International Biodeterioration & Biodegradation xxx (2012) 1e6

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier.com/locate/ibiod

Expanding the microbial monitoring toolkit: Evaluation of traditional and

molecular monitoring methods
Vic Keasler a, Brian Bennett a, Carrie Keller a, Pat Whalen b, Jim Cairns b, Renato M. De Paula a, *
a
b

Nalco Company, Energy Services, OFC Unconventional Production & Water Management, 7705 Highway 90A, Sugar Land, TX 77478, USA
LuminUltra Technologies Ltd., 440 King St., King Tower, Suite 630, Fredericton, New Brunswick, E3B 5H8 Canada

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 8 August 2011
Received in revised form
13 June 2012
Accepted 5 July 2012
Available online xxx

Evaluation of microbial populations in oileld systems is critical to understand the risk of microbiologically inuenced corrosion, reservoir and surface souring (hydrogen sulde production), and
biofouling. Although traditional culture based methods have dominated oileld microbial monitoring for
years, use of molecular tools is becoming more common for both eld and laboratory evaluations. The
implementation of these additional tools is in response to some of the disadvantages of culture-based
methods such as long incubation times and underestimation of actual microbial populations. The
current work provides a direct comparison of culture-based methods (serial dilution for sulfate reducing,
acid producing, and general heterotrophic bacteria) with molecular methods including adenosine
triphosphate (ATP) and adenosine monophosphate (AMP) quantication and quantitative polymerase
chain reaction (qPCR). The results demonstrate that these technologies provide nearly identical results in
untreated samples with known culturable organisms, but show some differences when used in the
context of a planktonic kill study. The pros and cons of each technology were addressed with respect to
their use in eld monitoring, laboratory monitoring, and microbial kill studies. The authors found that
none of the technologies described in this work provide an all-inclusive answer, but together they
provide signicant insight into the microbial population in an oileld system. In short, the authors
demonstrate that it is advantageous for oileld stakeholders to expand their microbial monitoring toolkit
with these new technologies to ensure sustainable, cost-effective operation.
2012 Published by Elsevier Ltd.

Keywords:
Microbiologically-inuenced corrosion
ATP
Monitoring

1. Introduction
For decades, the presence of microorganisms in different stages
of oil production has represented a constant concern to the
petroleum industry. Microbial activity can adversely affect oil
production in many ways. The presence of hydrogen sulde (H2S),
produced by sulfate-reducing microorganisms (SRM), reduces the
quality of oil and gas, increases the risks of corrosion of equipment
and poses a threat to workers due to its toxicity (Bastin et al., 1926).
Acid-producing Bacteria (APB) release acidic by-product that can
increase the corrosion rates on metal surfaces (Beech and Gaylarde,
1999) and general heterotroph bacteria (GHB) are directly involved
in biofouling (Dalton and March, 1998), which can lead to reduced
efciency of equipment operation and decreased oil recovery. To
circumvent problems associated with microbial activity, diligent
monitoring has been found to be a crucial step to maintain

* Corresponding author. Tel.: 1 281 263 7129; fax: 1 281 263 7848.
E-mail address: rdepaula@nalco.com (R.M. De Paula).

microbial numbers to levels at which they will neither compromise

the production and the integrity of the asset, nor become a safety
issue to operators (Schwermer et al., 2011).
In the past, enumeration and identication of bacteria in oileld
samples have been primarily accomplished by culture-based
methods (NACE Standard TM0194-2004, Field Monitoring of
Bacterial Growth in Oil and Gas Systems Houston, TX: NACE, 2004).
However, several caveats to this approach exist, including long
incubation times (up to 28 days for detection of SRBs) as well as an
inability to cultivate bacteria from extreme environments,
commonly found in oil producing reservoirs. This usually leads to
underestimation of the bacterial population present in the samples
and failure to tailor the appropriate microbial mitigation strategy
(Wilderer et al., 2002). A few non-culture based assays have been
developed for quick detection of microorganisms in the eld, but
they often present limitations in terms of accuracy or types of
samples that can be tested (Horacek and Gawel, 1988; Bitton and
Koopman, 1992). One of these methods rely on bioluminescent
ATP techniques, in which intracellular ATP provides energy to drive
the conversion of luciferin to oxyluciferin and light, in a reaction

0964-8305/$ e see front matter 2012 Published by Elsevier Ltd.

http://dx.doi.org/10.1016/j.ibiod.2012.07.002

Please cite this article in press as: Keasler, V., et al., Expanding the microbial monitoring toolkit: Evaluation of traditional and molecular
monitoring methods, International Biodeterioration & Biodegradation (2012), http://dx.doi.org/10.1016/j.ibiod.2012.07.002

V. Keasler et al. / International Biodeterioration & Biodegradation xxx (2012) 1e6

catalyzed by the rey luciferase enzyme. This method follows the

premises that all living organisms contain constant intracellular
ATP levels to maintain normal physiological activities (Beckers
et al., 1984). More recently, molecular techniques, including polymerase chain reaction (PCR) followed by denaturing gradient gel
electrophoresis (DGGE) (Ahn et al., 2002) and uorescence in-situ
hybridization (FISH) (Amann et al., 1990), have been developed to
provide more specic and accurate microbial characterization;
however these methods can only be performed in well-equipped
laboratory spaces.
An additional challenge encountered when using the available
microbial detection methods in the oilelds is the inability to
account for bacteria that are not actively growing. Frequently,
bacteria must change their metabolic status to survive unfavorable
conditions. This is achieved by a shift to an anabiotic (dormant)
state that allows the cells to survive for extended periods of time
without division and growth (Kaprelyants et al., 1993). In certain
environments, it is estimated that up to 80% of the bacteria in
samples from the wild appear to be metabolically inactive (Cole,
1999). Nevertheless, the dormancy state is reversible and most
cells can shift back to a fully active state when the appropriate
conditions for development are found (Weichart and Kjelleberg,
1996). Microbial dormancy contributes to high diversity levels in
most natural ecosystems by avoiding microbial death due to
unfavorable growth conditions (Jones and Lennon, 2010). Thus,
developing a methodology to enumerate active and inactive populations of bacteria is critical to acquire a complete understanding
of any microbial community.
In this study, we show the development of a new generation of
ATP/AMP quantication assay, which allows the detection of active
and inactive organisms. A comparison to other available microbial
detection methods is described. Additionally, we report how
chemical treatments, with several common oileld biocides, affect
the metabolic status of the microbial population. Our results indicate that detection of active and dormant microbial populations is
important to understand the risks posed to assets and the efciency
of chemical treatment.
2. Methodology

developed by LuminUltra Technologies Ltd., Canada, according the

manufacturers guidelines. In brief, uid samples were collected in
different locations throughout of the system, ltered through
0.2 mm equivalent lters to capture intact microorganisms and to
avoid interference from free ATP and AMP in the samples. Hydrocarbons were removed by an organic wash step with the 5 mL
Lumiclean reagent to prevent interference during the ATP reading.
Microorganisms present on the lter were then lysed with 1 mL of
Ultralyse reagent to collect intracellular ATP and AMP. The ATP
concentration was detected by mixing 100 mL of the diluted lysate
(1:10 dilution with Ultralute reagent) with 100 mL of reagent containing the enzyme luciferase, and the resulting light output was
measured using a handheld luminometer (l 420 nm). AMP levels
were determined via enzymatic conversion of AMP to ATP using
100 mL of Reagent-P, and subsequent measurement of ATP as
described above. ATP results are converted to fg/mL and then to
Microbial Equivalents based on the assumption that 1 fg ATP 1
Cell.
2.3. Bacterial DNA extraction and quantitative PCR
Samples containing microorganisms were collected from eld
uid samples, by ltration through 0.22 mm Durapore Membrane
Filters (Millipore). The sample volume varied from 20 to 60 mL and
was used to normalize the bacterial counts in different samples.
Bacterial genomic DNA was isolated from uid samples, collected in
the eld, using the UltraClean Soil DNA Isolation kit (MoBio e
Carlsbad, CA) according to manufactures instructions. A DNA fragment, encoding a portion of the bacterial 16S rDNA gene was
amplied using 25 mM primers 8F and 338R (Muyzer et al., 1993)
and 12.5 mL Sybr Green Real-Time PCR Master Mix (Invitrogen).
Amplication reactions were performed using an Applied Biosystems 7500 Real-Time PCR System. The PCR conditions were as
follows: 2 min at 50  C and 10 min at 95  C, followed by 40 cycles of
15 s at 95  C and 1 min at 60  C. A calibration curve was obtained by
using a serial dilution of a known concentration of positive control
DNA (bacterial 16S rDNA cloned pIDTSMART-AMP vector e IDT).
The CT values that were obtained from each sample were compared
with the standard curve to determine the copy number of
prokaryotic DNA present in the tested sample.

2.1. Bacterial enumeration by serial dilution and LB plating

2.4. Planktonic kill studies
Bacterial enumeration was performed according to NACE Standard TM0194-2004 (4) and bacterial numbers are reported as the
total number of sulfate reducing (SRB) or acid producing (APB)
bacteria per mL. Serial dilutions for SRBs and APBs were inoculated
into Modied Postgate (MPB) (Postgate, 1984) and Phenol Red
Dextrose (PRD) media, respectively. Culture bottles were incubated
for 14 or 28 days (PRD and MPB, respectively) in a Precision Model 4
Incubator (Thelco), at 30  C with no agitation. Positive bacterial
growth in the MPB medium was observed as a color change from
clear/white to black, indicating the presence of sulfate-reducing
bacteria (SRB). A positive result in the PRD medium was displayed by a color change in color from red to yellow, indicating the
presence of acid producing bacteria (APB) or a change in turbidity
indicating the presence of general heterotrophic bacteria (GHB). For
LB plating experiments a 5-fold dilution series of water and oil over
a 3-log range was prepared and plated in LB plates. The plates were
incubated at 33  C for 24 h and the CFU was determined by
counting.

Planktonic kill studies were performed as an evaluation of 3

different biocides: a common quaternary amine, THPS and glutaraldehyde. In brief, 100 mL of produced water samples were used to
test the kill efciency of the aforementioned biocides, at 100 and
300 ppm dosages. Samples were poured in 125 mL serum bottles,
capped with rubber caps and sealed with aluminum rings. Biocides
were added to the samples using 1 mL syringes. Samples were
incubated at 30  C for 4 h. After contact time, bacterial enumeration
was performed using serial dilution for SRB, APB and GHB and ATP/
AMP quantication assay. Serial dilutions were incubated for 14
days for detection of APBs and GHBs and 28 days for detection of
SRBs. Bacterial enumeration was calculated at the end of the
appropriate time and biocide efcacy determined. Additionally,
ATP and AMP measurements were performed in the same samples.
The AMP index was determined by the ratio of intra-cellular cAMP/
cATP. Experiments were repeated twice with similar results.
3. Results

2.2. Microbial enumeration by ATP and AMP quantication assay

Total microorganism quantication was performed via ATP
quantication, using the 2nd Generation ATP assay system

The development of non-culture based methods for microbial

detection has been of interest to many industries. The ability to
rapidly quantify different microbes offers an enormous advantage

Please cite this article in press as: Keasler, V., et al., Expanding the microbial monitoring toolkit: Evaluation of traditional and molecular
monitoring methods, International Biodeterioration & Biodegradation (2012), http://dx.doi.org/10.1016/j.ibiod.2012.07.002

V. Keasler et al. / International Biodeterioration & Biodegradation xxx (2012) 1e6

Table 1
Comparison of 1st generation ATP technology to 2nd generation ATP technology.
Potential problems

1st generation technology

2nd generation technology

Lower Limit of Detection (LLD)

Interference from hydrocarbon

w10 pg ATP/mL
Severe at any concentration e can
cause lower RLU results
Each order of magnitude increase in
TDS reduces RLU by the same value
2 steps required (Total ATP e Free ATP)
RLU

<0.5 pg ATP/mL
No interference e additional washing step
incorporated to eliminate impact
No interference e additional washing step
incorporated to eliminate impact
1 Step (Direct measurement)
pg ATP/mL

Interference from dissolved solids

Steps to measure intracellular ATP
Results reported in

towards mitigating problems commonly associated with uncontrolled microbial growth. A number of commercially available assay
kits, based on measurement of cellular ATP using bioluminescence,
are currently in use, especially for food processing hygiene applications and cooling water systems. However, several limitations, for
detection of microorganisms from oileld samples, were found
using these assays. Table 1 summarizes the most common problems observed with the use of this technology. These include
interference by hydrocarbons, elevated chloride levels, and chemical residuals, limitations regarding sample types (e.g. solids,
sludges, etc) and inability to detect intracellular ATP only. LuminUltra Technologies Ltd has developed a 2nd generation of ATP
quantication assay, in which many of the limitations have been
overcome to eliminate interfering molecules and extracellular ATP
(Table 1).
The two major changes incorporated into the new generation
ATP assay are depicted in Fig. 1. The samples are ltered through
a 0.2 mm equivalent membrane to capture the microbes on the
surface of the lter. This allows any free ATP to pass through and
avoid overestimation of microbial number during incubation with
the luciferase/luciferin reagent. The second modication includes
a wash with an organic solution that eliminates interfering particles
and hydrocarbon contaminants.
To test the accuracy of the new generation ATP assay,
a comparison with quantitative PCR was conducted. For this, 28
day-old sulfate-reducing bacteria (SRB) cultures were analyzed
simultaneously using both methods. The results indicated that
comparable bacterial numbers were obtained from the ATP and
qPCR methods (Fig. 2a). To determine the efciency of the assay for
use with oileld samples, the performance of the ATP kit was

compared to the traditional LB plating culture method using

organisms that are known to be able to be grown in culture
(Fig. 2b). Oil and water samples were used to test compatibility of
the assay with the most common types of oileld samples. The
results demonstrated an excellent correlation of microbial numbers
between the two methods, regardless the sample source. The
calculated correlation based on linear regression of the curves
showed values higher than 0.9, indicating that the data obtained by
ATP measurements closely trend those obtained by conventional
techniques.
The performance of the ATP assay was also compared to the
serial dilution and quantitative PCR methods (Fig. 3). Analysis of six
independent produced water samples from a system in North
America demonstrated that the values obtained with the ATP
quantication assay trended in the same direction as those determined by qPCR. In 5 out 6 cases, the qPCR assay detected a slightly
higher amount of organisms compared to the ATP method. This
may be due to the fact that DNA from dead and/or partly lysed cells
can be detected by qPCR. On the other hand, we observed that
microbial numbers obtained by serial dilutions were dramatically
decreased when compared to ATP and qPCR methods. This
conrms that, mostly, serial dilutions provide an underestimation
of bacteria counts. It is noteworthy that small differences between
the ATP and qPCR data were observed and this can be explained by
the presence of dormant organisms in the samples that cannot be
accounted for by the ATP assay, but are detected by the molecular
technique.
The ATP quantication assay was also used to enumerate
microbes in 8 producing wells located in the Barnett Shale region.
For each sample, ATP quantication and serial dilution was

Fig. 1. ATP extraction/quantication procedure. Total microorganisms are collected on a lter attached to a syringe. Hydrocarbon is removed by organic wash step to prevent
inaccurate ATP readings. Microorganisms present on the lter are lysed by the addition of a detergent and ATP is collected. The ATP is mixed with the enzyme luciferase, and the two
molecules interact to produce light which is captured by a handheld luminometer. The 2nd Generation ATP assay system was developed by LuminUltra Technologies Ltd., Canada.

Please cite this article in press as: Keasler, V., et al., Expanding the microbial monitoring toolkit: Evaluation of traditional and molecular
monitoring methods, International Biodeterioration & Biodegradation (2012), http://dx.doi.org/10.1016/j.ibiod.2012.07.002

V. Keasler et al. / International Biodeterioration & Biodegradation xxx (2012) 1e6

Table 2
Enumeration of microbes in a production system in the Barnett Shale. Microbial
enumeration, by serial dilution, ATP assay and qPCR, was performed on uid
samples collected from the well head of 8 different production wells. Total numbers
of microbes as determined by each technique are reported as number per mL of
uid.
Samples

Production
Production
Production
Production
Production
Production
Production
Production

Fig. 2. Comparison of microbial enumeration methods. A e Enumeration of sulfatereduction bacteria (SRB) by quantitative PCR and ATP measurements. B e ATP and
LB agar plating correlation. Results from the tests performed in oil and water samples
are plotted as ATP versus LB agar plating. A diamond: water samples, - rectangle: oil
samples. Trendlines were added for both the oil and water and R2 values calculated.

performed on-site and lters containing xed microbes were

analyzed by qPCR in the lab. Similarly as observed before, the
numbers obtained by serial dilution were signicantly lower when
compared to those detected by the other methods (Table 2).
Interestingly, in wells #1, #4 and #6 markedly numbers of microorganisms were detected by the ATP assay, although very low
microbial numbers were found using qPCR. This can be explained
either by the presence of Archaea or other microorganisms whose
unique DNA sequences are not detected by our qPCR assay. In the
well #5 very high numbers of SRBs were found using serial dilution
bottles despite the considerably low numbers obtained ATP and

Serial dilution

well
well
well
well
well
well
well
well

1
2
3
4
5
6
7
8

SRB

APB

10,000
10,000
100
1000
100,000,000
<LOD
100
10,000

<LOD
10,000
100
<LOD
100
<LOD
100
<LOD

ATP
quantication
66,240
2,725,179
2,435,423
50,799
314,305
47,434
181,111
8098

qPCR enumeration
Total Bacteria
1299
5,088,009
7,214,162
<LOD
991,016
<LOD
159,247
296,768

qPCR measurements. We conclude that this may have occurred

likely due to contamination during the inoculation of the culture
bottles or due to the presence of sulde or H2S that lead to a false
positive result.
The new ATP assay was used to monitor the implementation of
a new chemical program aimed to reduce the levels of microbial
activity in a production system at the Barnett Shale. The chemical
treatment program was designed to meet specic Key Performance
Indicators (KPIs), outlined in Table 3. The proposed program
allowed us to test another criterion concerning bacterial control in
oileld: the enumeration of dormant bacteria. The importance of
monitoring not only active, but also dormant populations of
microorganisms during all steps of oil production is critical because
very frequently, water from many sources are mixed and introduced in different environments where the conditions may or may
not favor the proliferation of microorganisms. When introduced in
hostile environments, most organisms will shut down their
metabolism and remain dormant until favorable conditions arise.
Thus, in some instances, detection of microbes based solely on the
ATP assay may lead to the erroneous assumption that no microorganisms are present in a particular location.
Low metabolic rates in cells are associated with a decrease in
ATP levels and concomitant increase in AMP amounts. Hence, the
ratio between cellular ATP to AMP, known as AMP index (AMPi),
can be used as a metabolic indicator for the microbial population
(Chapman et al., 1971). An increase in AMPi indicates a stressful
condition. On the other hand a decrease indicates an increase in the
populations health. In general, we have found that an index <0.1
indicates no stress, an index >1 indicates the presence of stress, an

Fig. 3. Enumeration in produced water samples. Enumeration was performed by qPCR, ATP quantication, and serial dilution method. qPCR results are a summation of total bacteria
and Archaea readings and serial dilution results are a summation of SRB, APB, and GHB readings. ATP results are converted to fg/mL and then to Microbial Equivalents based on the
assumption that 1 fg ATP 1 Cell. The experiment was performed twice and similar results were obtained. #1e6 represents the sample number.

Please cite this article in press as: Keasler, V., et al., Expanding the microbial monitoring toolkit: Evaluation of traditional and molecular
monitoring methods, International Biodeterioration & Biodegradation (2012), http://dx.doi.org/10.1016/j.ibiod.2012.07.002

V. Keasler et al. / International Biodeterioration & Biodegradation xxx (2012) 1e6

Table 3
Establishing control and treatment targets for each measurement. Prior to initiating
any experiments for evaluation of anti-microbial treatments, control and treatment
targets were established for each Key Performance Indicator (KPI). Good Control is
meant to convey the target KPI value for use in the eld, whereas the Treatment
Target conveys what performance is desired for successful one-time treatment.
These criteria are established based on experience in utilizing these tools in the eld.
Measurement method

Good control values

Treatment target

Serial dilution (CFU/mL)

cATP (pg/mL)
AMP index

100
100
1

4-Log reduction
95% reduction
5-Fold increase

index >3 represents a lethal stress while an index >10 indicates

that a lethal stress was applied for at least one day.
A planktonic kill study was performed to determine the efcacy
of different biocides on killing organisms present in a produced
water sample and whether the chemical treatment met the KPIs
established in Table 3. The efciency of the treatment was monitored by ATP/AMP quantication and serial dilution methods. The
results indicated that, at lower concentrations (100 ppm) all
biocides tested failed to reach the proposed KPI, as the microbial
reduction fell below the target 4 log reduction (serial dilution)
and 95% reduction (cATP) (Table 4). At this concentration, the
AMPi increased approximately 5x during treatment with a quaternary amine biocide (0.54) and about 3x during treatments using
THPS (0.31) and Glutaraldehyde (0.31) when compared to the
baseline (0.11) index. This indicates that at low concentrations, the
biocides evoked a mild stressful condition that can possibly be
explained by a shift of the microbial cells to a dormant state.
However, this condition is not classied as a lethal stress and the
cells could likely revert to normal actively growing state when the
biocide treatment ceased. At higher dosages (300 ppm), the KPIs
were accomplished more efciently as the bacterial numbers were
markedly reduced. The serial dilution method detected a >5-log
reduction for SRB, APB and GHB and the levels of cATP were
decreased >95% compared to the blank. Moreover, the AMPi
showed that the treatment with all 3 biocides induced a lethal
stress in the microbial cells (Table 4).
To determine how dormancy can affect the efciency of biocide
treatments, a new kill study was performed 3 weeks after the

conclusion of the rst kill study. Our hypothesis was based on the
assumption that, after a certain period of time, the microbial population would reach the stationary phase and the nutrients would
be exhausted from the medium. As consequence of the nutritional
starvation, microbial cells would change to a dormant state and
become more susceptible to an applied stress. The results shown in
Table 5 conrmed our hypothesis. Application of 100 ppm of the 3
biocides elicited a lethal stress in the population as indicated by the
AMPi (3.68), a log-reduction 5 in SRB, APB and GHB and 96%
decrease in cATP. Similar results were obtained with higher
concentration of the biocides. These ndings indicate that in
healthy populations, administration of low dosages of biocides may
not necessarily kill the organisms, but rather induce them into
a dormant state. Thus, determining the health of a microbial population may represent a key aspect of a chemical program in order
to identify the adequate biocide dosage to achieve complete kill.
4. Discussion
In this study we described the application of an improved
version of the ATP quantication assay to monitor microbial activity
in water samples collected in different oil production systems.
Previously developed ATP assays lacked the ability to specically
detect intracellular ATP and the results obtained were, quite often,
inated by the detection of free ATP. Moreover, the source of
samples that could be analyzed was signicantly limited due to
interference by organic materials. The new generation ATP assay
allows for the analysis of a broad spectrum of samples and the
quantication of active as well as dormant organisms. Our data
showed an excellent correlation between the ATP assay with
traditional and molecular methods when culturable organisms
were tested. The ATP assay can be used as the rst diagnostic tool to
identify the presence of microorganisms and assess risk in oileld
systems. When elevated microbial numbers are identied, samples
can be further analyzed using molecular techniques to acquire more
revealing and specic data about the types of organisms present. A
caveat with the improved ATP assays, similar to earlier versions, is
the lack of species differentiation. Because ATP is present in all living
cells, microbes other than bacteria can also be detected as well.

Table 4
Kill Study #1. Microbial reduction after biocide treatment. The following biocides: Quaternary amine, THPS and Glutaraldehyde were applied at 100 and 300 ppm.
Chemical

Serial dilution methods

SRB/mL

None (Blank)
Quaternary Amine 100 mg/L
Quaternary Amine 300 mg/L
THPS 100 mg/L
THPS 300 mg/L
Glutaraldehyde 100 mg/L
Glutaraldehyde 300 mg/L

1.00E
1.00E
0.00E
1.00E
1.00E
1.00E
0.00E

08
06
00
06
01
06
00

ATP and AMP methods

Log drop

APB/mL

e
2
8
2
7
2
8

1.00E
1.00E
0.00E
1.00E
1.00E
1.00E
1.00E

07
06
00
06
01
03
02

Log drop

GHB/mL

e
1
7
1
6
4
5

1.00E
1.00E
0.00E
1.00E
1.00E
1.00E
1.00E

07
06
00
06
02
04
02

Log drop

Effective?

cATP

% Drop

AMPi

Rise

Effective?

e
1
7
1
6
3
5

e
No
Yes
No
Yes
No
Yes

8728
2552
37
5703
100
2950
264

e
70.8
99.6
34.7
98.9
66.2
97

0.11
0.54
1.41
0.31
22.72
0.31
11.84

e
5
13
3
212
3
110

e
No
Yes
No
Yes
No
Yes

Table 5
Kill Study #2. Microbial reduction after biocide in 3-week old water samples. Dosages applied: 100 and 300 ppm.
Chemical

Serial dilution methods

None (Blank)
Quaternary Amine 100 mg/L
Quaternary Amine 300 mg/L
THPS 100 mg/L
THPS 300 mg/L
Glutaraldehyde 100 mg/L
Glutaraldehyde 300 mg/L

2.50E
1.00E
1.00E
1.00E
1.00E
1.00E
1.00E

SRB/mL

06
00
00
00
00
00
00

ATP and AMP methods

Log drop

APB/mL

e
6
6
6
6
6
6

1.00E
1.00E
1.00E
1.00E
1.00E
1.00E
1.00E

07
02
00
01
00
02
01

Log drop

GHB/mL

e
5
7
6
7
5
6

1.00E
1.00E
1.00E
1.00E
1.00E
1.00E
1.00E

07
02
00
02
02
02
01

Log drop

Effective?

cATP

% Drop

AMPi

Rise

Effective?

e
5
7
5
5
5
6

e
Yes
Yes
Yes
Yes
Yes
Yes

13,966
20
29
444
34
295
225

e
99.9
99.8
96.8
99.8
97.9
98.4

0.42
7.9
3.68
14.52
30.85
13.24
N/A

e
18.7
8.7
34.3
72.9
31.3
N/A

e
Yes
Yes
Yes
Yes
Yes
Yes

Please cite this article in press as: Keasler, V., et al., Expanding the microbial monitoring toolkit: Evaluation of traditional and molecular
monitoring methods, International Biodeterioration & Biodegradation (2012), http://dx.doi.org/10.1016/j.ibiod.2012.07.002

V. Keasler et al. / International Biodeterioration & Biodegradation xxx (2012) 1e6

The results from planktonic kill studies revealed that the

susceptibility of microorganisms towards chemical treatment with
biocides is greatly affected by the microbe metabolic status. It is
common that low dosages of biocide will stop growth of an
organism as the cells enter a dormant state to survive the harsh
conditions of the environment (Kim et al., 2009). Organisms may
enter in this state due to a combination of factors: to arrest division
to avoid damage to the genetic material caused by the biocide and/
or conserve energy to be used to pump toxic compounds out of the
cells. Furthermore, Gram-negative and Gram-positive bacteria
respond differently to different biocide by virtue of dissimilarities
in structure and composition of their outer membrane. In any case,
it is apparent that dormant cells are more susceptible to prolonged
stresses and the survival rate may be compromised upon extended
chemical applications. The new ATP/AMP assay allows the monitoring of distinct populations (active and dormant) and opens the
possibility for changes in the biocide treatment so that complete
microbial kill can be achieved by varying the dosages of the biocide
or the exposure time to the chemical.
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Please cite this article in press as: Keasler, V., et al., Expanding the microbial monitoring toolkit: Evaluation of traditional and molecular
monitoring methods, International Biodeterioration & Biodegradation (2012), http://dx.doi.org/10.1016/j.ibiod.2012.07.002