VARIATIONS IN BACTERIAL TRIPHOSPHATE VALUES DUE TO GENUS AND ENVIRONMENTAL CONDITIONS

by

Ruann Knox Hampson

Thesis submitted to the Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of

Master of Science

in

Food Science and Technology

APPROVED:

M. D. Pierson, Chairman

J. K. Palmer A. A. Yousten

December, 1986

Blacksburg, Virginia VARIATIONS IN BACTERIAL ADENOSINE TRIPOSPHATE VALUES

DUE TO GENUS AND ENVIRONMENTAL CONDITIONS

by

Ruann Knox Hampson

Committee Chairman: Merle D. Pierson Food Science and Technology

(ABSTRACT)

Variations in ATP content in three ground beef spoilage bacteria,

Lactobacillus brevis, Lactobacillus jensenii, and Pseudomonas sp. were investigated using the bioluminescence (luciferin-luciferase) assay.

Environmental factors (temperature, atmosphere, pH, aeration, and phase of growth), as well as differences among genera and species, were studied in relation to their effect on cellular ATP. Variations for each of the environmental factors and bacteria were shown statistically to be significantly different at the 0.05 level. The mean ATP/cell for each of the bacteria was 2.71 fg/cell (1..:_ brevis), 2.20 fg/cell (1..:_

jensenii), and 1.36 fg/cell (Pseudomonas sp.). For all three bacteria,

ATP/cell was lower and more stable throughout the culture's growth cycle at 3°c or in N2 . In general, ATP/cell increases from a lowest value in lag phase to a highest value in stationary phase. The effect of sonication on ATP/cell was tested for each bacterium at one set of factors. Sonication studies showed that L. brevis cells were clumping, especially in aged cultures. After sonication, ATP/cell remained relatively constant from lag through stationary phase. L. jensenii showed no signs of clumping and ATP/cell increased as the culture aged.

Sonication had a lethal effect on the Pseudomonas. Thus the ATP/cell for Pseudomonas increased dramatically as the culture aged. Guidelines concerning temperature, assumed ATP content for major contaminants, and sample handling must be followed in order to use the bioluminescence assay to estimate biomass in foods. Acknowledgements

This research project was funded by Packard Instruments, a division of United Technologies, Chicago, Illinois. I would like to thank their members for their financial support and suggestions.

I also would like to thank the members of my Graduate Committee, Dr.

Merle D. Pierson, Dr. James K. Palmer, and Dr. Alan A. Yousten, for their guidance and suggestions.

Many other members of the faculty and staff of the Food Science and

Technology Department also deserve my thanks for their assistance. I would like to give special thanks to John Chandler for his technical assistance.

Special thanks go to the Food Microbiology group -- Joan Hedrick,

Jeff Rhodehamel, Fred Cook, Rukma Reddy, Adnan Ismaiel, Rebecca Rorrer, and Marty Bucknavage -- for their support and encouragement.

To my husband, parents and friends, I give my love and thank them for all their emotional support (especially at three am), patience, and good humor.

iv Table of Contents

page Abstract . . . . ii

Acknowledgements iv

I. Introduction .1

II. Literature Review. .3

A. Biolurninescence. . 3

1. Mechanism. . .3

2. Factors Affecting Light Emission Color .5

3. Luciferase Specificity .6

4. Inhibitors .8

5. Applications of the Biolurninescence Assay. 10

B. .. 11

1. Biological Importance. 11

2. Biochemical Approach to ATP Content Determinations 13

3. ATP per Cell in Bacteria . 16

C. Bacterial Flora of Ground Beef 21

1. Natural Flora of Ground Beef 21

2. Spoilage in Aerobically Packaged Ground Beef 22

3. Spoilage in Vacuum Packaged Ground Beef. 23

III. Materials and Methods .. 25

A. Ground Beef Spoilage Organisms 25

1. Isolation. . . 25

2. Identification 26

B. ATP Determination. 26

v 1. Equipment. 26

2. Reagent Preparation. 27

a. Water. 27 b. Luciferin - Luciferase 27

c. Buffer 27

d. Extraction Reagent 28

e. Stock ATP Standard 28

f. ATP Standards. 28

g. Internal Standard. 28

h. Stock Phosphate Buffer 28

i. Butterfield's Phosphate Buffer 29

3. Instrument Program 29

4. ATP Calibration. 29

5. ATP Determinations of Unknown Samples. 30

c. Media Preparation. 30

1. Growth Media 30

2. Enumeration Media. 30

D. Experimental Protocol. 31

1. Factors Examined 31

2. Preliminary Growth Curve 31

3. Sampling Growth Curve. 33

4. Growth Curves with Sonication. 33

E. Statistical Analysis 34

IV. Results and Discussion 35

A. Spoilage Isolates. 35

vi B. Preliminary Growth Curve . . 37

C. Variations in ATP Content in Bacteria. 50

1. Statistical Analysis 50

2. Effect of Bacterium, Temperature, Growth Atmosphere, pH, and Aeration on ATP Content 52

D. Sonication Experiments 67 v. Summary and Conclusions. 80

Literature Cited 85

Vitae ...... 94

vii I Introduction

Millions of dollars are spent each day by the American public on fresh and processed meat and vegetable products. The food industry has the responsibility of supplying high quality food to the consumer that is nutritious as well as chemically and microbiologically safe. The food industry is regulated by federal, state, and local governments to operate under good manufacturing practices. More important than legal restrictions to the food industry should be the trust of the public in their products. For without public trust, the industry cannot survive in the marketplace.

Ground beef has become a common component of the American diet. By its very nature, ground beef has a microbial content of 105 or higher.

Examinations of ground beef for microbial load are routinely made by the food industry's quality control personnel. However, the techniques now used necessitate an undesirably long interim (24 to 72 hours incubation time) between sampling and the test's completion. Examinations of this type are both laborious and costly. For years the food industry has worked to develop techniques for the detection and enumeration of foodborne microorganisms that are rapid, reliable, and reproducible.

Bioluminometry shows promise of being a rapid method for estimation of bacterial loads in foods. In this method bacterial numbers are estimated by the measurement of adenosine triphosphate (ATP). In the bioluminescence assay luciferase reacts with luciferin and ATP in the presence of oxygen to produce light. This light can be measured and the

1 2

quantity used to determine the amount of ATP present. From this number the bacterial load of the food sample can be determined within an hour.

But how reliably can ATP be used to estimate bacterial numbers?

Karl (53) stated four assumptions that are made when ATP is used as an estimator of biomass. The first is that ATP could be easily extracted from cells and then measured. The second is that all living organisms contain ATP. Third, ATP is not associated with dead cells. The final assumption is that there is a fairly constant ratio of ATP to total cell carbon for all bacteria. It is this last assumption that has been the least studied. It is the purpose of this study to examine how bacterial

ATP content varies due to environmental factors (temperature, pH, atmosphere, aeration, and phase of growth) within as well as between genera and species. II Literature Review

A. Bioluminescence

1. Mechanism

Bioluminescence is the production of light by an enzyme catalyzed chemical reaction. Harvey (41) reviewed the various types of known luminescent organisms. Included in these are certain bacteria, fungi, sponges, snails, squid, fish, and fireflies. Firefly bioluminescence is the most studied bioluminescence system. McElroy, Seliger, and White

(71) determined the reaction mechanism of firefly bioluminescence. The reaction sequence can be swrunarized as follows (68):

1. LUCIFERIN +Mg-ATP + LUCIFERASE ->

LUCIFERASE-LUCIFERIN-AMP + PP. 1

2. LUCIFERASE-LUCIF~RIN-AMP + o2 -> LUCIFERASE-OXYLUCIFERIN * + co2 + AMP

3. LUCIFERASE-OXYLUCIFERIN* =>

LUCIFERASE + OXYLUCIFERIN + LIGHT

Luciferin must be present as the D(-) isomer in order to be biologically active (Fig. 1). When luciferin, luciferase, and oxygen are present in excess, ATP becomes the limiting reagent. Thus, the amount of light produced is directly related to the amount of ATP present. For this reason bioluminescence can be used as a sensitive method of assaying ATP

3 4

OXYLUCIFERIN

0 1-) LUCIFERIN

Figure 1 Chemical structures of D-(-)-luciferin, the active isomer of luciferin, and oxyluciferin, the oxidized product of the luciferin-luciferase reaction. 5

content (68). If ATP is present in excess, ATP and luciferin-AMP competitively inhibit Mg-ATP on the active site of luciferase (59,66).

High pyrophosphate levels also inhibit light production (59). Care must be taken to avoid these types of inhibition in the ATP assay.

Luciferase is a protein composed of two 50,000 dalton subunits (66).

Denburg, Lee, and McElroy (23) showed that two luciferin binding sites exist on luciferase. Each subunit has a luciferin and ATP binding site but there is only one site on the enzyme for Mg-ATP, the active form of the substrate (66,69). From all information available, McElroy and

DeLuca (66) hypothesized that only one subunit is enzymatically active.

Once Mg-ATP binds to this active site, conformational changes occur in the structure of luciferase (20).

2. Factors Affecting Light Emission Color

McElroy, White, and Seliger (71) showed that for Photinus pyralis, the luciferin-luciferase reaction with ATP emits a yellow-green light

(562 nm) when the reaction takes place at a neutral or alkaline pH

(optimum pH is 7.75 (97)). The peak emission wavelength varies according to the species of firefly used for luciferase isolation (6,70,86). If the reaction takes place under non-optimal conditions, a red light (614 nm) (which has a lower quantum yield than that of the yellow-green light) is emitted. This red shift occurs as the temperature rises above

23°C (71). The stereochemistry of attachment (67) as well as the presence of urea, zn2+, Cd2+, or Hg2+ (71) can also cause this shift to red light. Finally, the greater the polarity of a solvent, the 6

greater the red shift (71). Because of this, conditions must be optimized when a bioluminescence assay is being used.

3. Luciferase Specificity

How specific is luciferase for ATP? That is to say, when light is produced in the luciferin-luciferase assay, how sure can one be that ATP triggered the light production? Moyer and Henderson (73) found that the triphosphates in Table 1 caused light to be emitted. However, triphosphate (dATP) has the highest activity of the alternatives to ATP and it's activity is a fiftieth of ATP. All of the other nucleoside triphosphates have activities <0.1% of ATP. Moyer and

Henderson suggested possible contamination of the nucleoside triphosphates with minute amounts of ATP caused the low activity.

However, Holm-Hansen and Booth (46) found triphosphate (CTP) and triphosphate (ITP) to have the same amount of activity as

ATP. Lee et al. (59) also found dATP to have 2% of the activity of ATP with a portion of the light being red. McElroy and DeLuca (68) found adeonosine diphosphate (ADP) to be inactive in this reaction; whereas,

Holm-Hansen and Booth (46) reported a slight activity with ADP. Three

ATP analogs show some activity in the enzyme system. DeLuca et al. (19) showed that 3 iso-ATP and - ( -AMP) had activity. However, neither compares to the activity of ATP. When -AMP acts as the substrate, the resulting light emission is red. When 3 iso-

ATP acts as the substrate, the resulting light emission is partially 7

Table 1

Nucleotides tested for activity in the luciferin-luciferase reaction (73)

Adenosine triphosphate Deoxyadenosine triphosphate triphosphate triphosphate triphosphate triphosphate triphosphate triphosphate Deoxyinosine triphosphate triphosphate 8

red. Rosendahl, Leonard, and DeLuca (83) showed that lin-benzo-ATP also produces light, but at a reduced activity.

Th e second component necessary f or 1ig. h t pro d uction . is . Mg 2+ Lee et al. (59) found that Mg2+ is a requirement for light production. Mg 2+ must be complexed with ATP (see equation 1). However, other metals can take the place of magnesium in this reaction. In fact, manganese shows greater activity than does magnesium. The following list shows the relative activities of metals that can replace Mg:

Mn> Mg > Co,Zn,Fe > Cd> Ni > Ca> Sr (59)

4. Inhibitors

Care must be taken when using the bioluminescence assay. There are many molecules and compounds that can inhibit the enzymatic reaction.

Luciferin solutions must be kept free of oxidizing agent such as ferricyanide. These agents oxidize luciferin to dehydroluciferin.

Dehydroluciferin can react with Mg-ATP and luciferase as follows:

4. DEHYDROLUCIFERIN + Mg-ATP + LUCIFERASE =>

LUCIFERASE-DEHYDROLUCIFERIN + PP. i

This enzyme bound product acts as an inhibitor of luciferase by preventing the complex from reacting with oxygen to produce light. For the same reason, luciferin should be shielded from l.ight to avoid photo- oxidation (68). Intense colors are also reported to be inhibitory to the reaction (58). 9

As mentioned previously, there are some metal ions (Zn2+, Cd2+, and

Hg2+) which inactivate luciferase. Zinc is the least inhibitory whereas mercury is the most inhibitory. These ions react with critical

sulfhydryl groups in the active site of luciferase, thus decreasing

enzyme activity (59,71).

Anion inhibition of luciferase has also been reported. Denburg and

McElroy (24) reported the following ions to be inhibitory and their

relative effects:

SCN > I ,No3 > Br > Cl and Gilles et al. (39) described the following:

Cl04 > I > Cl > acetate

2 Sulfate (so4 -) is also a luciferase inhibitor (21). Thore (97) found numerous salts to be inhibitory as well as certain buffers (such as

HEPES/Cl and TRIS/Cl) and organic solvents (such as acetone and ethanol). Other inhibitory compounds are bovine serum albumin and Triton x-100.

Moyer and Henderson (73) found none of the triphosphates that they

tested (Table 1) to be inhibitory. Filippova and Ugarova (29) did find nucleotide bases, , and nucleotide 5'-mono-, 5'-di-, and 5'-

triphosphates to be inhibitors of luciferase isolated from the firefly

Luciola mingrelica. Thus, inhibitory effects will be dependent upon the

firefly species from which the luciferase was isolated. 10

5. Applications of the Bioluminescence Assay

For many years the luciferin-luciferase or bioluminescence assay for

ATP has been used in a variety of applications by experimenters of many disciplines.

The ATP content of many bacteria under a variety of conditions has been studied in many laboratories and will be discussed in section C.

There has also been much basic research on algae (7,44,89) and yeast

(78). Research on yeast also includes such applications as the estimation of yeast contamination of carbonated beverages (32) and fruit juices (92).

The bioluminescence assay has been used extensively for the estimation of bacterial numbers and the determination of bacterial growth in environmental, industrial, and clinical fields. Several studies have been made to determine the distribution of bacteria in aquatic environments (45,46,95) and in marine sediments (3,42).

Microbial growth was also measured in several different soil types (79).

Microbial biomass has likewise been estimated in compost (61) and wastewater (63).

Applying bioluminescence to the assessment of food quality has been widely studied. Sharpe (88) investigated bacterial contamination of many foods and the assay has been used to assess milk quality

(8,9,10,81,102). Other studies have attempted to predict the shelf life of meat as a result of its bioburden (54,64,90,91,93). Even brewery products have been tested for bacterial contamination by the bioluminescence assay (49). 11

Clinical applications are somewhat limited. Many studies have been conducted to determine the feasibility of using the assay to determine

the the presence of bacteriuria (65,85,98,99). Other studies have

investigated blood cultures (72) and dental plaque (82). Another

clinical application has been the use of the assay in conjunction with

antibiotics to determine if a bacterial strain is resistant or sensitive

to the antibiotic (4). Levin et al. (62) have suggested using this assay

in virus and cancer studies.

B. Adenosine Triphosphate

1. Biological Importance

Nucleotides must be present in every living and viable organism.

They serve four universal functions in cells: 1) regulation of the

cell's metabolism, 2) formation of intermediates in biosynthesis, 3)

synthesis of nucleic acids and 4) storage and transport of cellular

chemical energy (12,53). Adenosine triphosphate is central in these

roles, especially in cellular energy storage and transport.

Adenosine triphosphate (ATP) is made up of three main components

(Fig. 2). The nitrogenous base , is bound to the five carbon

sugar D-ribose. The ribose is bound to the three phosphate groups at

the 5' carbon of the ribose. The two phosphodiester bonds are known as high energy phosphate bonds due to their high standard free energy of hydrolysis, c0 '= -7.3 kcal/mole. It is through these high energy bonds 12

0- 0- 0-

-o-J-o-i-o-i-o-I I I 0 0 C)

OH OH

Figure 2 'lbe chemical structure of adenosine triphosphate (ATP). 13

that cells store and transport chemical energy (60).

Through the years, many studies have been undertaken to determine

ATP content within microbial cells. A variety of methods for reporting

ATP content, determined from the bioluminescence assay, are presented in the literature.

2. Biochemical Approach to ATP Content Determinations

One biochemical approach to reporting ATP content is to express the

ATP as adenylate energy charge or as a ratio to some other cellular measurements. Some of these cellular measurements include ATP/dry cell weight and ATP/volume of bacterial suspension (3,17,56). Both Karl (53) and Chapman and Atkinson (12) reviewed ATP concentrations within microbial cells and how these concentrations can vary due to their environment. The following summary expresses findings in relation to bacterium, growth phase, temperature, atmosphere, and availability of substrate.

The effect of growth phase has been studied or noted by several workers. Forrest (30) investigated the effects in Streptococcus faecalis. Forrest found that in the lag phase, the ATP pool level

(~g/mg cells) rose. At the beginning of the exponential or log phase, the ATP pool level progressively falls. Forrest also found that the composition of the medium has an effect on ATP pool levels. A mean pool level of 6.75 µg/mg cells during log phase exists in a complex medium.

In a semidefined medium with a phosphate buffer, the mean pool level of

5.1 µg/mg cells exists during log phase. When S. faecalis growth is 14

limited by an energy source (pyruvate instead of glucose), linear growth and a mean ATP pool level of 0.9 pg/mg cells occur. The ATP pool is not dependent upon growth rate. Forsberg and Lam (31) also found that the phase of growth had an effect on the bacterial ATP level. The ATP pool of Selenomonas ruminantium during early log phase, approximately 5

µg ATP/mg cells, decreases to approximately 2.5 pg/mg in late log and drops past 1 µg/mg in the stationary phase. Forsberg and Lam noted the same trend in six other rumen bacteria with pool sizes in the log phase ranging from 17.6 µg/mg to 1.8 pg/mg. The ATP pools of two rumen bacteria did remain nearly constant during log phase (2.6 µg/mg and 1.1

}lg/mg). Holms, Hamilton, and Robertson (47) likewise found the ATP pool size to be constant during the log phase of the growth cycle. The pool size ranges from 4.5 to 7.5 µmoles/g dry weight (2.3 to 3.8 µg/mg dry wt) in Escherichia coli. The range is due to changes of carbon and energy sources. Contrary to the findings of Forrest (30) and Forsberg and Lam (31), Knowles and Smith (57) found the ATP levels in Azotobacter vinelandii to be low in early log phase (approximately 1 nmoles ATP/mg dry wt or 0.5 µg/mg). The ATP level rises through the log phase and levels out in stationary phase at values of 4.8 to 6.2 nmoles ATP/mg dry wt (2.4 to 3.0 µg/mg). Huzyk and Clark (48), using chromatographic separation of nucleoside triphosphates and autoradiography, showed that concentrations in !..:_ coli are a function of the cell cycle. The ATP concentration rises to a maximum level just prior to cell division and then drops at cell division.

Knowles and Smith (57) investigated the effects of atmosphere 15

changes (aerobic to anaerobic growth) on ATP values. When the culture atmosphere is changed from air to N2 , ATP decreases to a level one- fourth of that of aerobically growing A. vinelandii (2 nmoles/mg or 1

µg/mg). Upon aeration of this anaerobic culture, the ATP content returns to the high levels of aerobic metabolism. Strange, Wade, and Dark (94) tested the effect of anaerobiasis on Aerobacter aerogenes. Under a nitrogen atmosphere, ATP content dropped from the aerobic level of 2.1

µg ATP/mg dry wt to the level of 1.0 p.g ATP/mg after one hour. Strange et al. also found that a similar drop occurred when the solute concentration of the medium increased. Cole, Wimpenny, and Hughes (15) also found a decrease in ATP for ~ coli upon anaerobiasis -from 12 to 6 nmoles ATP/mg dry cells (6.1-3.0 p.g/mg).

Starvation of bacteria also affects their ATP content. Strange et al. (94) investigated the effect of starvation on the ATP content of A. aerogenes at three temperatures - 20°c, 37°C, and 44°C. At 20°c the ATP content is steady at 2 pg/mg dry wt. AT 37°C ATP content rises initially but then falls to 1 ug/mg after 20 minutes. Finally, cells at

44°C have an increase in ATP from an initial level of 2.5 µg/mg to 6

µg/mg in approximately 2 minutes. After this time the ATP content drastically falls to less than 1 p.g/mg. Cell viability is not affected at 20°C and is affected only slightly at 37°C· however, at 44°C ' viability falls drastically. Starvation also decreases the rise in bacterial ATP when an anaerobic culture is transferred to an aerobic environment. Dawes and Large (18) studied the effect on the ATP levels in Zymomonas anaerobia due to starvation. ATP values in Z. anaerobia 16

fall as did those of A. aerogenes. ATP content decreases even more when cells are deprived of Mi+ Viability also decreases to a greater degree due to the absence of Mi+ Dawes and Large also studied the ability of starved cells to produce ATP upon the addition of a carbon source. When glucose is added to the medium, ATP content increases drastically and immediately (0 to almost 0.4 µg ATP/mg dry wt). ATP values rise further and then fall to the starved levels when glucose is depleted from the medium. When ethanol is added to starved cells in aerobic and anaerobic conditions, ATP values rise, with a greater rise in the aerated culture. Finally, Dawes and Large studied the ability of

Z. anaerobia, starved for 23, 47, or 143 hours, to produce ATP upon the addition of glucose. All three cultures retained the ability to synthesize ATP. However, after 15 days of starvation, only minimal amounts of ATP are synthesized. Bachi and Ettlinger (2) observed the drastic fall of ATP levels in Acetobacter aceti when starved of its energy source. ATP content falls from 7.6 moles/g dry wt (3.9 µg/mg dry wt) during log phase to nondetectable levels after a one hour of loss of an energy source. Cole et al. (15) found a similar drop of ATP in E. coli due to starvation. This drop is more pronounced under anaerobic conditions.

3. ATP Per Cell in Bacteria

If the bioluminescence assay is to be used as a rapid method for estimating bacterial numbers in foods, the assay must be proven as 17

reliable as the present system of enumeration. Quality control programs measure bacteria as colony forming units (CFU). Studies have been undertaken to determine the ATP content of bacteria.

There are only a few studies which investigated variations of

ATP/cell (or ATP/CFU) during the different phases of growth. Hamilton and Holm-Hansen (40) studied the ATP content in seven different marine bacteria. Four of the bacteria studied were Vibrio sp., Pseudomonas sp.,

Serratia sp., and Micrococcus sp. These bacteria have an ATP content of

1.3 to 4.0 fg/cell during the log phase. During early stationary phase,

the ATP content falls rapidly. After the stationary phase, ATP/cell

increases slightly. Nuzback et al. (76) investigated how ATP/cell varied through the growth cycle in rumen bacteria. Streptococcus bovis has 0.93 fg ATP/cell in early log phase but this concentration decreases

to 0.2 fg ATP/cell in the stationary phase. A mixed culture of rumen bacteria obtained from different animals was also studied. These cultures also show the decrease of ATP in the stationary phase.

However, ATP/cell varied widely between the two cultures. In the log phase one culture has an average concentration of 1.6 fg/cell while the second culture has 26 fg ATP/cell. These values fall to 0.3 and 0.6 fg

ATP/cell, respectively, in the stationary phase. D'Eustachio and Levin

(27) studied~ coli, Pseudomonas fluorescens, and Bacillus subtilis for variations of ATP content during the growth cycle. E. coli has a -10 constant ATP content of 1.45 x 10 pg/cell (0.145 fg/cell) throughout the growth cycle. P. fluorescens and B. subtilis have an average ATP -10 10 content of 1.6 x 10 µg/cell (0.16 fg/cell) and 2.82 x 10- µg/cell 18

(0.282 fg/cell). Both bacteria show a slight increase in cellular ATP during log phase and a decrease in the stationary phase.

Sharpe (87) stated that a bacterial cell's ATP content would be between 0.2 to 1.0 fg and this is dependent upon the size and metabolic state of the cell. Many investigators have studied the ATP content of various bacteria under a variety of environmental conditions.

Some of their findings are in Table 2. Unfortunately, growth conditions for these bacteria or the growth phase in which the bacteria were sampled were often times not stated. Thus, the comparison of ATP/cell may not be practical. Different extraction procedures would also affect the amount of ATP measured. It can be seen that the values for whatever reason, are not consistent. For instance, ATP/cell for ~ coli has been reported to be 0.8, 0.1, 0.41, 5.51, 1.07, and 2.0 fg

(5,13,16,25,26,63,75,98).

Theron, Prior, and Lategan (96) studied how temperature and media affected the ATP content of Enterobacter aerogenes. When~ aerogenes is stored at 2°c for 15 minutes and 2-8 days in a skim milk medium and a glucose mineral salts medium, the ATP levels in the cells fall to lower levels in the skim milk medium at every time period. If the cells are incubated at 30°c for 40 minutes before ATP/cell is measured, the values are much closer between the two growth media. Thus, environmental conditions do have an effect on the ATP content of a cell.

Several studies have involved the ATP content of mixed cultures in ground beef. Both Kennedy and Oblinger (54) and Stannard and Wood

(91,93) investigated the feasibility of using the bioluminescence assay 19

TABLE 2

ATP/cell for Selected Bacteria

ATP/cell (fg/cell) Ref Bacterium Avg Range Conditions Stated

63. Escherichia coli 0.8 0.3-1.7 25°C 63 Zoogloea ramigera 1. 3 0.31-3.08 35°C 63 Bacillus sp. 7.4 5.3-9.7 35°C

13 19 bacteria 0.028-0.89 Stationary (Selected) Phase

13 Aerobacter aero genes 0.28 13 Bacillus cereus O.ll 13 Escherichia coli 0.1 13 Pseudomonas fluorescens 0.31 13 StaEhylococcus aureus 0.64 13 Mycobacterium smegma tis 0.89

16 25 13 Bacteria 0.47 0.22-1.03 26 (Selected)

16 Aerobacter aero genes 0.24 16 Bacillus cereus 0.64 16 Bacillus subflava 1.03 16 Escherichia coli 0.41

75 Escherichia coli 5.51 a NB 37°C 75 CamEylobacter coli 2.06 b BB 37°C 75 CamEylobacter coli 2.37 c KB 37°C b 75 CamEylobacter jejuni 0.89 BB 37°C c 75 CamEylobacter jejuni l. ll KB 37°C 20

TABLE 2 (Cont'd)

ATP/cell for Selected Bacteria

ATP/cell (fg/cell) Ref Bacterium Avg Range Conditions Stated

88 Staphylococcus aureus 0.26-3.21 88 Lactobacillus acidophilus 1. 56-3. 62 88 Bacillus subtilis spores 0.018-0.046

5 9 Bacteria 0.026-2.2 (Selected) 5 Lactobacillus plantarum 2.0 5 Lactobacillus brevis 2.2 5 Pseudomonas fluorescens 0.96 5 Escherichia coli 1.07 5 Bacillus sp. 0.026 5 Aeromonas sp. 0.24

49 9 Bacteria 1. 3 0.010-2.4 25°C (Selected)

49 Aerobacter aero genes 1. 6 49 Bacillus cereus 2.2 49 Lactobacillus brevis 1.1 49 Flavobacillus proteus 0.01 49 Salmonella choleraesuis 2.4

98 Escherichia coli 2.0 37°C 98 Proteus sp. 0.5 37°C 98 Streptococcus sp. 1. 3 37°C a NB Nutrient Broth b BB Brucella Broth c KB Modified K Broth 21

to assess the quality of ground beef. Kennedy and Oblinger found bacteria to have a wide range of ATP/CFU 0.6 to 80.4 fg in bacteria.

However, the range is much narrower, 0.6 to 17.09 fg, when only those 7 samples with a bacterial load of 10 or greater are included. Stannard and Wood (91,93) found a smaller range of ATP values, 0.07 to 11.0 fg/CFU, in ground beef. The high ATP/CFU values are likewise found in samples with low levels of bacteria (less than 106). Both teams attributed these wide variations to either the lack of sensitivity of the instrwnent or to interference by nonrnicrobial ATP. Stannard and Wood also suggested the possibility of cell clwnping was masking the true

ATP/CFU values.

C. Bacterial Flora of Ground Beef

1. Natural Flora of Ground Beef

Animal muscle is usually considered to be sterile. However, the handling of the tissue during slaughter, processing, and packaging exposes fresh meat to several sources of contaminants. Due to the necessity of extra handling of ground beef and its greater surface area, bacterial levels are higher than in steaks or roast.

Many different bacteria are commonly associated with fresh meat.

These bacteria include:

Pseudomonas Acenetobacter-Moraxella Aeromonas Alteromonas Brochothrix thermosphacta 22

Bacillus Alcaligenes Lactobacilli Enterobacteriaceae Micrococcaceae Flavobacterium (1,11,51)

Ayres (1) found between 15-45% of the bacteria to be Pseudomonas and

Achromobacter (since reclassified to Alcaligenes, Acenetobacter-

Moraxella). Eribo and Jay (28) have reported that the incidence of

Acinetobacter spp. may be lower than has previously been reported.

2. Spoilage in Aerobically Packaged Ground Beef

For aerobically packaged meat at refrigerator temperatures (0-5°C)

the organisms that can utilize the available substrates the fastest and with the greatest affinity will dominate in the culture (or meat) (35).

These usually include Pseudomonas and Achromobacter (50,51). The

predominant spoilage organisms are Pseudomonas spp.

(1,33,37,50,52,55,80). Eribo and Jay (28) reported that Acenetobacter-

Moraxella are far less important to meat spoilage than previously

thought. Lactobacilli comprise less than 1% of these spoilage organisms

(80).

Jay (51) summarized the events that occur in meat as a result of microbial spoilage. The microbial flora on the meat stored at 5-7°C

change from a heterogeneous group to an almost homogeneous culture. The

spoilage process does not include significant proteolysis. Instead, once

the carbohydrate source in the meat is depleted, spoilage organisms use

amino acids or nucleotides in lieu of attacking larger proteins. When 23

bacteria use amino acids as an energy source, compounds such as ammonia, hydrogen sulfide, indole, and amines are produced. These by-products cause the pH of the meat to rise. It is these degradation products that produce· the off-odors and off-flavors typical of spoiled meat (35). The changes in beef due to spoilage will eventually result in an increase in

the hydration capacity of the beef proteins (51).

3. Spoilage in Vacuum Packaged Ground Beef

The flora of vacuum packed ground beef is quite different than that

of aerobically packed ground beef. Pierson et al. (80) reported that

there was little change in the number of fluorescent pseudomonads during

storage and the lactic acid bacteria make up 90% of the total number of bacteria. Enterococci are also present on the vacuum packed meat and

their numbers may increase slowly during storage. The number of

Microbacterium thermosphactum (~ thermosphacta), though high in numbers

initially, decrease in number during storage. The lactobacilli apparently inhibit competitors such as M. thermosphactum and

Enterobacter due to the production of an antimicrobial substance by

lactobacilli (38,74,84).

Spoilage in vacuum packed meat stored at low temperatures (between

0-5°C) has a much slower onset than in aerobically packed meat. After

exposing the meat to air, color and odor do not differ substantially

from that of fresh beef; although, a slight sour flavor develops after

10 days (80). This sour flavor results from the accumulation of short chain fatty acids produced by lactobacilli. B. thermosphacta causes 24

spoilage by the same mechanism in a lesser amount of time (36). These organic acids result in a decrease of the meat's pH (52). III Materials and Methods

A. Ground Beef Spoilage Organisms

1. Isolation

Twenty grams of retail ground beef was stored at s0 c in oxygen

permeable packages (in disposable petri dish covered with Reynolds

plastic wrap) and in vacuum packages (Cryovac p850).

One aerobic-and one vacuum-packed sample was selected after 3, 5, 7,

10, and 13 days storage. An 11 g sample of ground beef from each package was diluted in 99 ml of 0.1% peptone and homogenized in a Stomacher

(Tekmar Products). The homogenized sample was serially diluted in 99 ml

of 0.1% peptone and plated, using spread and pour plate methods, on MRS

agar, Trypticase soy agar (TSA), Gardner's medium (34), and Eosin methylene blue agar (EMB agar). TSA and Gardner's plates were incubated

at 20°C for 2 days. MRS and EMB plates were incubated at 35°C for 2 days.

Colonies were examined under a dissecting microscope. Isolates were

selected due to differing colony morphology and appearance on

differential media. All isolates were restreaked to assure colony purity

and a single isolated colony was selected for a stock culture. In all,

twenty-six colonies were isolated.

25 26

2. Identification

Each isolate was gram stained. The genus was determined using

Vanderzant and Nickelson's identification scheme (101).

Two of the more common genera isolated from aerobically packaged ground beef and one of the most common genus isolated from vacuum packaged ground beef were selected for further testing.

The Lactobacillus isolates were speciated using the identification method of the Virginia Polytechnic Institute Anaerobe Laboratory (43).

The Pseudomonas was confirmed using Thornley's medium 2A (100) as a

Hugh and Leifson oxidation-fermentation medium.

B. ATP Determination

1. Equipment

The Picolite 6200 (Packard Instrument Co., United Technologies) was used to determine ATP content of culture samples. The Picolite 6200 was interfaced with a Hewlett Packard 85 computer. The Hewlett Packard 85 was used to process and analyze raw data generated by the Picolite.

The Picolite's pump lines were cleaned thoroughly each day (or more often if blank readings were high). The pump lines were thoroughly flushed with boiling distilled, deionized water (approximately 200 ml) which had been filter sterilized (through a 0.22 µm filter) and then autoclaved (highly purified, low ATP water -- HP water). The pump lines were pumped dry and then primed with Picozyme F or Internal Standard. 27

The instrument was submitted to two chamber quality control checks each day. The first sampled the empty chambers to determine the inherent background of the chambers and to check for any abnormally high readings. The second quality control procedure sampled a chamber repetitively in order to determine the average with chamber background.

Pump volumes were checked periodically but when no abnormalities were noticed, daily checks were discontinued.

2. Reagent Preparation

a. Water

HP water was used to make all bioluminescence assay reagents.

b. Luciferin - Luciferase

A dehydrated form of highly purified luciferin-luciferase with Mg 2+

(Picozyme F, Packard Insturments) was rehydrated with 8 ml of HP water.

The solution was mixed gently and allowed to stand 30 minutes before use

(77).

c. Buffer

Hank's - Tris buffer was prepared by adding 1.818 g of Trizma HCl

(Sigma) and 0.417 g of Trizma Base (Sigma) to Hank's buffer (a 1 liter package) (Sigma). One liter of HP water was added to these compounds and the pH was adjusted to 7.75 + 0.2. The solution was stored under refrigeration (77). 28

d. Extraction Reagent

The extraction reagent was prepared by adding 0.125 g of Hyamine lOX (Sigma) and 0.38 g of disodium ethylene diamine tetraacetic acid

(EDTA) (Sigma) to 1 liter of Hank's - Tris buffer. The pH was adjusted to pH 7.75 ± 0.2. The solution was stored under refrigeration (77).

e. Stock ATP Standard

A dehydrated 10-5 molar stock of ATP (Picochec, Packard

Instruments) was rehydrated with 2 ml of Hank's - Tris buffer and mixed.

The solution was stored dehydrated under refrigeration (77).

f. ATP Standards

Picochec stock was serially diluted with extraction reagent to obtain ATP concentrations of 10-6 to 10-ll molar. Solutions were stored frozen (77).

g. Internal Standard

Internal standard was prepared by mixing 6.0 ml of Hank's - Tris buffer, 3.7 ml of HP water, and 0.3 ml of Picochec. The solution was stored frozen (77).

h. Stock Phosphate Buffer

A stock phosphate buffer (0.25 M) was prepared by mixing 34 g of

KH 2Po4 (Sigma) in 500 ml of HP water. The pH was adjusted to pH 7.2 with

NaOH, and the volume was adju~ted to 1 liter with HP water. The solution was stored under refrigeration (77). 29

i. Butterfield's Phosphate Buffer

The Butterfield's phosphate buffer (0.0003 M) was prepared by diluting 1.2 ml of 0.25 molar phosphate buffer in 1 liter of HP water, autoclaving, and storing at room temperature (77).

3. Instrument Program

The Picolite 6200 was programmed to operated in the following manner for both ATP calibrations and sample analyses:

For each sample or ATP concentration, 200 µl of enzyme was injected

into a vial holding the sample. After a five second delay, a fifteen

second count of the emitted light was made. This first count was

recorded as counts per second (CPSl). This count was followed by the

injection of 100 µl of internal standard, followed by a five second

delay. Then another fifteen second count of the emitted light was made

recorded as counts per second (CPS2).

A standard ATP curve was constructed. The ATP content of unknown

samples was determined from the ATP calibration curve (77).

4. ATP Calibration

Triplicate samples of three serial dilutions of ATP (e.g.

10-8 , 10- 9 Molar) plus three blank samples (extracting solution only) were analyzed. The counts for each set of three ATP samples and their

corresponding internal standards were each averaged. A ratio of the counts was determined using the following relationship: 30

Ratio ~ CPS2 / (CPSl - Blank)

A linear regression of the data was constructed using the log of the ratio versus the log of the ATP content. An ATP calibration was made each day that samples were analyzed (77).

5. ATP Determinations of Unknown Samples

Samples were analyzed in triplicate. The count ratio was computed and averaged. The log of this ratio was used to determine the ATP content of the sample (77).

C. Media Preparation

1. Growth Media

MRS broth (Difeo) (22), adjusted to a pH of 6.5 or 6.0 before autoclaving, was used for the growth of the lactobacilli. Nutrient broth

(BBL), adjusted to a pH of 6.7 or 6.0 before autoclaving, was used for the growth of the Pseudomonas sp.

2. Enumeration Media

Standard methods agar (BBL) was used for the enumeration of the

Pseudomonas sp. MRS agar (MRS broth+ 1.25% granulated agar (BBL)) was used for the enumeration of the lactobacilli. 31

D. Experimental Protocol

1. Factors Examined

Factors examined include bacteria, atmosphere, aeration, pH, temperature, and phase of growth. The variables within each factor are shown in Table 3. All combinations of these variables were examined.

2. Preliminary Growth Curve

A preliminary growth curve was monitored for each set of factors in order to estimate times of each growth phase. The preparation and environmental conditions used to prepare this curve were the same in the sampling experiments. Nephalo culture flasks (Belco) were prepared with the appropriate media. The flasks were stoppered with cotton or neoprene stoppers (depending on the atmosphere). The culture flasks were inoculated with 2.0 ml of culture and adjusted to an optical density of

1.0. The headspace of the culture flask was flushed with co2 or N2 for 30 or 60 seconds, respectively, when appropriate. The optical density was measured at 610 nm and monitored throughout the growth phase - from inoculation to late stationary phase. From this data, approximate times were selected for sampling each of the different phases. 32

Table 3

EXPERIMENTAL FACTORS TESTED

Factor Code Variable

Bacteria 5 Pseudomonas sp. 15 Lactobacillus brevis 17 Lactobacillus jensenii

Atmosphere A Air c Carbon Dioxide N Nitrogen

Temperature* 35 35°C 20 20°C 3 3°C

Aeration* SH Shaking ST Static pH R 6.5 or 6.7 B 6.0

Phase of Growth 0 Inoculum 1 Lag Phase 2 Log Phase 3 Early Stationary Phase 4 Late Stationary Phase

* 20°c, Shaking was not done 33

3. Sampling Growth Curves

Cultures and media were prepared as previously described. Duplicate cultures were prepared for each of the various combinations of experimental factors. The optical density was checked at the time determined from the preliminary growth curve. When the culture had reached the range of the desired growth phase, the flask was swirled to obtain a homogeneous mixture and 3.0 ml of the culture medium was withdrawn from the culture flask and placed in a sterile, disposable sample vial. All of the following inoculations were made using the EDP electronic digital pipette. The sample vial was vortexed and 0.1 ml of the culture was withdrawn and diluted in 9.9 ml of a 0.0003 M phosphate -2 buffer blank. From this 10 dilution, 0.3 ml was diluted in 0.9 ml of extraction reagent in a Sarstedt tube. This tube was vortexed, covered with Parafilm, labeled, and frozen at -16°c until ATP content was determined. The 10-2 dilution was then serially diluted 100-fold in

0.0003 M phosphate buffer and plated in duplicate at three appropriate dilutions. Lactobacilli were incubated at 35°C for 48 hours and

Pseudomonas sp. was incubated at 20°C for 48 hours. Finally, the pH of the culture medium in the original sample vial was determined.

4. Growth Curves With Sonication

In a separate experiment, samples taken in each growth phase were sonicated in order to determine if ATP per cell values were artificially high due to cell clumping. A Fisher sonic dismembrator was used at 35% 34

output for 15 and 30 seconds. The sample vial was immersed in an ice bath during 30 second sonication in order to avoid excess heat build up which might cause bacterial cell damage. Normal (unsonicated) controls were also run in parallel with sonicated samples.

E. Statistical Analysis

Data in these experiments was analyzed using the ANOVA procedure

(SAS Institute, Cary, NC). All analyses were conducted at the 0.05 level of significance unless otherwise specified. The pH was not included as main factor since the experiment was designed so that pH was determined by the medium used for the bacterium. For this reason, pH was "nested" within the bacterium and therefore was considered to be an interaction

term. A Duncan's multiple range test was performed on the main factors

to determine which variables of a factor differed significantly from

each other. IV Results and Discussion

A. Spoilage Isolates

There are a variety of bacteria associated with meat spoilage. Over

the years there has been some attempt to isolate and classify meat

spoilage organisms. Many studies were done prior to the institution of present day classification schemes (1) and often times spoilage bacteria were not identified past genus. Thus, there has been some confusion and

disagreement as to the identity of the major spoilage organisms of meat.

Because of this confusion as well as the non-availability of isolates of

spoilage bacteria from ground beef, bacteria from spoiled ground beef

were isolated for this study.

Aerobic and anaerobically packaged meat was sampled through 13 days

storage at 5°C. Initial isolation was made only on the basis of

d~fferential colony morphology and colony appearance on differential

media. It should be noted that this procedure was undertaken to isolate

representative ground beef spoilage organisms - not to isolate all of

the possible types of meat spoilage organisms. The identity of these

isolates is shown in Table 4.

Three organisms - two from aerobic packages and one from a vacuum

package - were selected for use in this study. Selection was based on how frequently the genus was isolated (i.e. was it a commonly isolated

organism). The organisms from aerobic packages selected for this study were #5 - a Pseudomonas and #17 - a Lactobacillus. The organism chosen

35 36

Table 4

Bacteria Isolated From Spoiled Ground Beef

Packaging Isolation Isolate Identification Condition Medium Number

Aerobic MRS Lactobacillus l,2,16,17,18a,23 Not Identified 3,22

TSA Pseudomonas 4,5,6,8,18b,19,20 Bacillus 7

Gardner's Brochothrix 21

EMB Aeromonas 24 Enterobacter 25

Anaerobic MRS Lactobacillus 9,10,11,15

TSA Streptococcus 12

Gardner's Brochothrix 13,14 37

from the vacuum package was #15 - a Lactobacillus.

The organisms isolated from the retail ground beef correlated well with the expected types of spoilage organisms. There were no

Acinetobacter sp. isolated. This agrees with the findings of Eribo and

Jay (28) that the importance of Acinetobacter as a spoilage organism in beef has been overestimated. The frequent isolation of lactobacilli in aerobically packaged beef is rather surprising (11,85). The Pseudomonas isolate was not identified beyond genus. Pseudomonas sp. produced ammonia from arginine, a key characteristic of pseudomonads (100), and was fluorescent on Pseudomonas F agar (Difeo).

The lactobacilli were characterized by the method of Holdeman, Cato, and Moore (43). Each isolate was confirmed to be a Lactobacillus.

Isolate 17 was identified as L. jensenii. Isolate 15 was identified as

L. brevis.

B. Preliminary Growth Curves

A growth curve was made for each bacterium at each growth condition.

Only selected growth curves are presented in Figures 3 - 13. Curves not shown for L. brevis, L. jensenii, and Pseudomonas sp. did not differ dramatically in form from those curves shown in the corresponding atmosphere. For instance, cultures grown at the lower pH (6.0) in air paralleled the matching curve at the higher pH in air, differing only in a slightly longer lag phase. Many trends can be seen from these graphs.

How these growth trends translate into variations of ATP/cell will be 1.•

1 .... ,~-==::O--o------0 1.2 ?'--~ ....,..:;;;." / ,...... ~ 1.0 / ,...... en /" /'" ~ .. /~)"' :;:>8 ,,~/ Q. 0 /•/ •• // /~/ v..> .... 00

.2

0 2 4 6 8 10 12 14 16 18 20 22 24 Time (hours)

Figure 3

Growth curve of~ brevis at 3S 0 c for pH 6.5. The growth curve is shown for three atmospheres and two types of aeration by the change in optical density (610 run) over time. Symbols: air (o); carbon dioxide (D); nitrogen (A); shaking culture (solid line); static culture (dashed line). 1.e ,J ---_,--· ----~~------~------4------,, ...... ' ,, , 1.2 .,,,, .. ---~-.... -, -....- ,' / / ,,' ,..' / ,, , ,,.... ,• ,,,,, / " .,,,,,,..,,,, ,,,,,,,,,,""" ~ 1.0+ / ,,,,, / ,,,,,,,. ,,..../r ,,.• J ...... // //"' / / , ,,. ,,. :§ I / / / 8- ...... / "/ ,'}/ w //// /'/ \0 // .... /~/ ,,,(;7 ?"' ,,,""""" _/ / .,,,"" .2+ .,,,..,,., / """'__/.-:'..a"'.. -- ,,.•"" . ... ,,. 0 ----_.,,,,. . 5 7 9 11 13 15 17 19 21 23 25 27 29 Time (hours)

Figure 4

Growth curve of 1..:.. brevis at 20°C in static culture. The growth curve is shown for three atmospheres and two values of pH by the change in optical density (610 nm) over time. Symbols: air, pH 6.5 (o); carbon dioxide, pH 6.5 (O); carbon dioxide, pH 6.0 (•); nitrogen, pH 6.5 {.6); static culture {dashed line). ,.. ,....

t.2

~ 1.0 cCD ~ •• -a(.) ~ a.. 0 •• ~ 0 ....

.2

0 2 4 6 8 10 12 14 TI me (days)

Figure 5

0 Growth curve of 1..:_ brevis at 3 C, in shaking culture, and pH 6.5. The growth curve is shown for three atmospheres by the change in optical density (610 run) over time. Symbols: air (o); carbon dioxide (D); nitrogen (A); shaking culture (solid line)...... ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-.

t.4 ~------· ~....------· t.2

~ t.o ~ g •• :g_ 0 .•

~ t-'

.4

.2

0 1 I f" f I I I I I I I I I I I I I I I I I I I I I I 2 4 6 8 10 12 14 16 18 20 22 24 Time (hours)

Figure 6

0 Growth curve of 1..:_ jensenii at 35 C and pH 6.5. The growth curve is shown for three atmospheres and in two types of aeration by the change in optical density (610 run) over time. Symbols: air (o); carbon dioxide (O); nitrogen(£:.); shaking culture (solid line); static culture (dashed line). '··r------

t.4 ~::r'~-----·------~------;;--- .,-;~ .,,,,,,, .,,,.,,, .,,,"" t.2 ~"" ""/ .,,, ,,""' / / // //, ~ t.o+ / // I'/ /// / / ,./ ~ .a+ /' // / g I / // ~ I/' _// I/ I" 8" .e+ / // ,' .i:-- e// / N /~ ..I/ .4+ // /r /1/ / .2+ .... -----/ _...st'" o I _ _,.4---_.,.-_. I I I I I I I I I I I I I I I I I I I I I I I I I I 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (hours)

Figure 7

Growth curve of~ jensenii at 20°C, pH 6.5, and static culture. The growth curve is shown for three atmospheres by the change in optical density (610 run) over time. Symbols: air (o); carbon dioxide (0); nitrogen (A); static culture (dashed line). 1.e

1.4

1.2

~ 1.0 ·ca c! •• -a(.) :i::J 0.. 0 •• ~ w ...

.2

0 2 4 6 a 10 12 14 TI me (days)

Figure 8

Growth curve of~ jensenii at 3°c and pH 6.5. The growth curve is shown for three atmospheres and in two types of aeration by the change in optical density (610 run) over time. Symbols: air (o); carbon dioxide (O); nitrogen (A); shaking culture (solid line); static culture (dashed line). ••

. 7 ,.,.,.,. ,.,....------· •• ,,...- ,,-" --- ~.a ,,-" ·-°' ,,-" ~ ,,-" - .... ,,-" ~ ,..-" 0.. ,,.,,. 0 .3 ,,-" po .,,..,,. po _,,Jlf"' .2 .,; .,,,.,,, ,.,.,.,.__ r____ _...... ------_..-;j;------· .1 .,..,.,.,.,.,.,. ",.,. 0 0 10 20 30 40 50 60 70 80 90 100 110 TI me (hours)

Figure 9

Growth curve of Pseudomonas sp. at pH 6.7, in air. The growth curve is shown for two types of aeration by the change in optical density (610 run) over time. Symbols: 3S 0 c (o); 20°c (O); shaking culture (solid line); static culture (dashed line). ,..

1.4

1.2

~ 1.0 ·-(I) c! c; .. ~ & •• ~ Vl

.4 ~ -- ...--- _.------~-- ~------·-- -- .2 ---- 0 ... ---- 2 4 6 8 10 12 14 TI me (days)

Figure 10

Growth curve of Pseudomonas sp. at 3 0 C, pH 6.7, in air. The growth curve is shown for two types of aeration by the change in optical density (610 nm) over time. Symbols: air (o); shaking culture (solid line); static culture (dashed line). ••• ,....

t.2

~ t.O C'I> ~ .B .. 8- •• .j:'- .... °'

.2

0 2 4 6 8 10 12 14 16 18 20 22 24 Time (hours)

Figure 11

Comparison of growth curves for h:_ brevis, h:_ jensenii, and Pseudomonas sp. at 35°c at pH 6.5, in static culture, in air. The growth curve is shown by the change in optical density (610 nm) over time. Symbols: 1.:.. brevis (o); h:_ jensenii (D); Pseudomonas sp. (.6). ,.. ,.... r

t.2+ //! ,,,,// f ... Ir ,,,, 8 .. ,, ~ ,, 0. .... 0 ,, .. ---""' ______•• ,, --~ .p.. ,,,, -- -...J .4+ ,,,, __ .. ------/p __.., -- .2+ --- ,..--//// _.. ---- 0 . 0 10 20 30 40 50 60 70 80 90 100 110 TI me (hours)

Figure 12

Comparison of growth curves for h:_ brevis, h:_ jensenii, and Pseudomonas sp. at 20°c, at pH 6.5, in static culture, in air. The growth curve is shown by the change in optical density (610 nm) over time. Symbols: h:_ brevis (o); h:. jensenii (O); Pseudomonas sp. (Ll). 1.e

1.4

Ml ------·------a :::~---0-:::::--.:::-:::--~ - -·

~ 1.0 en ,! •• ] 8" •• ~ 00 .... ------·-----',•------·------.2

0 2 4 6 8 10 12 14 TI me (days)

Figure 13

0 Comparison of growth curves for~ brevis, ~ jensenii, and Pseudomonas sp. at 3 C at pH 6.5 in air. The growth curve is shown for two types of aeration by the change in optical density (610 nm) over time. Symbols: ~ brevis (o); ~ jensenii (D); Pseudomonas sp. (A); shaking culture (solid line); static culture (dashed line). 49

discussed in the next section.

In general, .h:_ brevis (# 15) grew best at 35°C but also grew well at

20°c and 3°C (Fig. 3 -5). At 20°c the time required to reach late

stationary phase was approximately one and one-half times longer than at

35°C. At 3°C stationary phase of growth was reached in 11-14 days. ' Surprisingly for a Lactobacillus, the shaking cultures grew better

(faster) than static cultures at all temperatures. This strain of L. brevis was quite aerotolerant and grew best when shaken. The pH

except at 20°c in co2 , and in static culture - delayed the growth curve

only by a very small margin. In this one case, the lower pH enhanced

growth. Growth in different atmospheres was similar at each temperature

except at 3°c. While the best growth was in air at 35°C and 20°C, growth was by far the best in nitrogen at the lower temperature.

L. jensenii (# 17) grew best at 35°c but grew well at 20°C and 3°C

(Fig. 6 - 8). At 20°C the time to reach stationary phase was

0 approximately one and one half times longer than at 35°c. At 3 C, 10 to

13 days were required for growth to reach late stationary phase. Like L.

brevis, shaking cultures grew better than did static cultures (i.e. the

lag phase was shortened). The best growth was in air at 20°c and 35°C.

Interestingly, at 3°c L. jensenii in air appeared to exhibit some

inhibition by o2 and the culture in the nitrogen atmosphere had the 0 fastest growth at 3 C. The pH, as with L. brevis, resulted only in a

slight increase in the lag phase for the more acidic medium.

Since Pseudomonas is a genus of obligate aerobes, this culture was

not grown in co2 or N2 . Pseudomonas sp. (# 5) grew best at 3°c shaking so

and 20°c static (Fig. 9 - 10). The shaking cultures grew better than the static cultures since shaking provided more effective oxygen transfer to the medium.

Figures 11, 12, and 13 show a graphic comparison of the growth

0 0 curves of L. brevis, .h:_ jensenii, and Pseudomonas sp. at 35 C, 20 C, and

3°c. respectively. These graphs were made using the shaking culture at the pH specified for the medium.

C. Variations in ATP Content in Bacteria

1. Statistical Analysis

The data were analyzed for statistical significance due to treatment using an analysis of variance procedure. This procedure is used to determine if the variation of mean ATP/cell between treatments (factors) is due to experimental error or is in fact due to real differences in the mean ATP/cell. The analysis showed that ATP/cell was significantly different at the 0.05 level for all of these treatments. (The treatments in this case were bacteria, temperature, atmosphere, aeration, and phase of growth.) All factor interactions except those listed in Table 5 were also significantly different.

The mean ATP/cell value for all shaking cultures (2.51 fg) was significantly different (p ~ 0.05) from static cultures (2.40 fg). The mean ATP/cell for temperature - 3S 0 c (2.76 fg), 20°c (1.97 fg), and 3°c

(1.72 fg) - were significantly different (p ~ 0.05). Late stationary 51

Table 5

NONSIGNIFICANT INTERACTIONS OF MAIN EXPERIMENTAL FACTORS

Alpha - 0.05

Atmosphere * pH pH * Aeration Atmosphere * pH * Temperature pH * Temperature * Aeration pH * Temperature * Condition * Phase 52

(2.80 fg), inoculum (2.52 fg), early stationary (2.33 fg), and log phase

(1.90 fg) of growth were also significantly different (p ~ 0.05). The lag (1.90 fg) and log (1.90 fg) phase did not differ significantly from each other (p > 0.05). A Duncan's test for bacterium and atmosphere could not be done because some of the mean ATP values were artificially low due to inclusion of 0.0 fg/cell values. These zero values were necessary due to the lack of growth of Pseudomonas sp. in co 2 and N2 . Thus, no conclusions can be drawn statistically for the mean ATP/cell values for bacterium and atmosphere. The mean ATP/cell values for the bacteria were 2.71 fg for L. brevis, 2.20 fg for .h:_ jensenii, and 1.36 fg for Pseudomonas sp. The two lactobacilli values were significantly different (p ~ 0.05) from each other. The value for the Pseudomonas sp., while it is the true mean ATP value for the bacterium, cannot be commented on in the context of the Duncan's test. The mean ATP/cell values in different atmospheres were 2.59 fg in air, 2.37 fg in co 2 and

1.19 fg in N2 .

2. Effect of Bacterium, Temperature, Growth Atmosphere, pH, and Aeration on ATP Content

The effect that different atmospheres had on the three bacteria for each growth phase is shown graphically in Figures 14 through 16. Figure

14 shows the ATP content of .h:_ brevis in air, co 2 , and N2 . L. brevis had a higher ATP content in all phases of growth when it was in air. The cellular ATP values rose steadily from the lag phase to late stationary phase. The culture had an average ATP content of 4.22 fg/cell throughout 7---~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~--.

•

II c:- a; u ...... ::::,..oa• ~ (..) 3 ......

U1 ~ ~ VJ a I ...... / I

,

o..._~~~~~~--~~~~~~~--~~~~~~--ii--~~~~~~--~~~~~~~11--~~~~~~- 0 1 2 3 4 Phase of Growth Figure 14

Comparison of ATP/cell at each phase of growth for 1..:_ brevis in three atmospheres. Abbreviations are those defined in Table 2. Symbols: air (o); carbon dioxide (O); nitrogen (A). ~ ..... ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-.

~

C"' 8 ...... gt --@2 (.) ......

~ lJI ~ +--

1 I I

o--~~~~~~---~~~~~~~--~~~~~~~i--~~~~~~--~~~~~~~+-~~~~~~- 0 1 2 3 4 Phase of Growth Figure 15

Comparison of ATP/cell at each phase of growth for~ jensenii in three atmospheres. Abbreviations are those defined in Table 2. Symbols: air (o); carbon dioxide (0); nitrogen (.6.). 3.o .... ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

a.e

-::::::::- a.o 8 ...... :::::,Ot [ij 1.a u ......

VI ~ 1.0+ I ~ I VI

.a

o---~~~~~~_,.~~~~~~~t-~~~~~~....-~~~~~~--t~~~~~~~+-~~~~~~--4 0 1 2 3 4 Phase of Growth Figure 16

Comparison of ATP/cell at each phase of growth for Pseudomonas sp. in air. Abbreviations are those defined in Table 2. 56

it's growth. The culture grown in co2 also showed a rise in ATP content throughout the growth cycle but this value was about half of that when grown aerobically. The average ATP content from all phases was 2.12 fg/cell. The culture grown in N2 had the lowest average ATP content (1.79 fg/cell). The bacterial ATP content was less variable when grown in N2 but there was a slight rise in ATP content as the culture aged. Literature values of 2.2 fg/cell (5) and 1.1 fg/cell (49) for L. brevis are half that of the average in air; however, neither Bamgart et al. (5) nor Hysert et al. (49) stated growth conditions or growth phase for which these numbers were obtained.

L. jensenii (Fig. 15) had high levels of cellular ATP when grown in co2 as compared to in air. This is in contrast to the findings for L. brevis under the same conditions. In air, the ATP/cell dropped slightly in log phase and then rose in stationary phase. A similar drop in log phase occurred when h.:_ jensenii was grown in co 2. The cellular ATP also dropped slightly in late stationary phase. ATP content was the most stable in N2 just as was found for L. brevis. In N2 , the cellular ATP values rose slightly throughout the culture's growth cycle. Average ATP values for each atmosphere were 2.02, 2.63, and 1.78 fg/cell in air, co2 , and N2 , respectively. There are literature reports of 2.0 fg/cell (5) and 1.56 to 3.62 fg/cell (88) for L. plantarum and h.:_ acidophilus, respectively. These correspond well with 2.02 fg/cell, the average value for h.:_ jensenii. No values for h.:_ jensenii have been reported.

The ATP content for Pseudomonas sp. was highest during the lag phase and fell as the culture aged (Fig. 16). The average ATP content was 1.36 57

fg/cell. This value is higher than that for P. fluorescens (0.96

fg/cell) (5). Chappelle and Levin (13) obtained a value of 0.31 fg/cell

for P. fluorescens in the stationary phase. The Pseudomonas used had an

ATP value of 1.1 fg/cell during the stationary phase. Chappelle and

Levin's value is three times lower than the value obtained here. Perhaps

procedural or strain differences were sufficient to cause the large variation in the two values.

A change from aerobic to anaerobic conditions resulted in a drop in

the cellular ATP levels of L. brevis. This agrees with the findings of

Knowles and Smith (57) with A. vinelandii, although the decrease was not

quite as great. The decrease was of the same degree as reported by

Strange et al. (94) for ~ aerogenes and Cole et al. (15) in~ coli.

The drop in cellular ATP was not seen in L. jensenii. However, L.

jensenii is usually aerotolerant and growth enhanced by co2 . The variation in cellular ATP content can be seen in finer detail when separated by temperature, atmosphere, aeration, and bacterium (Fig.

17 - 23).

At 3, 20, and 35 0 C, L. brevis had the higher ATP content in the

aerobic, static cultures (Fig. 17). The values ranged from 1.04 to 13.92

fg/cell over the three temperatures. At 35°C the ATP content increased

through the stationary phase in both the shaking and static cultures.

The same increase of ATP content was present in static culture at 20°c.

At 3°C the ATP content was lower than at the other temperatures in all

phases except in the lag phase. ATP content declined in the log phase

and increased in the stationary phase for the shaking culture. In the 14--~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~--~~~~~~---.

12

10 -::::::- 11 ...... en a .:t:,

~ ...... • VI ~ CX> 4

2

o._~~~~~~--+~~~~~~~+-~~~~~~-+~~~~~~~+-~~~~~~-+~~~~~~---t 0 1 2 3 4 Phase of Growth Figure 17

Comparison of ATP/cell at each phase of growth for L. brevis in air, at three temperatures, and two - 0 0 0 types of aeration. Abbreviations are those defined in Table 2. Symbols; 35 C (o); 20 C (D); 3 C (.£\); shaking culture (solid line); static culture (dashed line). ·--~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-.

e ID /1,,P

...... // Q) .. /.? ...._(.) ~ .s ...._~ Ul ~2 '° ______~. .. ------~ 1

o.,,_~~~~~~--t,__~~~~~~-+-~~~~~~~1--~~~~~~-+~~~~~~~t-~~~~~~--1 0 1 2 3 4 Phase of Growth Figure 18

Comparison of ATP/cell at each phase of growth for ~ brevis in carbon dioxide, a~ three tewperature~, and two types of aeration. Abbreviations are those defined in Table 2. Symbols; 35 C (o); 20 C (O); 3 C (A); shaking culture (solid line); static culture (dashed line). ..,....~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-,

3

Ci> -u ...... ,.------0 ~ . ,g / .. ----- 2 // - Q) ,,.,,.~/ u ______-1!:1" / ...... ______// .. 0\ ~ 0

1

o ...... ~~~~~~~+-~~~~~~~t--~~~~~~--l1--~~~~~~--1~~~~~~~-t-~~~~~~~~ 0 1 2 3 4 Phase of Growth Figure 19

Comparison of ATP/cell at each phase of growth for L. brevis in nitrogen at three temperatures, and two - 0 0 0 types of aeration. Abbreviations are those defined in Table 2. Symbols; 35 C (o); 20 C (O); 3 C (.£\); shaking culture (solid line); static culture (dashed line). &---~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-.

...

/.. C- / / B / / """"- .3 / / £ / /~-=--==---- ...... /~ ~ --· """"-2 ...... // "' // °'I-' ~ -'A/ // --~/ --- .... t ------.....------

o.-.~~~~~~-1~~~~~~~--~~~~~~--1,__~~~~~~--~~~~~~~..,_~~~~~~- 0 1 2 3 4 Phase of Growth Figure 20

Comparison of ATP/cell at each phase of growth for L. jensenii in air at three temperatures, and two ~ 0 0 0 types of aeration. Abbreviations are those defined in Table 2. Symbols; 35 C (o); 20 C (O); 3 C (.6.); shaking culture (solid line); static culture (dashed line)...... ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-,

II

-==-"iii ... / / / --o (,) ·------/ / ...... / / / ~ / _.. Ill. 3 ''~-- ..... , ~ --- ...... -' ' - .... , ...... / r--- ', ---- ', 0\ / / r ', N ~2 ', / .,,,x, . __ ... __ ... ____ .... ~ / ', - fl!' ', --- t ~ --

o--~~~~~~~+-~~~~~~~.-~~~~~~--ii--~~~~~~--i~~~~~~~..... ~~~~~~~-t 0 1 2 3 4 Phase of Growth Figure 21

Comparison of ATP/cell at each phase of growth for L. jensenii in carbon dioxide at three temperatures, and two types of aeration. Abbreviations are those- defined in Table 2. Symbols; 35 0 C (o); 20 0 C (O); 3 0 C (~); shaking culture (solid line); static culture (dashed line). a...-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-.

"t \ .... \ \ \ \ C'" \ \ 8 \ \ "3 \ ,g \ \ \ \ ~ \ "2 \ °'w ~ ~---~-' ' ' ~-~--~~--- /"/ ------~ ~""' ...... ' ' '" ...... ______.... 1 .,,,""' ...... ' ' ~ .,,,.,,, ...... '~

o.._~~~~~~--ii--~~~~~~-+-~~~~~~--li--~~~~~~-+~~~~~~~+-~~~~~~~ 0 1 2 3 4 Phase of Growth Figure 22

Comparison of ATP/cell at each phase of growth for L. jensenii in nitrogen at three temperatures, and - 0 0 0 two types of aeration. Abbreviations are those defined in Table 2. Symbols; 35 C (o); 20 C (O); 3 C (A); shaking culture (solid line); static culture (dashed line)...... ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-.

8

-:::::::-- 84 ...._ £ ~ ...._~ /,,,...._ 0\ //// .i::-- ~2 // ·------:.L~~ - /~/ .A ---- ~ /.~/ .,,..,,. ' ------u---... --- -- // .,,..,,. ,,z / ' ' 1 ~,~ / ' ', ', '•------...______0--~~~~~~-+~~~~~~-+~~~~~~~1--~~~~~~+-~~~~~~~~~~~~~--1 0 1 2 3 4 Phase of Growth Figure 23

Comparison of ATP/cell at each phase of growth for Pseudomonas sp. in air at three temperatures, and two types of aeration. Abbreviations are those defined in Table 2. Symbols; 3S 0 c (o); 20°C (0); 3°c (~); shaking culture (solid line); static culture {dashed line). 65

static culture the ATP content declined through early stationary phase.

Figure 18 shows the growth of ~ brevis in co2 . As in air, ATP content was lowest at 3°C. The cellular ATP content for corresponding shaking and static cultures were very close for each temperature. The range in ATP values was 0.54 to 5.02 fg/cell in co2 . ATP content increased at all temperatures as the culture aged.

The range of cellular ATP in the presence of N2 was 0.99 to 2.55 fg/cell (Fig. 19). At all temperatures the ATP content in shaking and s t a ti c cult ures was very c 1 ose. Th e ATP va 1 ues were lowest a t 3oc.

Except for those cultures which were shaken at ATP content increased throughout the growth cycle. At 3°c the ATP values fell slightly from the lag to the log phase and then rose in the stationary phase.

L. brevis showed a more stable ATP content as the culture aged in a nitrogen atmosphere and the widest range of ATP content in an air atmosphere. The lowest and most stable cellular ATP values was at 3°c in each atmosphere. It is possible that cellular clumping occurred at 20°c and 35°C in the stationary phase. This is discussed in the following section. The rise in ATP from lag to late stationary phase is opposite to the findings of Forsberg and Lam (32) for S. ruminantium and of

Hamilton and Holm-Hansen (40) for marine bacteria. Those experimental factors which resulted in a rise of ATP content from lag to log phases correlate to the findings of Knowles and Smith (57) for A. vinelandii.

The growth of~ jensenii in air, co2 , and N2 are shown in Figures

20 to 22. When grown in air (Fig. 20), the ATP values ranged from 0.88 66

to 4.57 fg/cell. At each temperature, the shaking and static cultures were similar in ATP content. At 35°c cellular ATP increased in the stationary phase while it fell at 20°C. At 3°C the ATP content rose in the stationary phase of the static culture and fell in the stationary phase of the shaking culture.

ATP values of L. jensenii in co 2 (Fig. 21) were higher and had a wider range (0.94 to 5.12 fg/cell) than for cells grown in air and· N2 . Again shaking versus static growth had little effect on ATP content.

Values increased throughout the growth cycle at 35 0 C and rose only in the early stationary phase at 20°c. The ATP content at 3°c was very stable.

ATP values for L. jensenii grown under N2 ranged from 0.77 to 2.86 fg/cell (Fig. 22). Little difference was seen between shaking and static cultures. The ATP content rose through the stationary phase at 35 0 C. At

20°C cellular ATP was stable and at 3°C ATP levels rose in log phase and fell in the stationary phase.

In general ATP content for L. jensenii was at the lowest level at

3°C and the highest level at 35°C. Aeration made very little difference in cellular ATP content. The findings for L. jensenii, as for .h:_ brevis, do not agree with literature reports except the rise in ATP/cell from lag to log phase reported by Knowles and Smith (57). Hamilton and Holm-

Hansen (40) reported that ATP content falls in the stationary phase.

This was seen only at 3°C in a nitrogen atmosphere and at 3°c for a shaking culture in an air atmosphere.

The ATP content for Pseudomonas sp. grown at 3, 20, and 35 0 C ranged 67

from 0.16 to 5.17 fg/cell (Fig. 23). The 3°c shaking culture had higher

ATP values than did the static culture. The 20°c culture had a stable

ATP content. At 35°c the static culture's ATP level decreased as the culture aged and the ATP content increased into stationary phase in the shaking culture.

Little variation in cellular ATP was seen between a bacterium grown at two different initial pH values (data not shown). This stability was

seen for each bacterium in each atmosphere.

From the trends in ATP/cell presented above, it can be seen that

environmental conditions do affect cellular ATP content. The age of the

culture also affects ATP content as does the genus or species being

used. Table 2 shows some of the values reported in the literature for

many different bacteria. Many of these values seem low (<0.5 fg/cell) in

comparison to most of the values presented in this study. Most of these

low values were in older studies by D'Eustachio et al. (16,25,26) and

Chappelle and Levin (13). It is possible that the extraction methods

used at that time were not as complete as the method used in this study.

D. Sonication Experiments

The rise in cellular ATP between the log and the stationary phase of

growth could have been a result of cell clumping. Thus the assumption

that a colony forming unit was initiated by just one viable cell may not always be valid. With clumping, the plate count would be artificially low and ATP/cell would be artificially high. The phenomenon of cell 68

clumping has been cited by many authors. Both D'Eustachio and Johnson

(25) and Hamilton and Holm-Hansen (40) reported clumping of bacteria in pure cultures. When ATP is used to estimate bacterial numbers in foods, cellular clumping can also be a problem. Stannard and Wood (91,93) reported the possibility of clumping of bacteria from meats with low counts of viable microorganisms. The possibility was raised also for bacteria in urine specimens (98). To determine if clumping was occurring in the above experiments, one growth curve for each bacterium was repeated. The samples were sonicated in an attempt to disperse the cells.

L. brevis showed an increase in ATP/cell in the stationary phase

(Fig. 24). After the sample was sonicated for 15 or 30 seconds, ATP/cell decreased to a level similar to that found in the lag and log phases.

The plate counts for L. brevis increased after sonication in the stationary phase (Fig. 25) while total ATP (molar ATP) remained constant before and after sonication (Fig. 26). The increase in the plate count after sonication showed that clumping of cells was occurring, especially during the stationary phase. To illustrate this, in early stationary phase, sonication of the L. brevis culture for 30 seconds resulted in an 9 increase of the plate count from 1.2 x 10 CFU/ml to 2.1 x CFU/ml while the amount of molar ATP was essentially unchanged. Thus, due to the disruption of clumped cells, the ATP/cell dropped from 3.5 fg to 1.7 fg.

L. jensenii showed an increase in ATP/cell in the log and stationary phases both before and after sonication (Fig. 27). Both the plate counts e--~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-.

4

~ 8 ...... :s .::::;gt G:I ...... _2(..') -· -- °' ~ .;•" -- -- '° ~--- ,,,,,""' ------;:-<-_ .; ,,' 1 -·------,' -...,,-~~' ~ ·- -~------_.... --- ,,,,,-

o--~~~~~~--ii--~~~~~~--~~~~~~--11--~~~~~~--~~~~~~~P-~~~~~~- 0 1 2 3 4 Phase of Growth Figure 24

Effect of 0, 15, or 30 second sonication of a h:._ brevis culture on ATP/cell at each phase of growth. Abbreviations are those defined in Table 2. Symbols: Control for 15 seconds (o); 15 seconds sonication (•); Control for 30 seconds ([]); 30 seconds sonication <•>; control culture (solid line); sonicated culture (dashed line). 1000 --~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~--. ____ ...

-="" . E "' ... --- "" -~ 1® i ~ -->< ~c: ::I -...J 8 10 I 0 ~ a:

1 0 1 2 3 4 Phase of Growth Figure 25

Effect of 0, 15, or 30 second sonication of a L. brevis culture on the plate count at each phase of growth. Abbreviations are those defined in Tabl;-2. Symbols: Control for 15 seconds (o); 15 seconds sonication (•); Control for 30 seconds ([]); 30 seconds sonication (•); control culture (solid line); sonicated culture (dashed line). 10000

I ~ 1000

00 I < .-0 >< 100 'i::" CJ 0 ::::E - - - -...J - I-' ~ 10

1 0 1 2 3 4 Phase of Growth Figure 26

Effect of 0, 15, or 30 second sonication of a h:_ brevis on total ATP (Molar) at each phase of growth. Abbreviations are those defined in Table 2. Symbols: Control for 15 seconds (o); 15 seconds sonication (•); Control for 30 seconds (0); 30 seconds sonication <•>; control culture (solid line); sonicated culture (dashed line)...... ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~--.

... .,,,,..,,,,. .,,,,.• .,,,,..,,,,. ~"""""" -8 "-~ ~

"-2f1 .....,...... ~

t

0--~~~~~~--t~~~~~~~....-~~~~~~--i....-~~~~~~-+-~~~~~~~..... ~~~~~~-1 0 1 2 3 4 Phase of Growth Figure 27

Effect of 0, 15, or 30 second sonication of a 1.:_ jensenii culture on ATP/cell at each phase of growth. Abbreviations are those defined in Table 2. Symbols: Control for 15 seconds (o); 15 seconds sonication (•); Control for 30 seconds (0); 30 seconds sonication <•>; control culture (solid line); sonicated culture (dashed line). tOOO

"!::' E ...... too ~ .e, .&.. /'I/II- ~/ / / ...... / 0< ,,,,"" / • -->C / 1: / ::I / -..J / I VJ 8 10 / / ~ / a: '/

1 0 1 2 3 4 Phase of Growth Figure 28

Effect of 0, 15, or 30 second sonication of a ~ jensenii culture on the plate count at each phase of growth. Abbreviations are those defined in Table 2. Symbols: Control for 15 seconds (o); 15 seconds sonication (•); Control for 30 seconds (D); 30 seconds sonication <•>; control culture (solid line); sonicated culture (dashed line). 1000

CIO 100 I < .-0 >C 'i:'" CJ 0 ::::E ~/ ...... - 10 I I +:- ~

1 0 1 2 3 4 Phase of Growth Figure 29

Effect of 0, 15, or 30 second sonication of a 1;_ jensenii on total ATP (Molar) at each phase of growth. Abbreviations are those defined in Table 2. Symbols: Control for 15 seconds (o); 15 seconds sonication (•); Control for 30 seconds (O); 30 seconds sonication <•>; control culture (solid line); sonicated culture (dashed line). 75

(Fig. 28) and molar ATP (Fig. 29) were essentially the same before and

after sonication. For example, in early stationary phase, sonication of

the L. jensenii culture for 30 seconds resulted in little change in

plate count (8.0 x 108 CFU/ml to 8.7 x 108 CFU/ml) and no change in

molar ATP. Thus, ATP/cell showed little change after sonication (3.5 fg

to 3.2 fg). Therefore, no evidence of cell clumping was found and yet

the ATP content of the cells increased as the culture aged.

Pseudomonas sp. showed an unexpected reaction to sonication. While

the culture showed no rise in ATP/cell as the culture progressed through

the growth cycle, ATP/cell increased dramatically after sonicating for

15 or 30 seconds (Fig. 30). The plate counts (Fig. 31) decreased after

sonication, especially in the stationary phase, while molar ATP (Fig.

32) increased after sonication. To illustrate this, in early stationary

phase, sonication of the Pseudomonas sp. culture for 30 seconds resulted

in a decrease in the plate count (2.5 x 108 CFU/ml to 4.0 x 107 CFU/ml), 7 -6 while molar ATP increased (8.1 x 10- M to 1.3 x 10 M). Both of these

changes contributed to the dramatic increase of ATP/cell from 1.6 fg to

16.2 fg. Sonication may have injured the Pseudomonas and rendered it nonviable. This would account for the lower plate counts without

lowering the molar ATP. If the cells had been killed or disrupted, the molar ATP measurement should drop with sonication. However, molar ATP

increased aftersonication. There were two possible explanations for

this rise in total ATP. First, cellular diruption by sonication may have released more ATP from the cells than did the extracting reagent.

The second possibility was that although the cells were injured, the 17--~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~--.

18 /'4. / '\ Ut // ', 14 / '\ // '\ 13 / '\ / '\ -::::::-- 12 / ,.,...---~---· / '\ 11 /" 8 // / '\ ...... 10 ~ // '\ .:::::,..°' // / ' . ,. / / ' ~ I---?~ / 1" (.) .7 I / ----/ ...... I ,," ....i ~ 0\ cc .e •" / ,' ,/· 4 // 1,,/,' 3 // 2 /,,,' 1 ol rf' 1 1 1 1 I 0 1 2 3 4 Phase of Growth Figure 30

Effect of 0, 15, or 30 second sonication of a Pseudomonas sp. on ATP/cell at each phase of growth. Abbreviations are those defined in Table 2. Symbols: Control for 15 seconds (o); 15 seconds sonication (•); Control for 30 seconds (O); 30 seconds sonication (II); control culture (solid line); sonicated culture (dashed line). 10000

-==-E 1000 " ~

....~ 100 >< ~ / ·------· ~ ------..J 8 ~ ·------·------· -..J ~ 10

, 0 1 2 3 4 Phase of Growth Figure 31

Effect of 0, 15, or 30 second sonication of a Pseudomonas sp. culture on the plate count at each phase of growth. Abbreviations are those defined in Table 2. Symbols: Control for 15 seconds (o); 15 seconds sonication (•); Control for 30 seconds (O); 30 seconds sonication <•); control culture (solid line); sonicated culture (dashed line). 1000 --~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

co 100 I < 0 ->< -'i:" 0 0 2 ...._, '\..... ,\ - /// 10 I / I

t 0 1 2 3 4 Phase of Growth Figure 32

Effect of 0, 15, or 30 second sonication of a Pseudomonas sp. on total ATP (Molar) at each phase of growth. Abbreviations are those defined in Table 2. Symbols: Control for 15 seconds (o); 15 seconds sonication (•); Control for 30 seconds ([]); 30 seconds sonication <•>; control culture (solid line); sonicated culture (dashed line). 79

cellular metabolic activity was hegher in an attempt to repair the cell.

Thus, the amount of cellular ATP would be higher. These results suggested that the gram negative bacterium, Pseudomonas, was more sensitve to sonication than were the two gram positive lactobacilli.

Experiments were conducted to test the effect of sonication on each bacterium. Only optimum growth conditions for each bacterium at 35°C were used in the sonication tests. Due to time and material limitations, only three growth conditions were repeated to determine the potential effect of sonication on the ATP/cell. However, up to this time all bioluminescence assays have been conducted without sonicating the culture medium. These pure culture experiments used techniques similar to previous studies. Thus, these results showed the type of variation which would be found in the ATP/cell of bacteria in previous applications of the bioluminescence assay performed without including a sonication step.

Sonication experiments need to be performed for all of the experimental factors to determine where clumping may be interfering with the calculation of ATP/cell. For instance, is clumping occurring more in shaking or static cultures? Is clumping more prevalent at 3 0 C, 20 0 C, or

35°C? Does the type of atmosphere aid or inhibit cells from clumping?

Perhaps cell clumping depends on the bacterium in conjunction with other environmental conditions. From the initial sonication experiments, L. brevis cells did clump in a shaking culture while L. je~senii and

Pseudomonas sp. cells did not. 80

E. Summary and Conclusions

Isolates of bacteria from aged ground beef were made in order to select spoilage organisms of ground beef. Spoilage organisms were isolated from both aerobic-and vacuum-packed ground beef. L. jensenii and Pseudomonas sp. were selected as representative of those isolates from the aerobically packaged beef and L. brevis was selected as representative of those isolates from vacuum packaged beef.

ATP/cell was found to vary significantly within each of the treatments - bacteria, temperature, atmosphere, aeration, and phase of growth. It was determined that most of the interactions of these factors (or treatments) were also significantly different, including the

"nested" factor - pH.

L. brevis had an average ATP/cell of 2.70 fg. The highest average cellular ATP for L. brevis was in air. ATP content was usually higher in the static cultures rather than the corresponding shaken culture.

Cellular ATP content had less variation in all atmospheres at 3°C. ATP content was highest in late stationary phase and lowest in either lag or log phase in all conditions. Evidence of cell clumping by.!:..:_ brevis in a nitrogen atmosphere at 35°C in a shaking culture was seen.

L. jensenii had an average ATP/cell of 2.20 fg. The growth of.!:..:_ jensenii was enhanced in an atmosphere of carbon dioxide. In fact, the highest average ATP/cell was in carbon dioxide. Cellular ATP was highest at 35°c and lowest at 3°C in all atmospheres. ATP content was highest in late stationary phase and lowest in either lag or log phase in all 81

conditions. Evidence of cell clumping was not seen for h.:_ jensenii when grown in a nitrogen atmosphere at 35°C in a shaking culture.

The average ATP/cell for Pseudomonas sp. was 1.36 fg. The average cellular ATP content was highest in the lag phase of growth. However, when ATP/cell was viewed as a function of temperature, this generality did not hold true. ATP content was lowest at 35°C and had less variation for the 20°C static cultures and the 3°C shaking cultures. No evidence of cell clumping was seen for Pseudomonas sp. when grown in air at 35°c in a shaking culture. Sonication had a lethal effect on Pseudomonas sp ..

Thus, sonication is not the definitive solution for dispersing clumped cells and should not be used indiscriminatly as a step in the bioluminescence assay when susceptible cells, such as Pseudomonas sp., might be present.

The bioluminescence assay has been used recently to predict the shelf life of ground beef. A prediction curve of bacterial numbers from total ATP is seen in Figure 33. The linear slope of the line represents -1 -1 (ATP/cell) . (On the log scale presented, differences in (ATP/cell) are represented by separation between lines.) ATP/cell is assumed to be

1 fg (58). This study showed that the amount of ATP/cell varies between bacteria (genus and species) as well as a result of environmental conditions (atmosphere, aeration, temperature, and pH). Thus the prediction curve may underestimate the number of bacteria in the ground beef sample. This may be aggravated by the type of major contaminant, storage condition, or condition of the meat. If the ground beef has been temperature abused, the amount of ATP/cell will change. This is also the 11...-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~---.

// / .. 10 / .· / .. ·· / .. · / .·· e / .·· ...... // .. ··· ~ // ... ·· .e. . / ... ·· // .. ·· g / ... ·· ·c // Cl) .. ··· 1) • / .. ·· // .. ·· cB / ... ·· / 0 / .· ~ .. ·· ... ~' .· _g ~' .·· ~' .· 00 E 7 ~' .·· N ::I ,,,, .· ... · :z / .. ·· / .. ·· / .. · _§' ... ··· •

e ...... ~~~~~~~~~~-1-~~~~~~~~~~-+~~~~~~~~~~--i~~~~~~~~~~---1 -8 -6 -4 Log ATP (Molar) Figure 33

Effect of different values of ATP/cell on the predicted number of bacteria. Symbols: 1.0 fg (solid line); 2.5 fg (dashed line); 4.0 fg (dotted line). 83

case if the bacteria are allowed to incubate so that any injured cells have time to recover. If, for instance, the ATP/cell averages 2.5 fg, the predicted bacterial load will be lower and this prediction of the bacterial load would be still lower if ATP/cell averaged 4.0 fg (Fig.

33). Thus, the shelf life of the ground beef could be underestimated if the erroneous assumption of 1.0 fg as a constant is maintained. If the bioluminescence assay is to be accepted by food microbiologists as a reliable replacement of plate counts for determining microbial load in foods such as ground beef, the consequences of the variations in cellular ATP due to environmental conditions must be considered.

From the results in this study, it can be seen that the bioluminesce assay can be used as a reliable replacement of plate counts to estimate bacterial loads in foods if certain procedures are adopted. First, the food item - ground beef for example - should be stored at a refrigerator temperature. Samples should not be allowed to come to room temperature nor should any type of incubation step (to allow cellular repair or to allow enzymatic removal of nonmicrobial ATP) be performed at an elevated temperature. This is necessary since all experiments showed bacterial

ATP content to be the most stable at 3°c (refrigeration temperature).

Second, the assumption of 1 fg/cell should not be made. Type of packaging, kind of atmosphere, and predominant type of spoilage organism expected must be considered in order to make a good estimate of bacterial ATP content. If the ground beef is in aerobic packaging,

Pseudomonas would be expected to be the major spoilage organism. A value of 1.8 fg/cell should be used as an estimate ko ATP content. If 84

the' ground beef is in anaerobic packaging, Lactobacillus would be expected to be the major spoilage organism. A value of 1.4 fg/cell should be used to estimate bacterial ATP if a carbon dioxide atmosphere is present. However, if a nitrogen atmosphere is present, the lower value of 1.3 fg/cell should be used.

Finally, sonication should not be used in an effort to disperse cell clumps. Gram negative bacteria are sensitive to sonic vibaration and bacterial numbers can be overestimated by the bioluminescence assay and underestimated by plate counts. When the bioluminescence assay is used, cell clumping ceases to be a problem with biomass estimation as was seen with traditional plate counts.

The bioluminescence assay has been and can be a useful tool for estimating the number of foodborne microorganisms. However, with meat and other solid foods which would require homogenization before sampling, somatic ATP can interfer with estimatin bacterial numbers. A double filtration system has been developed to reduce the amount of ATP contaminating the assay (77). However, estimating the number of bacteria (or yeast) in beverages is not hindered by contamination ATP and the bioluminescence assay would be a very useful tool for determining biomass. In either food type, quality control measures for determining the number of microbial contaminants would require approximately one hour for the bioluminescence assay versus 24 to 72 hours for plate counts. One hour is much more acceptable and appealing to both food manufacturers and their quality control personnel. Literature Cited

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85 12. Chapman, A. G. and D. E. Atkinson. 1977. Adenine nucleotide concentrations and turnover rates. Their correlation with biological activity in bacteria and yeast. Adv. Microbial Physiol. 15:253-306.

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18. Dawes, E. A. and P. J. Large. 1970. Effect of starvation on the viability and cellular constituents of Zymomonas anaerobia and Zymomonas mobilis. J. Gen. Microbiol. 60:31-42.

19. DeLuca, M., N. J. Lgonard, B. J. Gates, and W. D. McElroy. 197~. The role of l,N -ethenoadenosine triphosphate and l,N - ethenoadenosine monophosphate in firefly luminescence. Proc. Natl. Acad. Sci. USA 70:1664-1666.

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