 2011). is called oxidation, while the process of adding electrons to an electron acceptorA chemical compound that accepts electrons transferred to it from another compound (based on USEPA 2011). is called reduction. Oxidation and reduction processes are often coupled and are called redox reactions. Bacteria conserve the energy released during redox reactions to generate a biochemically useful form of energy called adenosine triphosphate (ATP). The biochemical process of oxidizing an electron donor, generating ATP, and reducing an electron acceptor is called cellular respiration. Collectively, bacteria and other microorganisms can use a wide variety of chemicals as electron acceptors. Some of the most important of these processes in the environment are listed in Table D-1.

Table D-1. Major forms of electron donors and electron acceptors used by microorganisms. Adapted from Sullivan and Baross 2007.

Respiratory process	Electron donor	Electron acceptor	Product
Aerobic respiration	Various inorganic/organic compounds	oxygen (O2)	water (H2O)
Denitrification	Various inorganic/organic compounds	nitrate (NO3-) nitrite (NO2-) nitric oxide (NO) nitrous oxide (N2O)	nitrite (NO2-) nitric oxide (NO) nitrous oxide (N2O) nitrogen (N2)
Iron reduction	H2, organic compounds	ferric iron (Fe3+)	ferrous iron (Fe2+)
Sulfate reduction	H2, organic compounds	sulfate (SO42-)	hydrogen sulfide (H2S)
Acetogenesis	H2, organic compounds	carbon dioxide (CO2)	acetate
Methanogenesis   (ArchaeaMicroorganisms that are genetically distinct from bacteria. Methanogens are an example of archaea (www.biology-online.org, accessed online, 2013). only)	H2, organic compounds	carbon dioxide (CO2)	methane (CH4)
Fermentation	Organic compounds	No external acceptor; internal only	CO2, H2, acids, alcohols

 

All living cells use carbon obtained from their immediate environment to build the carbon-containing biomolecules (proteins, sugars, lipids, and nucleic acids) that are essential for growth. Carbon is the single most abundant element in biomolecules and represents approximately 50% of the dry weight of a bacterial cell. Some bacteria only use CO₂ as a carbon source while other bacteria can use thousands of different organic chemicals as carbon sources. In many instances, these carbon sources are contaminants found in the environment.

The four major biomolecules (proteins, sugars, lipids, nucleic acids) that make up all living organisms consist mainly of six major elements (CHNOPS). Carbon is the most common element found in biomolecules, while other major elements are required in lesser amounts. Nutrients like nitrogen (N), phosphorus (P), and sulfur (S) can often be obtained from minerals present in the soil and groundwater immediately surrounding the bacterium. The same is true for trace nutrients such as molybdenum (Mo), cobalt (Co), and iron (Fe).

Microbial metabolism includes all of the biochemical reactions that enable microorganisms to biochemically break down (catabolize) chemicals and to concurrently use the products of these reactions to make (anabolize) new lipids, proteins, polysaccharides, and nucleic acids required for growth. Bacteria and other microorganisms, including archaea and fungi, can grow on an enormous number of organic and inorganic chemicals. These chemicals include many compounds considered to be environmental contaminants. Understanding the microbial metabolism of contaminants helps environmental site managers to better understand the processes involved in natural and enhanced biodegradation processes and to improve the remediation of contaminated sites.

Many microorganisms can use oxygen as a terminal electron acceptor during the oxidation of organic compounds. During aerobic respiration, the organic electron donor is biochemically oxidized to carbon dioxide while oxygen (the electron acceptor) is reduced to water. Humans and other animals use this same respiratory process. Bacteria can use this respiratory process to biodegrade a wide range of contaminants such as petroleum hydrocarbons, methyl tertiary butyl ether (MTBE), and vinyl chloride (VC), among others. Some specialized bacteria can use oxygen as the terminal electron acceptor while oxidizing reduced inorganic compounds (such as Fe²⁺, H₂S, or NO₂^-) as electron donors. For example, iron-oxidizing bacteria such as Acidothiobacillus gain energy from oxidizing ferrous iron (Fe²⁺) to ferric iron (Fe³⁺) and by using oxygen as a terminal electron acceptor. This process is important in acid mine drainage because it affects the mobility of metals. Ammonia-oxidizing bacteria such as Nitrosomonas oxidize ammonia (NH₃) to nitrite (NO₂^-) using oxygen as a terminal electron acceptor. This process, called nitrification, is important in wastewater treatment processes to eliminate ammonia, which is toxic to fish and other organisms.

A wide range of anaerobic respiratory processes are important to bioremediationThe treatment of environmental contamination through the use of techniques that rely on biodegradation. Bioremediation has two essential components: biostimulation and bioaugmentation.. As with aerobic respiration, many forms of anaerobic respiration use organic contaminants as electron donors and oxidize these compounds to carbon dioxide. Although the biochemical steps involved are often different from those used by aerobic bacteria, petroleum hydrocarbons can often be degraded under anaerobic conditions by microorganisms that can use nitrate (NO₃^-) or sulfate (SO₄^2-) as terminal electron acceptors. Organisms that use nitrate as an electron acceptor generate compounds such as N₂O (nitrous oxide), NO (nitric oxide), and N₂ (nitrogen) and are called denitrifiers. Microorganisms that use and generate sulfate generated H₂S (hydrogen sulfide) are called sulfate-reducing bacteria (SRB).

Under anaerobic conditions hydrogen (H₂) is often generated during biodegradation processes. Hydrogen is also widely used as an electron donor by anaerobic bacteria. Some important microorganisms can also use H₂ as an electron donor. For example, acetate-generating (acetogenic) bacteria use H₂ as an electron donor and use energy from hydrogen oxidation to reduce CO₂ to acetate (CH₃COOH). Some microorganisms (methanogens) can also use H₂ oxidation to reduce CO₂ to methane (CH₄). This methane-generating activity is only found in archaea and not in bacteria.

Other bacteria can reduce inorganic chemicals under anaerobic conditions. For example, bacteria such as Geobacter use ferric iron (Fe³⁺) as an electron acceptor and reduce it to ferrous iron (Fe²⁺). Other bacteria can reduce metals such as manganese (Mn⁴⁺), arsenic (As⁵⁺), chromium (Cr⁶⁺), or uranium (Ur⁵⁺).

Dehalorespiration is another important type of anaerobic respiration for environmental contaminants. In this process, halogenated organic compounds are used as terminal electron acceptors. The halogenated compounds are sequentially reduced with the result that chlorine atoms are removed and replaced by hydrogen (reductive dehalogenation). An example of this process is reductive dechlorination of tetrachloroethylene (PCE) by Dhc. This organism uses H₂ as an electron donor and sequentially reduces PCE to trichloroethylene (TCE), to dichloroethene (DCE; both cis-, and trans- isomers of DCE, although cis- is much more commonly produced), to vinyl chloride (VC), and finally to ethene. Other examples of dehalorespiration include the reductive dechlorination of chlorobezenes, chlorinated bromines, and chlorinated phenols.

Microorganisms preferentially use electron acceptors that allow them to generate the most ATP during respiration.  If available, oxygen, which allows the maximal production of ATP, will be used first and the remaining electron acceptors will be used in a defined sequence based on their respective energy yields. This sequence is as follows:  O₂ > NO₃^- >Mn⁴⁺ > Fe³⁺ > SO₄^2- > CO₂. In a contaminated groundwater system containing readily biodegradable electron donors, the available electron acceptors will be rapidly consumed and the environment will tend to become methanogenic. Downgradient from the source area, the concentrations of dissolved electron donors will be lower and the available electron acceptors may not have been fully consumed. Under these circumstances, zones with different electron accepting processes will develop. These can be detected based on either depletion of the electron acceptor or detection of distinctive products of the prevailing electron accepting process (such as methane accumulation in methanogenic environments,or sulfide production in sulfate-reducing environments). Changes in subsurface redox potential and geochemistry (measured as either electron acceptors or metabolic byproducts) can indicate whether or not contaminant biodegradation is occurring.

No single type of bacterium can use all of the electron acceptors shown in Figure D-7, and many microorganisms only use one or two specific electron acceptors. Consequently, different electron acceptors tend to support the growth and activity of different bacteria with different metabolic capabilities.

 

 

Figure D-7. Predominant terminal electron accepting processes (TEAPS) within a dissolved contaminant plume as the plume migrates through the subsurface.

Source: AFCEE 2004. Naval Facilities Engineering Service Center (NFESC), and the Environmental Security Technology Certification Program (ESTCP). 2004. Principles and Practices of Enhanced Anaerobic Bioremediation of Chlorinated Solvents. Parsons Infrastructure & Technology Group, Inc., Denver, Colorado. August.

The term biodegradation is frequently used to describe the cellular metabolic processes that allow microorganisms to use a variety of organic compounds as carbon and energy sources for growth. In many cases, bacteria use the same organic compound (including some contaminants) as both their electron donor and carbon source. These organic compounds are broken down within the microbial cell via defined catabolic reactions. These sequential reactions are facilitated by various enzyme catalysts through a specific pathway.

In these pathways, the product of one enzyme-catalyzed reaction serves as the substrateAny substance that is acted upon by an enzyme. (reactant) for the next enzyme in the pathway throughout the process. The progressively smaller carbon-containing intermediates (metabolites) generated during catabolism have two eventual fates. Some metabolites are fully oxidized to terminal products and are excreted as waste products, such as CO₂. Other metabolites are used in biosynthetic (anabolic) pathways as the starting materials for the production of new biomolecules required for growth. Anabolic processes require large amounts of energy in the form of ATP. ATP is generated by redox reactions that ultimately lead to the reduction of the electron acceptor. The connection between catabolic and anabolic pathways, electron donors, electron acceptors, and energy (ATP) production is summarized in Figure D-8.

Figure D-8. Microbial metabolism and biodegradation.

Despite the structural diversity of organic compounds that microorganisms can metabolize, there are only six classes of enzymes that facilitate metabolic reactions. Certain classes of enzymes play a role in the biodegradation of certain classes of organic compounds. For example, many hydrocarbons are biodegraded by enzyme pathways that are initiated by oxygenaseAn enzyme that catalyzes the incorporation of molecular oxygen into a compound (based on Madigan et al. 2010). enzymes. These enzymes either introduce one or both atoms of oxygen (O) from molecular oxygen (O₂) into the hydrocarbon substrate. These enzymes are known as monooxygenases and dioxygenases, respectively. Table D-2 lists some examples of enzymes, the compounds they help degrade, and the gene that encodes the enzyme. Note the following in this table:

• The oxygenase enzymes are named after the compound that they help degrade. For example, benzene monooxygenase is the enzyme that initiates the pathway of benzene catabolism in organisms that can grow on benzene.
• Several hydrocarbons, such as toluene, can be oxidized by several structurally different oxygenases. Each of these enzymes introduces oxygen into a different position on the toluene molecule.
• Some hydrocarbons, such as straight chain n-alkanes, can be oxidized by more than one type of enzyme even though the products of these reactions are the same.
• Some contaminants, such as TCE, can be oxidized by enzymes that have other roles. For example, methane monooxygenase normally initiates the biodegradation of methane in aerobic methane-oxidizing bacteria (methanotrophs). Because the methane monooxygenase can react with compounds other than methane, methanotrophs can oxidize TCE even though they cannot grow on this contaminant. This lack of enzyme specificity underlies the process of cometabolism.

The enzymes described in Table D-2 are found in aerobic microorganisms and all require the presence of molecular oxygen. This requirement is an additional use for oxygen in aerobic organisms, beyond its use as a terminal electron acceptor.

Table D-2. Examples of oxygenase enzymes and some common contaminants.

Contaminant	Key enzyme	Relevant gene
benzene	benzene monoxygenase	bmo
toluene	toluene dioxygenase	tod
toluene	toluene-4-monoxygenase	tmo
xylenes	xylene monoxygenase	xyl
naphthalene	naphthalene dioxygenase	ndo
alkanes	alkane monoxygenase, alkane hydroxylase	alk
polychlorinated biphenyls	biphenyl dioxygenases	bph
vinyl chloride	alkene monooxygenase	etn
trichloroethylene	methane monooxygenase	mmo

In most cases, the enzymes listed in Table D-2 are also the first enzymes in pathways that allow bacteria to catabolize various contaminants. The full catabolic pathways for the contaminants listed in Table D-2 involve many other important enzymes. However, the initial reaction in a pathway is often the rate-limiting step and must facilitate reaction of otherwise unreactive compounds. For example, hydrocarbons are often unreactive compounds. However, they become relatively simple compounds to biodegrade once an oxygen atom has been introduced into a C-H bond to create an alcohol (for instance, in Figure D-9, a catechol compound is formed during aerobic respiration).

Figure D-9. Aerobic bacterial biodegradation of aromatic BTEX compounds.

Source: Adapted from the University of Minnesota Biocatalysis/Biodegradation Database (UM-BBD). Gao J, Ellis LBM, and Wackett LP (2010). “The University of Minnesota Biocatalysis/Biodegradation Database: Improving public access” Nucleic Acids Research 38: D488-D491. BTEX Metabolism Metapathway Map page author, Stephen Stephens. http://umbbd.ethz.ch/BTEX/BTEX_map.html  

Figure D-10 illustrates the steps involved in the biodegradation of toluene and the enzymes associated with each individual step. Mono- and Di-oxigenases are involved in the initial phase of toluene biodegradation leading to the opening of the aromatic ring.

 

 

Figure D-10. Toluene oxidation.

Source: University of Minnesota Biocatalysis/Biodegradation Database (UM-BBD). Gao J, Ellis LBM, and Wackett LP (2010). “The University of Minnesota Biocatalysis/Biodegradation Database: Improving public access” Nucleic Acids Research 38: D488-D491. Toluene Graphical Pathway Map (2) page author Dong Jun Oh. http://umbbd.ethz.ch/tol/tol_image_map2.html.

Figures D-11 and D-12 illustrate many of the key features of a typical biodegradation (catabolic) pathway. For example, the pathway is initiated by the activity of an oxygenase enzyme. The metabolites generated by the pathway are transformed into simpler compounds and the pathway ends with small simple metabolites that can easily be converted into carbon dioxide or used to start the synthesis of new biomolecules through anabolic pathways.

Biodegradation pathways in anaerobic microorganisms have similar characteristics to those described for aerobic pathways, except that molecular oxygen is not involved. The enzymes described in Table D-3 are found in anaerobic microorganisms and do not require the presence of molecular oxygen. For example, anaerobic degradation of aromatic compounds, such as toluene, is initiated by benzylsuccinate synthase in a similar manner that aerobic degradation is initiated by a toluene oxygenase (Figure D-11).

Table D-3. Examples of key enzymes involved in the anaerobic degradation of contaminants

Contaminant	Key enzyme	Relevant gene
toluene	benzylsuccinate synthase	bssA
perchloroethene	PCE reductase	pceA
trichloroethene	TCE reductase	tceA
vinyl chloride	VC-reductase	vcrA and bvcA

 

Figure D-11. Anaerobic bacterial biodegradation of aromatic BTEX compounds.

Source: Adapted from the University of Minnesota Biocatalysis/Biodegradation Database (UM-BBD). Gao J, Ellis LBM, and Wackett LP (2010). “The University of Minnesota Biocatalysis/Biodegradation Database: Improving public access” Nucleic Acids Research 38: D488-D491. BTEX Metabolism Metapathway Map page author, Stephen Stephens. http://umbbd.ethz.ch/BTEX/BTEX_map.html.

Unlike aerobic biodegradation, anaerobic biodegradation can also involve processes that use contaminants as electron acceptors. An example of this type of process is the pathway of PCE degradation to ethene by Dhc (Figure D-12). This organism uses a different reductase enzyme in each of the steps in this pathway.

Figure D-12. Sequential reductive dechlorination of PCE to ethene.

Source: AFCEE 2004. Naval Facilities Engineering Service Center (NFESC), and the Environmental Security Technology Certification Program (ESTCP). 2004. Principles and Practices of Enhanced Anaerobic Bioremediation of Chlorinated Solvents. Parsons Infrastructure & Technology Group, Inc., Denver, Colorado. August.

The ability of bacteria to biodegrade a specific contaminant is dictated by many factors including:

• environmental conditions, such as sufficient electron donor or acceptor, availability of water and other nutrients, temperature, and pH.
• characteristics of the contaminant, such as molecular structure, bioavailability, and toxicity
• genetic capability (do microorganism possesses the genes that encode the necessary enzymes required to degrade the contaminant?)
• nature of the contamination, such as concentration, and presence of co-contaminants

For example, even though bacteria may be present with the correct genes and metabolic capabilities required to degrade a specific contaminant, the contaminant may not be biodegraded due to the presence of a co-contaminant that inhibits biodegradation. 

Note that bacteria typically live as part of microbial communities that are typically characterized by a high degree of speciesThe lowest taxonomic rank, and the most basic unit or category of biological classification.(www.biology-online.org) interdependence. Metabolically similar microorganisms can be classified into groups called guilds (such as methanogens and SRB). The relationships between these guilds are important because the complete degradation of contaminants in the environment often involves the interactions of multiple guilds within a community and depends on syntrophic relationships. For example, the anaerobic biodegradation of BTEX compounds may involve microorganisms that initially degrade the compounds to intermediates, which then serve as substrates for additional groups of microorganisms.  Effective bioremediation approaches therefore need to account for not only the contaminant type, quantity, and bioavailability, but also the indigenous microbial communities at a site.

Molecular biology is the study of the essential molecules produced by living organisms such as those used in reproduction, energy generation, and cell structures. The main biomolecules relevant to biodegradation studies are nucleic acids, proteins, and lipids. Although obviously important to microorganisms themselves, polysaccharides are not particularly useful molecules to analyze for understanding biodegradation and the microorganisms involved in these processes.

Molecular biology overlaps with genetics (the study of the genes) and biochemistry (the study of the biomolecule structure, pathways, and metabolites). Molecular biology is a relatively young science (originating in the 1930s and 1940s), and did not become a distinct discipline until the 1960s, when scientists discovered the structure of DNA and how DNA sequences direct protein synthesis. For environmental scientists, the main uses of molecular biology are to identify or quantify contaminant-degrading microorganisms, determine the genetic capability of microorganisms, and describe microbial diversityMicrobial diversity can have many definitions but in this context generally refers to the number of different microbial species and their relative abundance in an environmental sample (Nannipieri et al. 2003). in the environment.

Until the introduction of molecular biology techniques, studies of microorganisms involved in biodegradation were often limited to determinations of the total numbers of microorganisms that could be grown or cultured under standard laboratory conditions. The shortcoming of this approach is that, despite many years of study, microbiologists can only grow a tiny fraction of the microorganisms present in the environment in the laboratory. Consequently, culture-dependent techniques such as heterotrophic plate counts drastically underestimate both the numbers of and types of microorganisms present in environmental samples. Most modern molecular biology techniques described in this document analyze biomolecules that are generated by microorganisms in the environment, and then use these indirect measurements to determine the abundance and activities of these microorganisms. These techniques typically do not require laboratory growth of microorganisms, therefore avoiding selective and inefficient culture-dependent processes.

Most molecular biology approaches used to characterize biodegradation processes analyze nucleic acids (DNA and RNA). One reason for analyzing nucleic acids is that nucleic acids are structurally homogeneous, (unlike proteins, which have different sizes and different chemical and physical properties). Although the individual nucleotide sequences in DNA molecules vary almost infinitely, these differences have limited effects on the techniques needed to extract, purify, and characterize the biomolecule from various sources. Human DNA behaves the same as plant DNA, bacterial DNA, or fungal DNA. Another reason to analyze nucleic acids is that, unlike the other biomolecules that are investigated in biodegradation studies (proteins and lipids), there are several powerful techniques that can be used to study DNA. Two of the most prominent techniques are the polymerase chain reactionMakes copies of a specific DNA sequence within a target gene of microorganisms that can be further analyzed. (Section 4) and automated DNA sequencing. These two key technical advances are described in the following two sections.

The polymerase chain reaction (PCR) is a routine molecular procedure that harnesses and directs the activity of DNA polymerase, a natural DNA-synthesizing enzyme. This enzyme “reads” the sequences of bases in a template DNA strand and can produce billions of identical copies of this sequence and its complementary DNA strand. The amplification is achieved through cycling a reaction mixture that contains template DNA and DNA polymerase through a carefully prescribed sequence of temperature changes and incubation conditions.

The PCR procedure has transformed the biological sciences and has many applications in environmental studies. PCR is particularly useful for generating large amounts of identical DNA, even if only small amounts of template DNA (or RNA) can be recovered from a sample. A remarkable feature of PCR is that it can specifically amplify one gene or DNA sequence—even when that target gene or sequence is present at extremely low concentrations in a DNA sample that contains many billions of other nontarget genes or DNA sequences.

DNA and RNA amplification specificity is made possible through the use of primersShort strands of DNA that are complementary to the beginning and end of the target gene and thus determine which DNA fragment is amplified during PCR or qPCR.. Primers are short pieces of DNA (~20 nucleotides) that are complementary to the beginning and the end of the section of DNA to be amplified by PCR. A typical PCR amplification reaction mixture contains billions of copies of these primers. During the amplification procedure, the primers bind to (anneal) to their complementary sequences and serve as initiation points for DNA polymerase to start synthesizing new DNA. Without primers PCR will not work; with poorly designed primers, a PCR amplification can generate large amounts of nontarget DNA. However with well-designed primers, PCR can selectively amplify genes of DNA sequences that are specific for individual species of microorganisms.

A typical PCR amplification requires the presence of the following materials:

• Template DNA: This DNA contains the nucleotide sequence of interest (target DNA). The template DNA can be any form of DNA extracted from a sample.
• A heat-stable DNA polymerase: DNA polymerase is an enzyme that identifies existing nucleotide sequences in single-stranded DNA and concurrently synthesizes a complementary strand. A heat-stable form of the enzyme is used because PCR employs repeated high temperature cycles as part of the amplification process.   
• Deoxyribonucleotides: DNA is a polymer of four deoxyribonucleotides building blocks (A, T, C, and G). These compounds are added to a PCR mixture as deoxyribonucleotide triphosphates (dNTPs) and are used by DNA polymerase to synthesize new DNA.
• Primers: Primers are short, single-strand DNA molecules that bind specifically to the target DNA sequence in template DNA. The primers are used to direct the DNA polymerase to the section of DNA that is to be amplified.

DNA amplification using PCR is conducted on a small scale (<50 µl) in sealed microtubes. These microtubes are incubated in an automated thermocycler that can very quickly and accurately change the reaction temperature of the PCR mixture within the microtubes. A typical PCR program consists of the following steps:

1. Denaturation: The reaction mixture is heated to ~95°C to melt any double-stranded DNA into a single-stranded form.
2. Annealing:  The reaction mixture is cooled to 50-65°C to allow the primers to anneal (bind) to the single-stranded DNA template. The annealing temperature is critical in determining the specificity of the DNA amplification and may vary depending on the specific primers used.
3. Extension/elongation: The reaction temperature is raised to about 75°C. During this step the DNA polymerase binds to a DNA-attached primer and synthesizes a new DNA strand that is complementary to the target DNA sequence in the template DNA.

After each cycle, the number of copies of the target sequence is doubled and continues to increase exponentially throughout the reaction time course (25-40 cycles). Figure D-13 describes this process.

Figure D-13. PCR Process steps.

Source: USEPA 2004.

The amplification of DNA using PCR has many applications in environmental diagnostics. In some cases, PCR can simply be used to detect the presence or absence of a particular target sequence or gene or it is used simply to generate sufficient DNA to conduct other types of molecular analyses. In other cases, the basic PCR process has been modified so that the numbers of target sequences in a template DNA sample can be accurately and quickly determined, called qPCR. More information related to qPCR and its applications can be found in Section 4.

The second key technology that underlies molecular biology is the determination of the linear order of nucleotides (bases) in DNA molecules (DNA sequencing). This sequence of nitrogen-containing bases (A, T, C, and G) determines which amino acids are incorporated into proteins. This result in turn defines the type of reaction that an enzyme can catalyze. Consequently, the nucleotide sequence of genes can reveal what type of contaminant-degrading activities might occur for a specific organism or microbial communityThe microorganisms present in a particular sample.. DNA sequencing technologies are rapidly changing. Recently, several high-throughput systems have been developed including pyrosequencing (such as 454 sequencing) and ion semiconductor sequencing (for example, Ion Torrent™ technology). PyrosequencingA common high throughput DNA sequencing approach that uses light-emitting enzyme couple systems to detect pyrophosphate released when one nucleotide is attached to another. This is a well-established DNA sequencing approach that regulators, consultants and others in environmental site management are likely to encounter. detects light generated from enzymes that use pyrophosphate released when a base is added to a growing DNA molecule while ion semiconductor sequencing detects protons (H+) released during the same process. Further advances in DNA sequencing are emerging and will continue to dramatically decrease the cost and concurrently increase the use of large scale DNA sequence analysis for characterizing microbial communities and determining the likely activities of member organisms.

Like all other living cells, the flow of “information” in a microorganism goes from genes (DNA) to mRNA to proteins. As technologies become available for the rapid amplification and sequencing of DNA, many molecular studies in the environmental arena focus on detecting specific DNA molecules. The detection of particular genes or DNA sequences is then used to predict or interpret the results of other more conventional analyses.

One limitation of PCR is that the primers required in this procedure simply define the start and end of the stretch of DNA to be amplified and do not provide any information about the sequence of nucleotides between these two points. Some common checks on PCR amplification products (amplicons) are to determine whether the product is the correct predicted size (number of base pairs) and whether it has the correct physical properties (for instance, melting temperature). However, the most thorough analyses typically sequence the PCR amplicons to determine their nucleotide sequence. 

Molecular biology studies also detect specific genes through the process of hybridization. Hybridization describes the non-covalent bonding that occurs between two strands of nucleic acids. The strength of this bonding is dictated by the degree to which the two strands are complementary. If two strands are highly complementary (for example, every T in one strand has a matching A in the other strand) the degree of hybridization will be strong. If the two strands are dissimilar, the degree of hybridization will be limited.  This intrinsic ability of nucleic acids to form stable hybrids enables the primers used in PCR to amplify specific genes. Hybridization is also exploited in several other EMDs, including fluorescence in situ hybridization (FISH)Detects the presence of targeted genetic material in an environmental sample and estimates the number of specific microorganisms or groups of microorganisms. and microarrays.

Two different types of genes are of interest in molecular studies of biodegradation processes: 16S ribosomomal RNA (rRNA) genes and functional genes (the genes that encode for enzymes involved in specific biodegradation processes).

The analysis of 16S rRNAA subunit of the ribosome composed of ribonucleic acid (RNA).  The RNA sequence is used to classify and identify microorganisms (e.g. genus and species). has emerged as an important focus in biodegradation studies, as well as in more general studies of microbial ecology. Ribosomes in bacteria are made up of two major components: the small (30S) and the large (50S) subunit. The small ribosome subunit contains several proteins as well as a single RNA molecule which is known as the 16S rRNA. The equivalent molecule in eukaryotic microorganisms such as fungi is called 18S rRNA. The 16S rRNA is a useful genetic target in bioremediation studies because the molecule is relatively easy to extract and purify from an environmental sample. This molecule also contains enough nucleotide sequences for microorganisms to be differentiated from each other. With the advent of PCR, microbiologists now focus on the gene that encodes this RNA, but the principle underlying the analysis remains the same.

The 16S rRNA serves as a molecular clock and undergoes changes in nucleotide sequence at a rate comparable to the rate at which bacteria evolve and differentiate with new capabilities. Consequently, analyses of changes in 16S rRNA nucleotide sequences can quantify how related one bacterium is to another. Bacteria typically only have one copy of this gene in their genome, and it is only transmitted when one cell divides into two—which simplifies this analysis.

In studies of biodegradation processes, analysis of 16S rRNA genes has many uses. In some cases, the number of copies of a specific 16S rRNA gene can be measured using qPCR. Conversely, an analysis of all of the 16S rRNA sequences present in a sample can be used to define which types of microorganisms are present and how the composition of a microbial community changes in response to a treatment or contaminant. The presence and number of organisms with a particular 16S rRNA sequence can also be determined by techniques such as FISH.

The central argument often made in analyses of 16S rRNA sequences is that a high degree of similarity between two 16S rRNA sequences (>97%) implies that the two microorganisms are closely related at the species level. It is often further assumed that a high degree of sequence similarity implies the two species have similar, if not identical, metabolic capabilities. However, there are a growing number of examples of organisms that have identical 16S rRNA genes sequences but have distinctly different metabolic capabilities. This realization has led to a progressive increase in interest in detecting and quantifying functional genes in environmental samples.

Functional genes encode enzymes involved in specific biochemical processes. Analysis of functional genes can therefore describe what biodegradation processes an individual microorganism or microbial community might be capable of, without providing any real evidence of which bacteria the gene came from. 

Enzymes are the actual biomolecules that catalyze biodegradation reactions. Even though a gene is detected or quantified in an environmental sample, the corresponding enzyme has not necessarily been produced by the microorganisms within the sample nor is this enzyme necessarily fully functional.

Some EMDs, such as enzyme activity probes (EAPs), detect specific enzyme activities in environmental samples. Enzyme analyses measure the potential for a given reaction (such as a key step in the degradation of a contaminant) in a given environment at a given time, so the results are useful to measure whether a reaction will occur and to evaluate the impacts of different management options on the potential for that reaction. EAPs can also be applied in the field to determine in situ rates of some biodegradation processes.

Phospholipid fatty acid (PLFA) analysis is useful for estimating the amount of microbial biomass in a sample that is metabolically active. Unlike DNA, microbial phospholipids rapidly decompose after microorganisms die. Therefore, PLFA analysis is an accurate quantification of live microorganisms in a sample. PLFA analyses can also be used to identify broad groups of metabolically active microorganisms as a fingerprinting technique for characterizing microbial community dynamics. Finally, some microbes modify specific PLFAs when stressed, so lipid analysis can provide some information on the health of the microbial community. The most recent development in PLFA analysis has been to combine this approach with stable isotope probing (SIP)A synthesized form of the contaminant containing a stable isotope (e.g., ¹³C label) is added. If biodegradation is occurring the isotope will be detected in biomolecules (e.g., phospholipids, DNA). to verify degradation of a contaminant. In this method, a contaminant labeled with a stable isotopeTwo atoms with the same number of protons but a different number of neutrons. (such as ¹³C) is added to a culture or environmental sample. After an exposure period, the lipids are recovered and analyzed.  If the label is found in the lipids, then the compound was degraded and organisms incorporated it into their membrane’s biomolecules.

CSIA is already a versatile, widely used EMD that can detect both biological and chemical transformations of contaminants. CSIA currently characterizes the isotopic composition of a whole contaminant rather than characterizing the isotopic composition of atoms in particular positions within a contaminant. Deuterium nuclear magnetic resonance (²H-NMR) has the potential to contribute to source determination of MTBE and other hydrogen-containing compounds, such as chlorinated solvents that are only partially chlorinated. For example, ²H-NMR could tell if the VC present in a groundwater sample is the biodegradation product of TCE or PCE.

Another growing field for isotopic analyses is its application to metals such as chromium, copper, lead, and uranium. This analysis uses a technique that measures the total isotopic ratioThe concentration of the heavy isotope divided by the concentration of the light isotope. for all species that contain the element of interest called multi-collector inductively-coupled-plasma mass-spectroscopy (MC-ICP-MS). Much like CSIA, this technique could be used to analyze fate and transport of metals, as well as in forensic applications.

Aqueous mineral intrinsic biogeochemistry analysis (AMIBA) is a suite of analyses that provides a molecular-level examination of the geochemistry fundamental to biogeochemical transformation. More information is available about the AMIBA analyses and about collecting these samples in Technical Protocol for Enhanced Bioremediation Using Permeable Mulch Biowalls and Bioreactors (AFCEE, 2008).

Metagenomics, metatranscriptomics, metabolomics, proteomics, and high-throughput sequencing tools are among the emerging techniques that will impact the understanding of biodegradation processes in the environment. These techniques analyze the structure and functioning of entire microbial community rather than individual microorganisms. Many of these techniques are being developed as the result in advances in high-throughput methodologies which enable multiple samples to be rapidly analyzed.

Metagenomics is the analysis of the genome (complete DNA sequence) of multiple organisms. In the environmental field, metagenomics analyzes the genome of all of the organisms within a specific microbial community. This analysis is useful because biodegradation of contaminants often involves the activities of multiple microbial types operating as a community. The movement towards whole community sequencing recognizes that virtually no microorganism exists in nature as a pure culture and that interactions between organisms as part of sometimes complex communities is the norm rather the exception. Metagenomic analysis of contaminated environments may prove that particular contaminants lead to the establishment of microbial communities with consistent functions and representative species. However, association of a particular species or gene with environmental processes will remain tenuous without further compelling information that links these sequences to functions in heterogeneous environments.  Advanced DNA sequencing methodologies such as pyrosequencing are facilitating DNA sequencing of microbial communities.

Metatranscriptomics is the application of high-throughput DNA sequencing approaches to determine the transcriptional activities of entire microbial communities. Understanding the transcriptional responses of entire microbial communities to environmental conditions and perturbations can potentially provide important insights into the factors that control the activities of individual strains with required degradation capabilities.

Metabolomics is the analysis of the entire suite of small metabolites that are generated by microorganisms during their normal functioning. In many instances, current analyses of metabolites focus on individual compounds or groups of related compounds. The aim of metabolomics studies is to obtain a comprehensive understanding of all major metabolites within a sample at any given time. Metabolomics studies have been successfully developed to understand the function of cells in pure culture, where concentrations of metabolites can be high. Because these metabolites cannot be amplified in the same way that nucleic acids can, the development of comprehensive metabolomics approaches must capitalize on increased sensitivities and resolution of analytical approaches, such as mass spectrometry. Applications of metabolomics to contaminant biodegradation studies can potentially provide valuable information related to the metabolic status of entire microbial communities involved in, and required for, contaminant biodegradation.

Proteomics determine the protein expression patterns of microorganisms. This type of analysis comes closest to directly analyzing the functional capabilities of a microorganism because it detects and quantifies all of the proteins and enzymes currently present within the microorganism. Since protein-based enzymes catalyze the vast majority of the biochemical reactions within a cell, the proteomic profile of a bacterium effectively indicates what biochemical processes are being completed at any given time or when growing under a specific set of conditions. However, individual microorganisms can have several thousand different proteins present at any given time and the relative concentrations of these proteins may vary by many orders of magnitude. Like metagenomics, environmental proteomics extends the analysis from a single type of organism to an entire microbial community in an environmental sample. Similarly, advances in proteomic analyses will require refinements and extensions of the capabilities of mass spectrometers along with corresponding databases that will allow protein fragment data to be identified and assigned to specific proteins and enzymes. Because mass spectrometry can resolve the isotopic composition of protein fragments, environmental proteomics may be combined with stable isotope probing approaches that will enable functioning organisms and their enzymes to be identified.

Established and emerging EMDs can generate enormous amounts of data. Limitations to using some of these emerging techniques include handling the enormous amounts of data generated, extracting useful information from these data, and communicating these findings in effective and meaningful ways. Many of these approaches will require the development of specialized bioinformatic tools and effective curated databases. Not all emerging technologies will gain traction within the commercial market since for some, the cost of development is larger than the potential benefits. On the other hand, some methods have already been commercialized (such as pyrosequencing) and have been incorporated into certain applications.

The EMDs described in this document have eliminated the need to physically isolate and culture microorganisms to understand their distribution and activities in contaminated environments. This contribution has been powered in large part by laboratories' ability to extract and amplify nucleic acids (DNA and RNA) using techniques such as PCR and to use related amplification techniques such as qPCR to quantify individual genes and organisms in environmental samples. A second group of emerging technologies is moving in the opposite direction, toward the “meta” techniques that aim to analyze the genomes and metabolic capabilities of individual microbial cells.

While efforts continue to improve methods that culture and isolate microorganisms, the development of single-cell analytical approaches is an alternative approach that precludes the need to isolate organisms through culture-based approaches. For example, individual cells can be identified and isolated using flow cytometers, which are microfluidics platforms than can be used to study the activities of individual bacterial cells. The genomes of individual cells can also be sequenced after amplification of DNA using less-biased and non-thermal amplification technologies such as multiple displacement amplification.