METAGENOMICS: THE SCIENCE OF BIOLOGICAL DIVERSITY

By K. J. Shelswell

(August 2004)

Biological Diversity

For approximately 4.5 billion years, the Earth has been evolving from a barren volcanic landscape into the vibrant globe full of life that it is today. The first forms of life, small microorganisms, have been found in fossils from 3.5 billion years ago. Around 1.5 billion years ago, motile microorganisms emerged allowing life to migrate to different environments with different environmental conditions like increased exposure to ultraviolet radiation or higher temperatures. Microorganisms began to evolve with the changing environmental conditions of the planet.

These new environmental conditions, acting as selective pressures, drove the evolutionary process. They forced new species of organisms to evolve that were better suited to survival under particular environmental pressures. The evolution of new species generates biological diversity, which is represented by the number of different species in an environment. Over time, the evolutionary process has led to the development of more complex life forms such as trees, fish, and humans. A simple example of biological diversity is a comparison between a human and a monkey (see Figure 1).

Figure 1. The visible differences between a human & a monkey.

Both species (human and monkey) are eukaryotes, multicellular, vertebrates, mammals, and primates. However, significant differences between humans and monkeys are immediately visible such as body hair and arm length. Other differences such as amino acid synthesis cannot be perceived by casual observation. The basis of this biological difference lies in the organization and expression of the genetic material of each species.

Genetic Diversity

DNA is responsible for encoding the physical characteristics of an organism. Differences in DNA sequences between organisms create genetic diversity. These changes are also responsible for the subtle differences (such as hair colour, eye colour, or height) between organisms of the same species. This genetic diversity is able to manifest itself as biological diversity through the structure, organization, regulation, and expression of DNA. These effects determine how organisms develop physically, assimilate nutrients, interact with the environment, and even, in some cases, how they behave (see Figure 2).

Figure 3. How genetic diversity affects biological diversity.

It is these properties of genetic diversity that support the effective stability of natural environments. Multiple biological and non-biological components interact through intricate nutrient cycling webs to create a macroscopic global environment. This global environment can be imagined as a pyramid in which the entire structure depends on each of the small blocks that are used to create and support the larger structure. For this reason, it is often useful to examine the environmental impacts of small ecological components such as microorganisms. As noted above, microorganisms are believed to be the origins life on the planet. This theory is supported by the fact that they display the highest degree of biological diversity.

Metagenomics

To understand the biochemical processes of life, it is often easier to study them in a simple system (like a microorganism) instead of a complex one (like humans). Microorganisms have many of the same properties as more complex organisms such as amino acid biosynthesis. They also contain unique properties such as the ability to degrade waste products. As a result, the genetic and biological diversity of microorganisms is an important area of scientific research. Unfortunately, scientists are able to grow less than 1% of all microorganisms observable in nature under standard laboratory conditions. This leaves scientists unable to study more than 99% of the biological diversity in the environment. Metagenomics is a new field combining molecular biology and genetics in an attempt to identify, and characterize the genetic material from environmental samples and apply that knowledge. The genetic diversity is assessed by isolation of DNA followed by direct cloning of functional genes from the environmental sample.

Metagenomics is described as “the comprehensive study of nucleotide sequence, structure, regulation, and function”. Scientists can study the smallest component of an environmental system by extracting DNA from organisms in the system and inserting it into a model organism. The model organism then expresses this DNA where it can be studied using standard laboratory techniques.

Metagenomics is employed as a means of systematically investigating, classifying, and manipulating the entire genetic material isolated from environmental samples. This is a multi-step process that relies on the efficiency of four main steps (see Figure 3). The procedure consists of (i) the isolation of genetic material, (ii) manipulation of the genetic material, (iii) library construction, and the (iv) the analysis of genetic material in the metagenomic library.

Figure 3. The standard steps of a metgenomics experiment.

The first step of the procedure is the isolation of the DNA. First, a sample is collected that represents the environment under investigation because the biological diversity will be different in different environments. The samples contain many different types of microorganism, the cells of which can be broken open using chemical methods such as alkaline conditions or physical methods such as sonication. Once the DNA from the cells is free, it must be separated from the rest of the sample. This is accomplished by taking advantage of the physical and chemical properties of DNA. Some methods of DNA isolation include density centrifugation, affinity binding, and solubility/precipitation.

Once the DNA is collected, it is manipulated so that it can be used in the model organism. Genomic DNA (the genetic material of an organism) is relatively large so it is cut up into smaller fragments using enzymes called restriction endonucleases. These are special enzymes that cut DNA at a particular sequence of base pairs. The enzymes move along the long fragments until they recognize these sequences where they cut both strands of the DNA. This results in the smaller, linear fragments of DNA depicted in Figure 2. The fragments are then combined with vectors. Vectors are small units of DNA that can be inserted into cells where they can replicate and produce the proteins encoded on the DNA using the machinery that the cells use to express normal genes (see Figure 3). The vectors also contain a selectable marker. Selectable markers provide a growth advantage that the model organism would not normally have (such as resistance to a particular antibiotic) and are used to identify which organisms contain vectors and which ones do not.

The third step is to introduce the vectors with the metagenomic DNA fragments into the model organism. This allows the DNA from organisms that would not grow under laboratory conditions to be grown, expressed, and studied. The DNA inserted in the vector is transformed into cells of a model organism, typically Escherichia coli. Transformation is the physical insertion of foreign DNA into a cell, followed by stable expression of proteins. It can be done by chemical, electrical, or biological methods. The method of transformation is determined based on the type of sample used and the required efficiency of the reaction. The metagenomic DNA in the vectors are all in the same sample initially but the vectors are designed so that only one kind of DNA fragment from the sample will be maintained in each individual cell. The transformed cells are then grown on selective media so that only the cells carrying vectors will survive. Each group of cells that grows is called a colony. Each colony consists of many cloned cells that originated from one single cell. These samples of cells containing all of the metagenomic DNA samples on vectors are called metagenomic libraries. Each colony can be used to create a stock of cells for future study of a single fragment of the DNA from the environmental sample.

The fourth and final step in the procedure is the analysis of the DNA from the metagenomic libraries. The expression of DNA determines the physical and chemical properties of organisms so there are many potential methods of analysis. A phenotype is the physical attribute associated with expression of a gene. An example of metagenomic analysis would be to look for an unusual colour or shape in the model organism. An aspect of the phenotype that is not readily observed is chemical reaction. The chemical properties of the expressed metagenomic DNA can be examined by performing chemical assay on products created by the model organism. This would investigate whether the model organism gained an enzymatic function that it was previously lacking such as use of an unusual nutrient source for growth under conditions that limit normal nutrient availability.

Metagenomic libraries are typically used to search for new forms of a known gene. First, the metagenomic DNA is inserted into a model organism that lacks a specific gene function. Restoration of a physical or chemical phenotype can then be used to detect genes of interest. A genotype is the specific sequence of the DNA and provides another means of analyzing the metagenomic DNA fragment. The sequence of the bases in the DNA can be compared to databases of known DNA to get information regarding the structure and organization of the metagenomic DNA. Comparisons of these sequences can provide insight into how the gene products (proteins) function.

Genotypic analysis is usually performed after phenotypic analysis. A typical metagenomic analysis involves several subsequent rounds of the procedure in order to definitively isolate target genes from environmental samples and to effectively characterize the information encoded by the DNA sequence. The information gained from the metagenomic procedure provides information regarding the structure, organization, evolution, and origin of the DNA and can be used in scientific applications for the benefit of society and the environment

Applications of Metagenomics

Many microorganisms have the ability to degrade waste products, make new drugs for medicine, make environmentally friendly plastics, or even make some of the ingredients of food we eat. By isolating the DNA from these organisms, it provides us with the opportunity to optimize these processes and adapt them for use in our society. As a result of ineffective standard laboratory culture techniques, the potential wealth of biological resources in nature (like microbes) is relatively untapped, unknown, and uncharacterized. Metagenomics represents a powerful tool to access the abounding biodiversity of native environmental samples. The valuable property of metagenomics is that it provides the capacity to effectively characterize the genetic diversity present in samples regardless of the availability of laboratory culturing techniques. Information from metagenomic libraries has the ability to enrich the knowledge and applications of many aspects of industry, therapeutics, and environmental sustainability. This information can then be applied to society in an effort to create a healthy human population that lives in balance with the environment. Metagenomics is a new and exciting field of molecular biology that is likely to grow into a standard technique for understanding biological diversity.

Information References

1.Kimball J. Kimball’s Biology Pages: Taxonomy.

2. Jasper S. University of Texas: Life Sciences.

3. Vogel TM, Nalin R (2003). Sequencing the metagenome. ASM News 69(3):107. pH meter

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A pH meter. Notice the temperature and pH probes on the right.

A pH meter is an electronic instrument used to measure the pH (acidity or basicity) of a liquid (though special probes are sometimes used to measure the pH of semi-solid substances). A typical pH meter consists of a special measuring probe (a glass electrode) connected to an electronic meter that measures and displays the pH reading.

A simple pH meter with its probe immersed in a mildly alkaline solution. The two knobheads are used to calibrate the instrument. Contents

 1 The probe  2 The meter  3 Calibration and use  4 Types of pH meters  5 History  6 Building a pH meter  7 See also  8 References  9 External links

The probe

The pH probe measures pH as the activity of hydrogen ions surrounding a thin-walled glass bulb at its tip. The probe produces a small voltage (about 0.06 volt per pH unit) that is measured and displayed as pH units by the meter. For more information about pH probes, see glass electrode. The meter

The meter circuit is fundamentally no more than a voltmeter that displays measurements in pH units instead of volts. The input impedance of the meter must be very high because of the high resistance — approximately 20 to 1000 MΩ — of the glass electrode probes typically used with pH meters. The circuit of a simple pH meter usually consists of operational amplifiers in an inverting configuration, with a total voltage gain of about - 17. The inverting amplifier converts the small voltage produced by the probe (-0.059 volt/pH in basic solutions, +0.059 volt/pH in acid solutions) into pH units, which are then offset by seven volts to give a reading on the pH scale. For example:

 At neutral pH (pH 7) the voltage at the probe's output is 0 volts. 0 * 17 + 7 = 7.  At alkaline pH, the voltage at the probe's output ranges from > 0 to +0.41 volts (7 * 0.059 = 0.41). So for a sample of pH 10 (3 pH units from neutral), 3 * 0.059 = 0.18 volts), the output of the meter's amplifier is 0.18 * 17 + 7 = 10.  At acid pH, the voltage at the probe's output ranges from -0.7 volts to < 0. So for a sample of pH 4 (also 3 pH units from neutral, but in the other direction), 3 * +0.059 = +0.18 volts, the output of the meter's amplifier is -0.18 * 17 + 7 = 4.

The two basic adjustments performed at calibration (see below) set the gain and offset of the inverting amplifier. Calibration and use

Calibration with at least two, but preferably three, buffer solution standards is usually performed every time a pH meter is used, though modern instruments will hold their calibration for around a month. One of the buffers has a pH of 7.01 (almost neutral pH) and the second buffer solution is selected to match the pH range in which the measurements are to be taken: usually pH 10.01 for basic solutions and pH 4.01 for acidic solutions (It should be noted that the pH of the calibration solutions is only valid at 25°C). The gain and offset settings of the meter are adjusted repeatedly as the probe is alternately placed in the two calibration standards until accurate readings are obtained in both solutions. Modern instruments have completely automated this process and only require immersing in each solution once, or at worst, twice.

The calibration process correlates the voltage produced by the probe (approximately 0.06 volts per pH unit) with the pH scale. After calibration, the probe is rinsed in distilled, deionized water to remove any traces of the buffer solution, blotted with a clean tissue to absorb any remaining water which could dilute the sample and thus alter the reading, and then quickly immersed in the sample. Between uses, the probe tip, which must be kept wet at all times, is typically kept immersed in a small volume of storage solution, which is an acidic solution of around pH 3.0. Alternatively, the pH 7.01 calibration solution can be used, but this results in a need for more frequent calibration. In an emergency, tap water can be used, but distilled or deionised water must never be used for longer-term probe storage as the relatively ionless water 'sucks' ions out of the probe, which degrades it.

Occasionally (about once a month), the probe should be cleaned using pH-electrode cleaning solution; generally a 0.1 M solution of Hydrochloric Acid (HCl) is used [1], having a pH of about one. Types of pH meters pH meters range from simple and inexpensive pen-like devices to complex and expensive laboratory instruments with computer interfaces and several inputs for indicator (ion- sensitive, redox), reference electrodes, and temperature sensors such as thermoresistors or thermocouples. Cheaper models sometimes require that temperature measurements be entered to adjust for the slight variation in pH caused by temperature. Specialty meters and probes are available for use in special applications, harsh environments, etc. Pocket pH meter are readily available today for a few tens of dollars that automatically compensate for temperature. History

The first commercial pH meters were built around 1936 by Radiometer in Denmark and by Dr. in the . While Beckman was an assistant professor of chemistry at the California Institute of Technology, he was asked to devise a quick and accurate method for measuring the acidity of lemon juice for the California Fruit Growers Exchange (Sunkist). Beckman's invention helped him to launch the Beckman Instruments company (now ). In 2004 the Beckman pH meter was designated an ACS National Historical Chemical Landmark in recognition of its significance as the first commercially successful electronic pH meter.[2] Building a pH meter

Because the circuitry of a basic pH meter is quite simple, it is possible to build a serviceable pH meter or pH controller with parts available at a neighborhood retailer. (pH probes, however, are not so easily acquired and must usually be ordered from a scientific instrument supplier.) For a walkthrough of how to build the simplest possible pH meteror a detailed description of how to build a pH meter/pH controller, see The pH Pages. The application note for the LM6001chip at the National web site also has a very simple demonstration circuit. Although the application note is for a specialty IC, serviceable pH meters can be built from any operational amplifier with a high input impedance, such as the common and inexpensive National Semiconductor TL082 or its equi

pH measuring

pH is a measure of the acidity or alkalinity of a solution. Aqueous solutions at 25°C with a pH less than seven are considered acidic, while those with a pH greater than seven are considered basic (alkaline). When a pH level is 7.0, it is defined as 'neutral' at 25°C + − because at this pH the concentration of H3O equals the concentration of OH in pure + [1] water. pH is formally dependent upon the activity of hydronium ions (H3O ), but for + very dilute solutions, the molarity of H3O may be used as a substitute with little loss of [2] + + accuracy. (H is often used as a synonym for H3O .) Because pH is dependent on ionic activity, a property which cannot be measured easily or fully predicted theoretically, it is difficult to determine an accurate value for the pH of a solution. The pH reading of a solution is usually obtained by comparing unknown solutions to those of known pH, and there are several ways to do so. The concept of pH was first introduced by Danish chemist S. P. L. Sørensen at the Carlsberg Laboratory[3] in 1909. The name pH has been claimed to have come from any of several sources including: pondus hydrogenii, potentia hydrogenii (Latin),[4] potentiel hydrogène (French), and potential of hydrogen (English).[5]

Contents

 1 Definition  2 Explanation  3 Calculation of pH for weak and strong acids  4 Measurement  5 pOH  6 Indicators  7 Seawater  8 Body fluids  9 See also  10 References  11 External links

Definition pH (per hydron or per hydrogen, also power of the hydrogen) is defined in International Standard ISO 31-8: Quantities and units – Part 8: Physical chemistry and molecular physics, Annex C (normative): pH. International Organization for Standardization, 1992. operationally as follows. For a solution X, first measure the electromotive force EX of the galvanic cell where

F is the Faraday constant; R is the molar gas constant; T is the thermodynamic temperature.

Defined this way, pH is a dimensionless quantity. Values pH(S) for a range of standard solutions S, along with further details, are given in the relevant IUPAC recommendation[6]. pH has no fundamental meaning as a unit; its official definition is a practical one. However in the restricted range of dilute aqueous solutions having an amount-of- dissolved-substance concentrations less than 0.1 mol/L, and being neither strongly alkaline nor strongly acidic (2 < pH < 12), the definition is such that

+ + where [H ] denotes the amount-of-substance concentration of hydrogen ion H and γ1 denotes the activity coefficient of a typical univalent electrolyte in the solution. Explanation

Visual representation of the pH scale.

Another visual representation of the pH scale.

In simpler terms, the number arises from a measure of the activity of hydrogen ions (or their equivalent) in the solution. The pH scale is an inverse logarithmic representation of hydrogen proton (H+) concentration. Unlike linear scales, which have a constant relationship between the item being measured (H+ concentration in this case) and the value reported, each individual pH unit is a factor of 10 different than the next higher or lower unit. For example, a change in pH from 2 to 3 represents a 10-fold decrease in H+ concentration, and a shift from 2 to 4 represents a one-hundred (10 × 10)-fold decrease in H+ concentration. The formula for calculating pH is:

+ + Where αH denotes the activity of H ions, and is dimensionless. In solutions containing other ions, activity and concentration will not generally be the same. Activity is a measure of the effective concentration of hydrogen ions, rather than the actual concentration; it includes the fact that other ions surrounding hydrogen ions will shield them and affect their ability to participate in chemical reactions. These other ions change the effective amount of hydrogen ion concentration in any process that involves H+.

In dilute solutions (such as tap water), activity is approximately equal to the numeric value of the concentration of the H+ ion, denoted as [H+] (or more accurately written, + [H3O ]), measured in moles per litre (also known as molarity). Therefore, it is often convenient to define pH as:

For both definitions, log10 denotes the base-10 logarithm, therefore pH defines a logarithmic scale of acidity. For example, if one makes a lemonade with a H+ concentration of 0.0050 moles per litre, its pH would be:

A solution of pH = 8.2 will have an [H+] concentration of 10−8.2 mol/L, or about 6.31 × −9 + −9 + 10 mol/L. Thus, its hydrogen activity αH is around 6.31 × 10 . A solution with an [H ] concentration of 4.5 × 10−4 mol/L will have a pH value of 3.35.

In solution at 25 °C, a pH of 7 indicates neutrality (i.e. the pH of pure water) because water naturally dissociates into H+ and OH− ions with equal concentrations of 1×10−7 mol/L. A lower pH value (for example pH 3) indicates increasing strength of acidity, and a higher pH value (for example pH 11) indicates increasing strength of basicity. Note, however, that pure water, when exposed to the atmosphere, will take in carbon dioxide, some of which reacts with water to form carbonic acid and H+, thereby lowering the pH to about 5.7. Neutral pH at 25 °C is not exactly 7. pH is an experimental value, so it has an associated error. Since the dissociation constant of water is (1.011 ± 0.005) × 10−14, pH of water at 25 °C would be 6.998 ± 0.001. The value is consistent, however, with neutral pH being 7.00 to two significant figures, which is near enough for most people to assume that it is exactly 7. The pH of water gets smaller with higher temperatures. For example, at 50 °C, pH of water is 6.55 ± 0.01. This means that a diluted solution is neutral at 50 °C when its pH is around 6.55 and that a pH of 7.00 is basic.

Most substances have a pH in the range 0 to 14, although extremely acidic or extremely basic substances may have pH less than 0 or greater than 14. An example is acid mine runoff, with a pH = –3.6. Note that this does not translate to a molar concentration of 3981 M; such high activity values are the result of the extremely high value of the activity coefficient while concentrations are within a "reasonable" range [7]. E.g. a 7.622 + molal H2SO4 solution has a pH = -3.13, hydrogen activity αH around 1350 and activity + coefficient γH = 165.4 when using the MacInnes convention for scaling Pitzer single ion activity coefficient [7].

+ Arbitrarily, the pH is − log10([H ]). Therefore,

+ pH = − log10[H ] or, by substitution,

.

The "pH" of any other substance may also be found (e.g. the potential of silver ions, or pAg+) by deriving a similar equation using the same process. These other equations for potentials will not be the same, however, as the number of moles of electrons transferred (n) will differ for the different reactions. Calculation of pH for weak and strong acids

Values of pH for weak and strong acids can be approximated using certain assumptions.

Under the Brønsted-Lowry theory, stronger or weaker acids are a relative concept. But here we define a strong acid as a species which is a much stronger acid than the + hydronium (H3O ) ion. In that case the dissociation reaction (strictly + − + − HX+H2O↔H3O +X but simplified as HX↔H +X ) goes to completion, i.e. no unreacted acid remains in solution. Dissolving the strong acid HCl in water can therefore be expressed:

HCl(aq) → H+ + Cl−

This means that in a 0.01 mol/L solution of HCl it is approximated that there is a concentration of 0.01 mol/L dissolved hydrogen ions. From above, the pH is: pH = −log10 [H+]: pH = −log (0.01) which equals 2.

For weak acids, the dissociation reaction does not go to completion. An equilibrium is reached between the hydrogen ions and the conjugate base. The following shows the equilibrium reaction between methanoic acid and its ions:

HCOOH(aq) ⇌ H+ + HCOO−

It is necessary to know the value of the equilibrium constant of the reaction for each acid in order to calculate its pH. In the context of pH, this is termed the acidity constant of the acid but is worked out in the same way (see chemical equilibrium):

Ka = [hydrogen ions][acid ions] / [acid]

−4 For HCOOH, Ka = 1.6 × 10

When calculating the pH of a weak acid, it is usually assumed that the water does not provide any hydrogen ions. This simplifies the calculation, and the concentration provided by water, 1×10−7 mol/L, is usually insignificant.

With a 0.1 mol/L solution of methanoic acid (HCOOH), the acidity constant is equal to:

+ − Ka = [H ][HCOO ] / [HCOOH]

Given that an unknown amount of the acid has dissociated, [HCOOH] will be reduced by this amount, while [H+] and [HCOO−] will each be increased by this amount. Therefore, [HCOOH] may be replaced by 0.1 − x, and [H+] and [HCOO−] may each be replaced by x, giving us the following equation:

Solving this for x yields 3.9×10−3, which is the concentration of hydrogen ions after dissociation. Therefore the pH is −log(3.9×10−3), or about 2.4.

Measurement

Representative pH values Substance pH

Hydrochloric acid, 10M -1.0 Lead-acid battery 0.5 Gastric acid 1.5 – 2.0 Lemon juice 2.4 Cola 2.5

Vinegar 2.9 Orange or apple juice 3.5 Tomato Juice 4.0 Beer 4.5 Acid Rain <5.0

Coffee 5.0 Tea or healthy skin 5.5 Urine 6.0 Milk 6.5 Pure Water 7.0

Healthy human saliva 6.5 – 7.4 Blood 7.34 – 7.45 Seawater 7.7 – 8.3 Hand soap 9.0 – 10.0 Household ammonia 11.5 Bleach 12.5 Household lye 13.5

pH can be measured:

by addition of a pH indicator into the solution under study. The indicator color varies depending on the pH of the solution. Using indicators, qualitative determinations can be made with universal indicators that have broad color variability over a wide pH range and quantitative determinations can be made using indicators that have strong color variability over a small pH range. Precise measurements can be made over a wide pH range using indicators that have multiple equilibriums in conjunction with spectrophotometric methods to determine the relative abundance of each pH-dependent component that make up the color of solution[citation needed], or

by using a pH meter together with pH-selective electrodes (pH glass electrode, hydrogen electrode, quinhydrone electrode, ion sensitive field effect and others).

by using pH paper, indicator paper that turns color corresponding to a pH on a color key. pH paper is usually small strips of paper (or a continuous tape that can be torn) that has been soaked in an indicator solution, and is used for approximations.

The lowest and highest ends of the pH scale do not oxidize. The middle of the scale is what oxidizes, such as water and blood.

As the pH scale is logarithmic, it does not start at zero. Thus the most acidic of liquids encountered can have a pH as low as −5. The most alkaline typically has pH of 14. Measurement of extremely low pH values has various complications. Calibration of the electrode in such cases can be done with standard solutions of concentrated sulphuric acid whose pH values can be calculated with the Pitzer model[7].

As an example of home application, the measurement of pH value can be used to quantify the amount of acid in a swimming pool. pOH

There is also pOH, in a sense the opposite of pH, which measures the concentration of OH− ions, or the basicity. Since water self ionizes, and notating [OH−] as the concentration of hydroxide ions, we have

(*) where Kw is the ionization constant of water. Now, since by logarithmic identities, we then have the relationship: and thus

This formula is valid exactly for temperature = 298.15 K (25 °C) only, but is acceptable for most lab calculations. Indicators

The Hydrangea macrophylla blossoms in pink or blue, depending on soil pH. In acidic soils, the flowers are blue; in alkaline soils, the flowers are pink.

An indicator is used to measure the pH of a substance. Common indicators are litmus paper, phenolphthalein, methyl orange, phenol red, bromothymol blue, bromocresol green and bromocresol purple. To demonstrate the principle with common household materials, red cabbage, which contains the dye anthocyanin, is used.[8] Seawater

In chemical oceanography pH measurement is complicated by the chemical properties of seawater, and several distinct pH scales exist[9].

As part of its operational definition of the pH scale, the IUPAC define a series of buffer solutions across a range of pH values (often denoted with NBS or NIST designation). These solutions have a relatively low ionic strength (~0.1) compared to that of seawater (~0.7), and consequently are not recommended for use in characterising the pH of seawater (since the ionic strength differences cause changes in electrode potential). To resolve this problem, an alternative series of buffers based on artificial seawater was developed[10]. This new series resolves the problem of ionic strength differences between samples and the buffers, and the new pH scale is referred to as the total scale, often denoted as pHT. The total scale was defined using a medium containing sulphate ions. These ions + 2− − experience protonation, H + SO4 ⇌ HSO4 , such that the total scale includes the effect of both protons ("free" hydrogen ions) and hydrogen sulphate ions:

+ + − [H ]T = [H ]F + [HSO4 ]

An alternative scale, the free scale, often denoted pHF, omits this consideration and + focuses solely on [H ]F, in principle making it a simpler representation of hydrogen ion + [11] + concentration. Analytically, only [H ]T can be determined , so [H ]F must be estimated 2− − * using the [SO4 ] and the stability constant of HSO4 , KS :

+ + − + 2− * −1 [H ]F = [H ]T − [HSO4 ] = [H ]T ( 1 + [SO4 ] / KS )

* However, it is difficult to estimate KS in seawater, limiting the utility of the otherwise more straightforward free scale.

Another scale, known as the seawater scale, often denoted pHSWS, takes account of a further protonation relationship between hydrogen ions and fluoride ions, H+ + F− ⇌ HF. + Resulting in the following expression for [H ]SWS:

+ + − [H ]SWS = [H ]F + [HSO4 ] + [HF]

However, the advantage of considering this additional complexity is dependent upon the abundance of fluoride in the medium. In seawater, for instance, sulphate ions occur at much greater concentrations (> 400 times) than those of fluoride. Consequently, for most practical purposes, the difference between the total and seawater scales is very small.

The following three equations summarise the three scales of pH:

+ pHF = − log [H ]F + − + pHT = − log ( [H ]F + [HSO4 ] ) = − log [H ]T + − + pHSWS = − log ( [H ]F + [HSO4 ] + [HF] ) = − log [H ]SWS

In practical terms, the three seawater pH scales differ in their values by up to 0.12 pH units[9], differences that are much larger than the accuracy of pH measurements typically required (particularly in relation to the ocean's carbonate system). Since it omits consideration of sulphate and fluoride ions, the free scale is significantly different from both the total and seawater scales. Because of the relative unimportance of the fluoride ion, the total and seawater scales differ only very slightly.

Body fluids

The pH of different body fluids varies with function and other factors. Mostly it is a tightly regulated system to keep the acid-base homeostasis.

pH in body fluids [12]

Fluid pH gastric acid 0.7 lysosome 5.5

granule of chromaffin cell 5.5 Neutral H2O at 37°C 6.81 cytosol 7.2

CSF 7.3 arterial blood plasma 7.4

mitochondrial matrix 7.5 exocrine secretions of pancreas 8.1

Chemical Waste Disposal Questions

Where do I get the multi-part white "Laboratory Waste Tag" and the yellow "Laboratory Waste Accumulation" stickers from?

These are both available at:

 Given Mailroom (in a box across from the mailboxes)  HSRF 221 (dark room on shelf)  Stafford Mailroom  Rubenstein Laboratory (mail desk)  Colchester Research Facility maintenance office  Cook (Room 243, Chem Stockroom)  Marsh Life Science (Biology Stockroom) All labels and stickers are available through Environmental Safety as well. To get some, e-mail us at mailto:[email protected] and tell us how many you need. When does my waste get picked up?

Generally waste is picked up from main campus locations on Fridays. Other locations, such as the Colchester Research Facility or the Rubenstein Ecosystem Laboratory, pick- ups occur monthly.

According to EPA regulation, it must be removed from your laboratory within 30 days of the date on the white waste tag. If you find waste that has been in your laboratory longer than that or which is in your way, please let us know by sending us the top copy of the multi-part white "Laboratory Waste" tag or entering the information from the tag on line at http://esf.uvm.edu/tags_entry/ How do I dispose of broken glassware?

Broken laboratory glassware that is not contaminated with hazardous materials should be placed in a sealed cardboard box for disposal. If necessary, you can order the large cardboard containers for broken glass from a vendor such as VWR. When the box is full, close all sides and tape the lid securely closed with wide clear or duct tape (NOT ‘Scotch’-type tape from a desk dispenser), then label the box as Trash. Custodians will dispose of the boxes as trash if they are adequately closed and labeled.

If broken glassware is contaminated with an acutely hazardous material (see the Laboratory Chemical Inventory form for identification of these materials), or grossly contaminated with any hazardous material, it should be placed in a sealed and leak proof container that will not be punctured or torn by the glass, labeled with the appropriate hazard warning(s) and a laboratory waste tag. Notify Environmental Safety (ES) for pick- up.

For example, you could use a cardboard box within a tightly sealed plastic bag with the label, phenol contaminated debris/broken glass on the multi-part white Laboratory Waste tag. If the broken glassware is contaminated with biologically hazardous material, you could place the sealed cardboard box in a red bio-waste bag and call for pick up by ES staff. How do I dispose of empty chemical containers?

-Containers that held air or water reactives, stench, or highly hazardous materials, such as carcinogens, teratogens, mutagens, acutely hazardous (toxic) materials must remain closed and be tagged for pickup through the hazardous waste disposal system. See the UVM HCOC (Hazardous Chemicals of Concern) list attached to the Chemical Use Planning form to identify these chemicals at http://esf.uvm.edu/uvmemp/01chemuse.pdf. - Empty containers that held other chemicals such as corrosives, flammables or other toxics not referenced above MUST be thoroughly rinsed with water and the label MUST be defaced. The rinse liquid can go down the drain. Please do not place empty containers in the fume hood to evaporate.

-Empty glass containers that held chemicals are not recyclable and can be reused as laboratory waste containers or go in the trash. Make sure they are thoroughly rinsed, labels defaced, and clearly labeled as ‘Trash’. Custodians will dispose of them for you.

-Metal cans should be placed in recycling bins. Make sure they are thoroughly rinsed, labels defaced, and if clearly labeled ‘For Recycling’ custodians will manage them for you.

-You MUST deface the labels of all empty containers for either trash or recycling or to save them in your lab for future use. If the labels are not defaced then storage rules apply according to the label information. Use a thick black marker to cover the chemical name and all hazard information.

Where should I store chemical waste?

Chemical waste is best stored with compatible stock chemicals: Flammables in the Flammables cabinet, Corrosives in the Corrosives cabinet with like corrosives, Reactives with reactives, etc.

Chemical waste must be labeled with the yellow Laboratory Waste Accumulation sticker while the container is being filled. Once full, fill-out a multi-part white Laboratory Waste tag and attach it to the container. For liquid waste, the container must be placed in secondary containment.

Do not store waste on the floor, except for 5 gallon containers of solid or liquid toxics. These 5 gallon containers must be in secondary containment. Do not place the 5 gallon containers in aisle spaces. Make sure they are labeled fully and clearly with the yellow Laboratory Waste Accumulation sticker to assist Environmental Safety Technicians during pick up and to protect custodians during trash removal.

If you have a designated "Chemical Waste Storage" cabinet, be sure to separate your chemicals accordingly. All liquid wastes must have seconday containment in case of leaks or spillage. Can I store my chemical waste in the fume hood? No. Please store chemical waste with compatible stock chemicals and keep your hood space available for work. Every additional item in your fume hood has a negative effect on the hood’s ability to effectively capture and exhaust chemical fumes. Is it okay to use a ‘Ziploc’ plastic bag for storage of chemically contaminated items?

Dry chemically contaminated debris can be collected in a closed container such as a closed zip-loc bag, if the bag is not degraded by the chemical over time. It must be labeled with the UVM yellow Laboratory Waste Accumulation sticker if you are accumulating the material over time. Ultimately, it must be tagged with our multi-part white Laboratory Waste tag for pick-up and disposal by Environmental Safety staff. If it contains no free liquid, spill clean up material may also be placed in a closed ziploc bag and tagged with the white multi-part Laboratory Waste tag.

Never use a biohazard bag to store chemical debris. What do I do when I break a piece of mercury containing equipment?

Mercury is very difficult to clean up completely and can release vapors that can create toxic levels at room temperature. A surprisingly small amount of mercury can create a significant concentration of mercury in the room air. Therefore, do not try to clean it up yourself.

First, turn off the equipment to prevent further release of mercury vapors and have all personnel leave the room. Close the door, place a sign to avoid re-entry, and call the ESF at 656-5400. We will respond as soon as possible to complete the clean up and monitor the room air for mercury vapors. How can I make sure my chemical waste doesn’t get picked up mistakenly as trash?

The chemical wastes most likely to be confused with trash are the 5 gallon buckets of solid, toxic waste that are allowed to be stored on the floor. Make sure these buckets are clearly labeled with the yellow Laboratory Waste Accumulation stickers and these stickers are visible at all times. Use clear bags as a liner inside this clearly labeled bucket. Don’t remove the bag and set it aside, unlabeled, for any reason and never put the lid underneath the bucket. Can I use the Environmental Safety (ES) supplied Chemical Spill Kit on all chemical spills? There are 2 styles of spill kits that have been supplied by the ES:

1) The older version consists of an absorbent powder, a brush for sweeping it effectively over the spilled chemical, nitrile gloves, instructions and multi-part laboratory waste tags. The spill kit is specifically designed for use by lab personnel in cleaning up small spills (1 liter or less); the absorbent powder is compatible with most chemicals, with the exceptions of hydrofluoric acid and metallic mercury.

2) After March, 2007, we were not able to obtain additional absorbent powder and so have substituted gray absorbent pads (this also eliminates the need for the brush in the kit). The pads are compatible with all chemicals including hydrofluoric acid, but do not work with metallic mercury spills. They still have a maximum capacity of about 1 liter.

Note that neither absorbent removes any hazards associated with the spilled chemicals and that the clean up materials must be treated as chemical waste.

Call Police Services at 911 for help with any spill of chemicals that have corrosive, flammable or toxic vapors or that are in quantities large enough to preclude clean up by lab workers. Do I have to use the Environmental Safety (ES) waste labels and tags or can I create ones of my own?

You must use the label and tag supplied by ES because they include information required by government regulation. This includes the yellow Laboratory Waste Accumulation sticker if you are accumulating chemical waste over time and the multi-part white Laboratory Waste tag when you are ready to dispose of an accumulation container. Can I mix my acids with some alkaline liquids to neutralize them and pour them down the drain?

Please do not treat any of your hazardous chemicals in order to dispose of them down the drain or in the trash. There are regulatory implications to any drain disposal of hazardous materials. Contact us at mailto:[email protected] if you have questions about proper disposal of chemical or other waste. Are there any chemicals that I should not mix together for disposal?

Yes. Do not mix any chemicals for disposal without checking with Environmental Safety staff first. In many cases, mixing chemicals together results in a much higher disposal cost and sometimes makes it impossible to dispose of a material. Acutely hazardous chemicals and mercury should never be mixed with other chemicals. Can I put chemical waste in red Biohaz bags?

No, please use only CLEAR bags to collect any waste or debris contaminated with chemicals. Label them with the “Laboratory Waste Accumulation” sticker while collecting waste. When full, complete and attach the multi-part white “Laboratory Waste” tag for disposal. Can I dispose of any chemicals or buffers down the sink?

At UVM, sink disposal of hazardous laboratory chemicals is forbidden. According to the Burlington Sewer Use Ordinance, solutions with a pH equal to or less than 5.0 or greater than or equal to 10.5 should not be discharged into the sewer system. In addition any laboratory chemical that exhibits an ignitable, toxic or reactive characteristic, is a dye, has a strong odor, or has a high viscosity is prohibited from drain disposal.

In addition, biologically benign solutions such as those containing sugars and salts and no other hazardous material may be disposed of down the sink drain.

If you have specific questions about whether a chemical is suitable for sink disposal, contact Environmental Safety staff at mailto:[email protected]. My laboratory is moving. What should I do with my waste chemicals?

Please do not tag each individual container. Set the chemicals to be disposed of aside in your laboratory and place a note on the collection that says “For Environmental Safety (ES) disposal”. Contact us at mailto:[email protected] to schedule a time for ES technicians to sort the waste chemicals into groups based on their hazard class and Department of Transportation regulations. Then you will be able to use one tag per group of chemicals and save yourself and us the time and effort of managing a large number of tags. I had a chemical spill. What should I do?

If it is a spill you cannot manage on your own, all workers should leave the lab, place a DO NOT ENTER sign on the door and call ESF at 6-5400. Have the following information available:

1) Your name, building and room number

2) A phone number to reach you at

3) The name of the chemical spilled 4) How much spilled

5) Where the spill is

6) If anyone is hurt

Environmental Safety (ES) staff can then assess the situation and decide whether it can be safely cleaned up by laboratory personnel with a Spill Kit or if an ES technician or contractor should do the cleanup. If in doubt call the ES for advice. I am cleaning out a freezer full of old samples. What should I do with them?

It depends if they are biological samples or chemical samples. Before you thaw the samples, call us to have an Environmental Safety technician visit to help you sort through the containers. It may take us several days to arrange to pick up waste materials. What do I do with an unknown chemical?

Use the multi-part white “Laboratory Waste” tag and label it as “unknown”. If you suspect it is a particular chemical you may give this information but the primary label remains “unknown”. Environmental Safety technicians will test these a few times per year for proper disposal. I’m trying to decide which chemicals I should cull from my stocks. How do I decide when a chemical is old?

You should sort through them and dispose of the ones you haven’t used in about a year and those that are beyond their expiration date. Chemicals which do not flow freely are not likely used in the future and are best disposed of. How do I dispose of dry waste debris?

Dry waste debris may be collected in a ziploc bag or another type of closed container and then tagged with the multi-part white “Laboratory Waste” tag for disposal.

It has taken me more than a year to fill my 5 gallon container with liquid waste. Is this appropriate? No. According to government regulations, chemical wastes should be removed at least once a year. Your container size is too big if it takes you a year to fill; contact us at mailto:[email protected] for assistance in selecting another container.

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