Vaccari D.A., Strom P.F., Alleman J.E. Environmental Biology for Engineers and Scientists

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Example 11.4 If an average of 41 colonies is counted per ﬁlter when 10 mL of a 10

3

dilution of the sample is ﬁltered, what was the concentration of CFUs in the original

sample?

Answer

CFUs=mL ¼ 41 colonies=plate  10 mL 10

3

¼ 4:1  10

CFU=mL

Membrane ﬁltration can be especially helpful in enum erating microorganisms present

at low concentrations in water. However, particulate material present in the sample can be

an interference.

Most Probabl e Number Techniques Suppose a sample that originally contained ﬁve

bacteria per milliliter was diluted 10- and 100-fold (Figure 11.13). Then ﬁve tubes of

media were each inocula ted with 1 mL of the sample, another ﬁve tubes were each inocu-

lated with 1 mL of the 10-fold dilution, and another ﬁve tubes were each inoculated with

1 mL of the 100-fold dilution (a total of 15 tubes inoculated, ﬁve at each of the three dif-

ferent concentrations). Further, assume that each cell could grow if it is placed in a tube.

Which of the tubes are likely to show growth? Since on average the tubes receiving 1 mL

of undiluted sample receive ﬁve bacteria each, it is likely they will all grow, although

there is a statistical possibili ty that one or more tubes may not get any bacteria (and

will not grow), whereas others will probably get more than ﬁve. However, for the

10-fold dilution, on average each tube gets 0.5 cell. Of course, a tube cannot receive

0.5 cell, so that some tubes will get one or more cells and be ‘‘positive’’ (show growth),

whereas others will not get a cell and will thus be negative. Probably two or three of the

10 x dilution

inoculate

incubate

sample

Figure 11.13 MPN method schematic. Positive tubes are shown as dark after incubation.

308

QUANTIFYING MICROORGANISMS AND THEIR ACTIVITY

tubes will end up positive. At the 100-fold dilution, on average only 0.05 cell is added to

each tube, and hence all may be expected to be negative.

The pattern of positive tubes that results (in this case, perhaps 5-3-0) is an indication of

the cell numbers originally present. Through a mathematical procedure, in fact, the most

probable number (MPN) of cells originally present resulting in that pattern can be esti-

mated. These values have been tabulated for several different numbers of replicates (e.g.,

3, 5, and 10 tubes per dilution), and also for different dilution factors (e.g., two fold as

well as 10-fold serial dilutions). Note that a positive or negative result in each tube is used

to arrive at a quantitative estimate of cell numbers. The estimate from a pattern of 5-3-0 is

an MPN of 8.0 mL

1

of the original sample. (A response of 5-2-0 gives an MPN of 5.0,

the starting value used in the example.)

An obvious disadvantage of this approach is that it produces a statistical estimate rather

than an actual count. For example, with the 5-3-0 example, there is a 95% likelihood

that the actual number is between 3.0 and 25, an undesirably broad range. On the other

hand, the MPN is potentially useful for a wide variety of samples and can be adapted for

many types of microorganisms, including some for which other methods have not been

successful.

Turbidity and Absorbance A method that can be used in laboratory cultures, especially

pure cultures, grown on soluble (and preferably colorless) media is measurement of

turbidity (cloudiness) or absorbance. Turbidity is the amount of light dispersed 90



from the path of incident light passing through a material. It is measured with a turbidi-

meter using a photocell at right angles to the light path. Absorbance is the reduction in the

transmission of light along the light path and may occur due to both dispersion and

absorption of light. Abs orbance is also called optical density (OD) and is usually mea-

sured with a spectrophotometer (also called a spectrometer). Absorbance is used more

often than turbidity because spectrometers are more commonly available than turbidi-

meters.

If a small amount of microbial suspension is placed in a sample tube, its turbidity, or

the amount of light dispersed toward the photocell, is proportional, up to a point, to the

number of particles in the suspensio n. Similarly, for absorbance, the amount of light that

passes through the suspension will be inversely proportional to the concentration of organ-

isms, provided that particle size does not change. This is a form of Beer’s law (absorbance

is proportional to concentration), which is the basis of most quantitative spectrophotome-

try (although in this case a substantial amount of the light is refracted rather than actually

absorbed). Thus, increases in either turbidity or absorbance can be used as a surrogate

measure of growth, or correlated through use of a calibration curve to microbial counts

obtained by other methods.

One advantage of using turbidity or absorbance is that they are not destructive of the

culture. In fact, special ﬂasks with sidearms are available so that the turbidity of a pure

culture can be determined over time without the need to open the ﬂask and risk contam-

ination. However, if particle size increases, through ﬂocculent growth or ﬁlament elonga-

tion, turbidity and OD will underestimate the cell count or biomass. Also, this approach

generally cannot be used with environmenta l samples.

Counting Viruses and Bacteriovores Viruses are too small to see under any light micro-

scope for direct counting. Also, since they grow only wi thin cells of other organisms, they

COUNTS OF MICROORGANISM NUMBERS 309

cannot be cultured directly like bacteria. Thus, alter native methods are needed to enumer-

ate them.

If a suspension of the bacteria Escherichia coli is spread over the surface of a plate

containing an appropriate medium, a uniform bacterial lawn will develop. If at the

same time that the culture is added, a sample containing coliphage (viruses that attack

E. coli) is included, ‘‘holes’’ in the lawn will develop as the viruses infect and kill

cells and then spread to neighboring cells. These clear areas, where the bacteria have

been lysed by the phage, are referred to as plaques (Figure 11.14). The number of

such plaques is then an estimate of the number of plaque-forming units (PFUs) present

in the volume of sample added. Similarly, other phage can be enumerated using appropri-

ate host bact eria for the lawn. Analogous to CFUs, it is often expected that each PFU

stems from a single initial phage.

Many animal and plant viruses can be counted in similar ways using appropriate host

cell cultures. In these cases, however, individual infected cells may be counted rather than

plaques. Alternatively, an MPN type of procedure might be used, or if necessary, the

development of infection in exposed whole organisms.

Plaque formation can also be used to count some organisms that feed on bacteria, such

as amoeba. Host cell lysis caused by Bdellovibrio, the bacteriovorous proteobacteria

(Section 10.5.6), also forms plaques.

11.5.3 Relationship between Numbers and Mass

The size of a population of microorganisms can be expressed on the basis of their number

or their mass. These are, of course, expected to have some relationship. In fact, if the size

and shape of a particular microbe are known, its mass can usually be estimated easily.

Example 11.5 A rod-shaped bacterial cell is 0.8 mm in diameter and 2 mm in length.

What is its mass?

Figure 11.14 Sketch of petri dish with bacteriophage plaques. (Plate contains 43 PFUs.)

310

QUANTIFYING MICROORGANISMS AND THEIR ACTIVITY

Answer The rod shape can be approximated by that of a cylinder, so that its volume,

V, can be estimated as

V ¼ pr

h ¼ pð0:4 mmÞ

ð2 mmÞ1:0 mm

1cm



¼ 1:0  10

12

Most cells have a density only slightly greater than that of water, perhaps 1.05 to

1.10 g/cm

, so that this cell would have a mass of 1  10

12

g. Since cells are typically

about 85% water, this would be a dry weight of about 1:5  10

13

g per cell.

If 10

of the rods in Example 11.5 were present in a sample, this could then be estimated

to correspond to about 1 mg of the wet weight of cells (10

cells  10

12

g/cell ¼

3

g ¼1 mg). Alternatively, if the mass of these cells in a sample was known, the numbers

present could be estimated.

Most bacteria probably have wet weights in the range 0:2to3 10

12

g and dry

weights of 0.3 to 4  10

13

11.5.4 Surface Area/Volume Ratio

The surfaces of prokaryotic cells are not just protective wrappings. They often are highly

active regions that may be required to transport incoming substrates, remove outgoing

products and waste s, shed excess heat, regulate internal osmotic pressure, and support

ﬂagella. Thus, the surface area/volume (or mass) ratio ðA

=VÞ of prokaryotic cells

plays an important part in their survival and metabolic success.

=V is not commonly measured, but it is important to realize that it is very dependent

on cell size. For cocci, for example, assuming that they are spherical with radius r,

4pr

4=3pr

Thus, this ratio is highest in small organisms and will decrease in an inversely

proportional way with size. In fact, this is a factor that limits the size of single-celled

organisms.

11.6 MEASURING MICROBIAL ACTIVITY

In some cases it may be desirable to quantify the general or speciﬁc activity of microor-

ganisms in a system. This may then be used to estimate microbial mass or numbers, or

may be of intere st in its own right. What usually is meant by microbial activity is the rate

at which microbially mediated reactions are occurring. Thus, the rate of activity might be

determined by measuring the rate of disappearance of any of the reactants or the appear-

ance of any of the products. In some cases, it may also be measured by looking at the rate

of enzymatic activity in a sample.

MEASURING MICROBIAL ACTIVITY 311

11.6.1 Aerobic Respiration

A generalized overall reaction for aerobic respiration of organic material can be expressed

organic matter þ O

! CO

þ H

O þ new biomass þ energy

Analogous reactions can be developed for inorganic substrates such as ammonia and

hydrogen sulﬁde, which are used by lithotrophic organisms. The rate of change of each

of the six quantities in this equation in theory could be used to measure the rate of aerobic

microbial activity. This is typically done by making repetitive measurements of disappear-

ance or accumulation over time. However, such repeated sampling may inadvertently

affect the system itself. Alternatively, multiple replicate systems can be established

initially and one or more sacriﬁced at each different sampling time. A disadvantage of

this approach is the inherent variability between ‘‘replicate’’ systems.

Organic Matter Disappearance In some situations the microbial activity on a particular

compound is of interest. If this compound can be measured, its disappearance over time

will be a direct indication of activity. However, proper controls are necessary to ensure

that the activity is biological rather than the result of other mechanisms, such as volati-

lization, adsorption, or chemical transformation. Killed controls, in which any organisms

present or added are destroyed by heat, ultraviolet radiation, or chemical means, are often

best for such techniques. Also, disappearance of the parent compound does not neces-

sarily mean that it is being completely biodegraded; some compounds are merely trans-

formed to metabolic products that then persist and accumulate (see ‘‘Biodegradation’’ in

Section 13.1.3).

Alternatively, some general measure of organic material might be used, and its disap-

pearance monitored. Biochemical oxygen demand (BOD), chemical oxygen demand

(COD), and total organic carbon (TOC) are three such measures commonly used for

this purpose and are discussed further in the section ‘‘Quantiﬁcation of Organic Carbon’’

in Section 13.1.3.

Measurements of organic material are often of great importance for measuring extent

of disappearance but typically are not convenient for measuring rates. Good rate measure-

ments usually require frequent sampling, which may be disruptive to the system or

demand large numbers of replicate systems for sacriﬁce. In most cases the need for che-

mical analysis also places practical limits on the number of samples that can be processed.

Oxygen Utilization In the headspace of a closed system of ﬁxed volume and const ant

temperature, oxygen removed by respiration is replaced by an almost equal molar amount

of carbon dioxide produced. However, carbon dioxide, a weak acid, is easily removed by

absorption with an alkali, such as KOH solution. The pressure in the system will then drop

as oxygen is consumed. Alternatively, if the pressure and temperature of the system are

held constant, the volume will decrease. In both cases, frequent measurements can be

taken without the need for expensive or disruptive analyses. Such methods are referred

to as respirometric techniques.

Early respirometers typically required manual measurements of changes in pressure.

Many modern systems can collect data automatically every few seconds, if desired, allow-

ing careful monitoring of the pattern of oxygen utilization (Figure 11.15). In one device,

312 QUANTIFYING MICROORGANISMS AND THEIR ACTIVITY

for example, oxygen is replace d automatically each time a slight pressure change occurs,

and the amount of oxygen supplied is recorded.

With the development of dependable electrochemical dissolved oxygen (DO) probes,

it has also become common to measure the oxygen uptake rate (OUR) directly in liquid

culture. The liquid is ﬁrst aerated, if needed, and then the drop in DO over time is measured

and reported in milligrams of DO uptake per liter per minute (or hour). This may be

further normalized to the biomass present, usually measured as SS or VSS, to give a speciﬁc

OUR (SOUR), in units such as milligrams of DO uptake per minute per gram of biomass.

In systems in which the air ﬂows through continuously, it may be possible to determine

oxygen utilization by measuring the difference in the entrance and exit concentrations of

oxygen in the airstream. Similarly, in continuous-ﬂow liquid systems it may be possible

to measure oxygen uptake by measuring changes in DO concentrations.

Production of Carbon Dioxide The carbon dioxide produced by respiration might be

measured directly (e.g., with an infrared spectrophotometer) or absorbed in an alkali

and measured by titration with acid. For an aerated system, the exit gas might be bubbled

through KOH solution in several ﬂasks in series to be sure that it is all removed. Special

biometer ﬂasks (also known as Bartha ﬂasks after their inventor, Richard Bartha) with

sidearms containing KOH can also be used (Figure 11.1 6). Measurements of CO

can be

particularly useful because they indicate mineralization of organic matter. Especially if

appropriately

C-radiolabeled test compounds are available,

production gives

clear evidence of their complete biodegradation.

Production of Water In most aqueous systems the amount of metabolic water pro-

duced is too small to measure com pared to the amount of water already in the system.

New Biomass Many of the methods described in Section 11.4 might be used to deter-

mine biomass over time. Alternatively, counts (Sections 11.5.1 and 11.5.2) might be used

and converted (Secti on 11.5.3), if necessary, to biomass. However, the relationships

0 2 4 6 8 10 12 14

Time (hr)

Oxygen Uptake (mg/L)

lean

rich

technical

reagent

Figure 11.15 Example of respirometry data. Oxygen uptake with various grades (reagent,

technical, lean, and rich) of monoethanol amine (MEA). (Data courtesy of Y. Lam, R. M. Cowan,

and P. F. Strom.)

MEASURING MICROBIAL ACTIVITY 313

between new biomass produced and other aspects of microbial activity may be complex.

These are discussed more in Section 11.7.

Energy Some of the energy released by metabolic activities is captured and used for

growth and other cell functions; the rest is given off as heat. Special instruments

known as calorimeters can sometimes be used to measure this release of heat experimen-

tally. The amo unt of heat released per mass of oxygen utilized is 14,000 J/g O

for oxi-

dation of a wide variety of organic compounds. Also, in some systems of interest to

environmental engineers and scientists, such as composting, sufﬁcient heat is released

that it may be possible to measur e temperature changes or other signs of heat production

as a means of determining the rate of activity [see the discussion of equation (16.5)].

11.6.2 Anaerobic Systems

In anaerobic systems it may also be possible to measure the disappearance of organic

material or of electron acceptors such as nitrate or sulfate. Alternatively, monitoring

the appearance of particular products such as ethanol or acetate may be feasible (if they

accumulate). In methanogenic systems, the rate of formation of gaseous products can be

measured volumetrically if they can be trapped in a gas collection column (Figure 11.17).

This will typically be about 65% methane and 35% carbon dioxide (with traces of other

gases). The total can be measured, or the carbon dioxide can be removed by scrubbing

with KOH.

soil

KOH

solution

plug

needle

trap

stopcock

Figure 11.16 Biometer ﬂasks.

314

QUANTIFYING MICROORGANISMS AND THEIR ACTIVITY

11.6.3 Enzyme Activity

The amount of activity of a particular enzyme or group of enzymes in a sample can serve

as an indication of the number and activity of microorganisms present. Probably the most

widely used is the dehydrogenase assay, in which a tetrazolium salt is converted to a

colored formazan product that is extracted and quantiﬁed. Other general enzyme assays

include those for proteases, phosphatases, and esterases. Particular activities of interest,

such as cellulose (cellulases), starch (amylases), or chitin (chitinases) degradation or

nitrogen ﬁxation (nitrogenases), also can be quantiﬁed.

11.7 GROWTH

Some microorganisms can reproduce asexually by budding, and many eukaryotes have a

method of sexual reproduction. However, most microorganisms commonly reproduce

asexually by binary ﬁssion, and that is the means of growth that we focus on here. In

this process, a single ‘‘parent’’ cell physically splits into two genetically identical

(in the absence of mutations) ‘‘daughter’’ cells. Since the original cell does not ‘‘die’’

in this process, terms such as age and life span have a different meaning than with plants

and animals.

11.7.1 Exponential Growth

If they were able to grow freely without outside constraints (and no removal or death),

cells growing by binary ﬁssion would follow the pattern depicted in Figure 11.18.

Under these conditions the time between each successive split, producing a new

incubator

reactor

tubing

clamp

overflow

collection

vessel

gas

collection

column

gas

Figure 11.17 System for anaerobic gas production measurement.

GROWTH 315

‘‘generation’’ of daughter cells, is constant and is called the generation time ðt

Þ. Since

the nu mber of cells doubles each generation, the same period also is commonly referred to

as the doubling time ðt

Þ. This type of growth pattern is referred to as exponential

growth. Note that whereas the time period is constant, the number of new cells added

during that period doubles each time.

Most people do not have an intuitive feeling for exponential growth. As a simple men-

tal exercise, picture folding a standard sheet of paper in half, then in half again, and again.

Imagine that you could continue folding it in half 50 times. How tall would the folded

stack of paper then be: a meter? 100 m ( a football ﬁeld)? New York to San Francisco

(5000 km)? After ﬁrst trying to guess, cal culate an approximate answer by assuming

that the paper is 0.1 mm thick and doubles each time it is folded (or 0.1 mm 2

(Note:2

¼10

Minimum Doubling Times Under ideal growth conditions, microorganisms will

achieve their most rapid growth and hence their minimum t

. This value is inherent to

the particular strain of organism (for the system in which it is growing), and there is in

fact a tremendous range of minimum doubling times in the microbial world. Some exam-

ples are shown in Figure 11.19.

Many common fast-growing bacteria (e.g., Escherichia coli) can have generation times

of only 20 minutes. A few, such as the thermophile Bacillus stearothermophilus (found,

for example, in composting piles), grow even more rapidly, with minimum generation

times of around 10 minutes. This is about 1,000,000 times faster than a typical human

generation of approximately two decades!

Prokaryotes in general have faster growth rates than eukaryotes, with their greater

complexity. However, there are also slow-growing bacteri a. Organisms tend to adapt to

181624

Time

Doubling

Exponential

Growth

Pattern

Time

Doubling

Time

Doubling

Time

Doubling

Figure 11.18 Theoretical microbial growth schematic.

316

QUANTIFYING MICROORGANISMS AND THEIR ACTIVITY

their environments, so that bacteria living in systems where substrates become available

slowly will tend to be slow growing. Also, bacteria utilizing substrates that release little

energy may have minimum doubling times of several hours or even days. Such substrates

can include hard-to-degrade organic substances as well as some inorganic energy sources.

For example, the chemolithotroph Nitrobacter winogradskyi derives only 17 kcal of

energy per mole of NO



-N that it oxidizes to nitrate, as compared to the 600 kcal/

mol potentially available to E. coli from the oxidation of glucose. This is reﬂected in

the 18-hour minimum generation time of N. winogradskyi (Figure 11.19).

Modeling Exponential Growth If one cell begins growing exponentially, and cell num-

ber ðNÞ is plotted vs. time, initially N increases in discrete steps: 1–2–4–8–16–32–64–

(Figure 11.20). After a period of time, the cell divisions would no longer be synchronized

precisely, so that the intermediate numbers (such as 50) would be present for brief peri-

ods. Still, only whole numbers of cells can occur (not 50.2), making the plot a series of

small steps. However, once high enough numbers are reached, the rate of increase would

appear to be smooth because the steps are so small.

On the other hand, biomass increases as a continuous function (except on the molecu-

lar level). Thus, biomass is theoretically more amenable than cell number to mathematical

expression and is the focus of our discussion. In practice, howe ver, it is also usually perfectly

acceptable to use the same sorts of expressions with N, except when counts are very low.

Rather than biomass itself, more commonly it is biomass concentration ðXÞ that is

considered. This typically might be on a mass per volume basis for liquid s (e.g., mg/L

in water or media) and gases (mg/m

of air), or on a mass per mass basis for solids

(mg/kg of soil or sludge).

0.1 1 10 100

Escherichia coli

Bacillus subtilis

Clostridium botulinum

Rhodospirullum rubrum

Anabaena cylindrica

Nitrobacter winogradsky

Scenedesmus quadricauda

Asterionella formosa

Euglena gracilis

Ceratium tripos

Tetrahymena geleii

Paramecium caudatum

Leishmania donovani

Giardia lamblia

Saccharomyces cerevisiae

Monilinia fructicola

Generation Time (hrs)

Fungi

Algae

Protozoa

Bacteria

Fungi

Algae

Protozoa

Bacteria

Figure 11.19 Representative microbial generation times under optimal conditions.

GROWTH 317