weftec-2005-dissolved-atp–a-new-process-control-parameter-for-biological-wastewater-treatment

7/29/2019 WEFTEC-2005-dissolved-atpa-new-process-control-parameter-for-biological-wastewater-treatment

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Dissolved ATP A New Process Control Parameter for

Biological Wastewater Treatment

J. E. Cairns*, P. A. Whalen, P. J. Whalen, D. R. Tracey, R. E. Palo

LuminUltra Technologies Ltd.

440 King Street, King Tower, Suite 630

Fredericton, New BrunswickCanada, E3B 5H8

[email protected]

ABSTRACT

ATP (Adenosine Triphosphate) is the primary energy molecule in all living cells.Numerous researchers have concluded that ATP monitoring of biological processes has

the potential to be valuable for process improvement and troubleshooting. However, moststudies have not used methods that distinguish between extracellular or dissolved ATP

and the ATP contained only within microorganisms. While developing ATP assayreagents and protocols that facilitate easy analyses and that have been optimized

specifically for wastewater treatment, it was discovered that samples from several

biological wastewater treatment systems contained significant levels of dissolved ATP. Asurvey of seven different treatment sites conducted during routine operations found that

dissolved ATP content ranged from 0.7 to 73 % of the total sample ATP. A stress index

was formulated based of the ratio of dissolved ATP to total ATP, referred to as theBiomass Stress Index. It was found both in laboratory and full-scale reactors that as

stresses such as sub-optimal pH, anoxia, toxicity, and nutritional deficiencies were

applied to the microbial populations, the stress index increased. The stress index can beused to solve problems and enable continuous process improvement. For accurateestimation of viable biomass, dissolved ATP measurement is essential. After correction

for dissolved ATP content, ATP can be used more effectively for process control such as

in adjusting food to microorganism ratio, sludge age, and nutrient additions.

KEYWORDS

ATP, adenosine triphosphate, activated sludge, wastewater treatment, process control,

biomass, dissolved ATP, total ATP, cellular ATP, biomass stress index, LuminUltraTM

,

tATPTM, dATPTM, cATPTM, BSITM, biological monitoring.

INTRODUCTION

As early as thirty-five years ago, the value of monitoring ATP (adenosine triphosphate) in

biological waste treatment was recognized (Paterson et al., 1970). More recently,Archibald et al (2001), in a study using a suite of respirometric tests on mixed liquor


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from paper mill activated sludge processes, concluded that ATP measurements provided

a useful monitor of the proportion of viable cells and a toxicity indicator in an activatedsludge process.

The continuing scientific interest in ATP monitoring of biological waste treatment

processes is not surprising. As the keystone of metabolic activity (Lehninger, 1982), mostof the energy within microorganisms is stored and transmitted via ATP. ATP is produced

as microbial food is consumed and is subsequently utilized for cell maintenance and thesynthesis of new cells and biochemicals.

Furthermore, ATP can be easily measured with high specificity by the firefly luciferaseassay. The reaction is as follows:

lightinoxyluciferPPiAMPluciferinOATPluciferaseMg

+++ ++

++

2

Where,ATP = Adenosine triphosphate

AMP = Adenosine monophosphate

PPi = pyrophosphateMg++ = Magnesium ion

The chemical energy produced from the breakdown of ATP is converted into lightenergy. Each molecule of ATP consumed in the reaction produces one photon of light.

This light output can be quantified using a luminometer within a matter of seconds.

Archibald et al. (2001) note that pulp and paper mill wastewaters contain many non-

biological solids that are poorly or non-biodegradable, which can accumulate in the flocof a biological waste treatment process. Doubtless, this occurs in other wastewater

treatment systems. Therefore, conventional measurements such as mixed-liquorsuspended solids (MLSS) or mixed-liquor volatile suspended solids (MLVSS) can

provide misleading information about the amount of viable biomass in the reactors.

Furthermore, these measurements do not distinguish between living and dead cells.Because ATP is produced only by living cells, its measurement can overcome these

difficulties and provide an opportunity for superior control of such fundamental operating

issues such as food to microorganism ratio, sludge age, and nutrient feed.

Although ATP is vital to all wastewater treatment microorganisms and the measurement

process described is simple, ATP has not been routinely adopted as a process parameterin operating wastewater treatment plants. Possible reasons for lack of routine use include

the following:

Instability of reagents; Ineffective or cumbersome ATP extraction techniques for wastewater treatment

bioreactor samples;

Lack of test protocols optimized for wastewater treatment bioreactors;


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Insufficient monitoring guidelines.Furthermore, it is frequently assumed that ATP is only found within living cells.

Typically, during ATP analyses, samples from waste treatment plants are immersed into

an extraction agent such as boiling buffer (Paterson et al, 1971 ), organic solvents

(Lefebvre, 1988), proprietary surfactant solutions, or acid solvents (Archibald, 2001) withno separation of the microorganisms from the liquid portion of the sample. Thus, if the

sample included and extracellular ATP, it would not be distinguished from ATP

contained within the living cells (i.e. intracellular ATP).

In 1999, our organization began a program to optimize the ATP assay application for

monitoring biological wastewater treatment processes. Based on prior experience withbiocide treatment of contaminated industrial water systems, we were aware that in

environments that are lethal to microorganisms, significant amounts of extracellular or

dissolved ATP can be created and maintained for a period of time. Because of thisexperience, measurement of dissolved ATP was included as a focal point in the project.

METHODOLOGY

Tests were conducted on samples from laboratory reactors and full-scale operations.

Laboratory bench-scale tests were typically performed by collecting mixed-liquorsamples from the reactor of a municipal activated sludge wastewater treatment plant and

adding them to a two-liter vessel fitted with an air-stone connected to an air pump for

aeration. Samples were aerated for at least 60 minutes prior to the initiation of anexperiment. In special cases, samples were placed in vessels that were not aerated but

kept sealed. At various time intervals, sub-samples were removed for ATP analyses. Full-

scale plant operations were monitored by collecting grab samples at various timeintervals and locations throughout the plant. ATP analyses were conducted on sub-samples removed from the sample containers, usually as soon as the samples had been

brought to the plant laboratory.

tATPTM (Total ATP intracellular ATP plus extracellular ATP content) analyses were

performed by adding a sub-sample of wastewater to an ATP-releasing agent and mixing.

The mixture was then diluted and assayed for ATP using the bioluminescent fireflyluciferase test. dATPTM (Dissolved ATP extracellular ATP only) was measured by

allowing the sample to settle, taking a sub-sample of the supernatant and diluting it with

an ATP-stabilizing reagent. The diluted sub-sample was then assayed for ATP. All ATPanalyses were performed using reagents designed and optimized for the wastewater

treatment application, manufactured by LuminUltraTM Technologies Ltd. The light

produced in the luciferase reaction was measured in a luminometer (either Turner

Designs Model 20e or Kikkoman Lumitester C-100).

RESULTS AND DISCUSSION


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At the outset of this project, the first few steps taken involved the measurement of

tATPTM

and dATPTM

in mixed-liquor samples collected from the reactor of an activatedsludge plant. To our surprise, we discovered that dissolved ATP was present in samples

taken during the normal plant operation.

Furthermore, when we subjected the samples to stressful conditions, we discovered thatthe dissolved ATP content of the sample increased. In one experiment, for example, the

population of the mixed-liquor sample was subjected to an alkaline stress. This was doneby raising the pH from neutral to pH 10.5 in increments of 0.5 units every 30 minutes.

After each incremental adjustment, dATPTM and tATPTM were measured.

0.0

100.0

200.0

300.0

400.0

500.0

600.0

7.36 8.00 8.50 9.00 9.50 10.00 10.50

pH

tATPTM(ng/mL)

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

dATPTM(ng/mL)

dATPTM

tATPTM

Figure 1 tATPTM and dATPTM Response to Alkaline pH Stress

The results of the experiment are shown in Figure 1. As anticipated for an unfavorable

environment, the total ATP of the sample decreased as the pH was raised to a highly

alkaline level, indicating a drop in the proportion of living biomass. At the same time,this was accompanied by a simultaneous increase in dissolved ATP. While it was not

surprising that dissolved ATP might be detected when the sample pH was raised to a

level that is known to be inhospitable for typical microbial growth, significant increasesoccurred even when the pH was merely in a sub-optimal region (pH 8-9).

Observations of this kind led to the development of a stress index based on the dissolved

fraction of sample ATP that was measured by dATPTM, hereto in referred to as BSITM(Biomass Stress Index).

%100= TMTMTM

tATPdATPBSI

Where,


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BSITM

= Biomass Stress Index

tATPTM

= Total ATPdATPTM = Dissolved ATP

0

200

400

600

800

1000

1200

7 7.5 8 8.5 9 9.5 10 10.5 11

pH

PercentChangefromNeutralpH

tATPTM

cATPTM

BSITM

Figure 2 cATP

TMand BSI

TMResponse to Alkaline Stress

Figure 2 shows the results of the data from Figure 1 graphed to show the sensitivity of

BSITM

compared to simply the total sample ATP. At the most extreme pH, BSITM

showed a relative change of almost double that for tATPTM

measurements. Even at pH9.5, the total ATP had decreased approximately 2 times, while the BSITM increased by

approximately 4 times.

Another parameter shown in Figure 2 is cATPTM

(Cellular ATP intracellular ATPonly). cATPTM is calculated as follows:

TMTMTM dATPtATPcATP =

Where,

cATPTM

= Cellular ATPtATPTM = Total ATP

dATPTM

= Dissolved ATP

This is another advantage of accurate assessment of the dissolved ATP component itsuse in directly calculating the proportion of ATP contained only in living cells, therefore

providing a relative measure of biomass concentration as cATPTM

. Figure 2 shows that

under normal conditions, tATPTM

and cATPTM

relative changes are closely related, butcATPTM becomes more sensitive to changes in living biomass concentration.


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Figure 3 shows the results of a similar experiment except that the pH adjustments were

decreased to make the sample acidic.

-200

0

200

400

600

800

1000

1200

1400

1600

4 4.5 5 5.5 6 6.5 7 7.5

pH

PercentChangefromNeutralpH

BSITM

tATPTM

Figure 3 tATP

TMand BSI

TMResponse to Acid Stress

Through these results, it can be seen that the BSITM

was considerably more sensitive topH than the measurement of total ATP content.

Food and nutrient deficiency and heat shock laboratory stress tests (data not shown) alsodemonstrated that monitoring dATPTM helped to reflect these unfavorable conditions.

However, another set of experiments involving anoxic stress of aerobic organisms

showed that BSITM does not display a universal sensitivity to stressors. Anoxic

conditions were created by adding BOD (2000 mg/L glucose) and sealing the containerholding the mixed liquor sample. A number of sub samples were subjected to this

treatment so that each data point could be obtained from an individual sub sample (i.e.

such that oxygen would not be re-introduced to the treated sample throughout theexperiment).

Figure 4 below shows that during the first 6 hours of this experiment, the BSITM

responded with approximately the same sensitivity as the total sample ATP.


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-5

-4

-3

-2

-1

0

1

2

3

4

1 2 3 4 5 6

Time of Anoxic Conditions (hr)

RelativeChangefromInitialCondition

BSITM

tATPTM

Figure 4 tATPTM and dATPTM Response to Short-Term Anoxic Stress

For a 24 hour exposure, the change in tATPTM

measurement was found to be moresensitive (Figure 5).

-25

-20

-15

-10

-5

0

5

0 5 10 15 20 25 30


RelativeChangefromInitialCondition

tATPTM

BSITM

Figure 5 tATPTM and dATPTM Response to Long-Term Anoxic Stress

It is believed that this occurred because of an increase in ATP-degrading enzymes

associated with the prolonged stressor. Therefore, there appear to be some types of

stresses in for which the stress index may be less informative than others. However, ineither case, ATP monitoring was an excellent tool for stress detection. Figure 6 is a graph

showing the peak in BSITM

that is not so apparent in Figure 5. The importance of this will

be seen when field-scale results are discussed.


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-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0 5 10 15 20 25 30


RelativeChangefromInitialCon

dition

BSITM

Figure 6 BSI

TMRelative Response to Long-Term Anoxic Stress

A control test (aerobic) was also conducted at this time. Figure 7 shows how favorable

conditions can results in a decrease in BSITM

.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 5 10 15 20 25 30


BSITM(%)

BSITM

Figure 7 BSI

TMChange in Control During Anoxic Stress Test

For practical application, it is necessary to determine if dissolved ATP is present in all

types of field operations and can be used for monitoring stress beyond the controlledenvironment of the laboratory.


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To determine if the measurement of dATPTM

was typical in biological wastewater

treatment processes a variety of plants were sampled and analyzed. Table 1 summarizesselect results. Dissolved ATP was detected in every sample, regardless of the type of

wastewater being processed, the design of the plant, or the form of biological respiration

(i.e. aerobic or anaerobic). Moreover, in more than half of the plants, dissolved ATP

represented a major proportion of the total sample ATP at one time or another.

Table 1 Biomass Stress Index (BSITM

) Results at Select Industrial & Municipal Sites

Type of Plant Type of BioreactorBSI

TM(%)

Minimum Maximum

MunicipalSewage

Activated Sludge 0.7 36

Paper Mill Aerated Lagoon 1.0 44

Paper Mill Series of Aerated Lagoons w/Combined Air & Pure Oxygen

3.0 73

Food ProcessingPlant

Covered Anaerobic Lagoon 1.4 14.5

Food ProcessingPlant

Biological Nutrient Removal Reactor 0.1 4.7

Paper Mill Upflow Anaerobic Sludge Blanket 7.7 40

Paper Mill Activated Sludge 0.1 0.9

It is obvious from this summary that facilities encounter a wide range of biomass stress

levels. It was also found that the dissolved ATP concentration can increase quitesuddenly. Figure 8 shows an example for an aerated basin treatment system of a pulp and

paper mill in which dissolved ATP rose to become the predominating form of ATP

within one day.


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0%

10%

20%

30%

40%

50%

60%

70%

80%

11/2/2004 0:00 11/2/2004 12:00 11/3/2004 0:00 11/3/2004 12:00 11/4/2004 0:00 11/4/2004 12:00

BSITM(%)

Cell 2A

Cell 2C

Cell 2B

Figure 8 BSITM Monitoring in Aerobic Lagoon Treating Pulp & Paper Effluent

As a corollary, the data collected during this monitoring period can be used to show thatmeasuring only total ATP could be misleading for estimations of the amount of viable

biomass in the system. Figure 9 shows the same time period as above, with tATPTM

and

cATPTM

data from the Cell 2A sample point.

0

40

80

120

160

200

11/2/2004 0:00 11/2/2004 12:00 11/3/2004 0:00 11/3/2004 12:00 11/4/2004 0:00 11/4/2004 12:00

cATPTMortATPTM(ng/mL)

Cell 2A tATPTM

Cell 2A cATPTM

Figure 9 cATP

TMMonitoring in Aerobic Lagoon Treating Pulp & Paper Effluent

Accounting for the dissolved ATP component allows a better assessment of theconcentration of living, viable biomass than does the tATPTM alone during stressful

periods. As discussed previously, cellular ATP has potential to provide an estimate of

viable biomass that is superior to MLSS or MLVSS.


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The following figures show examples of how the BSITM

can reveal stress in full scaleoperations.

0

0.5

1

1.5

2

2.5

22-Feb-04 29-Feb-04 7-Mar-04 14-Mar-04 21-Mar-04 28-Mar-04

ReactorDissolvedOxygen(mg/L)

0

5

10

15

20

25

30

35

40

45

ReactorBSITM(%)

BSITM

Dissolved OxygenConcentration

Figure 10 BSI

TMResponse to Dissolved Oxygen Deficiency

Figure 10 shows the results from an aerated lagoon treating pulp and paper wastewater.

In this example, aeration was insufficient to maintain a consistent supply of oxygen and,

when the DO dropped to 0.2 mg/L, the dissolved ATP increased to 38% of the total ATP.Again, as observed in the lab experiment, this high stress index was only maintained

during the onset of the stress.

0

0.02

0.04

0.06

0.08

0.1

Feb-0

4

Mar-0

4Apr-0

4

May-0

4

Jun-0

4Jul-0

4

Aug-0

4

Sep-0

4

ReactorIn

fluentNutrient/CODRatio

0

5

10

15

20

25

30

35

40

45

Re

actorBSITM(%)

Period 1

R = -0.09

Period 2

R = -0.38

BSITM

Nutrient-to-CODRatio

Figure 11 BSI

TMResponse to Macronutrient Deficiency


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Figure 11 displays data collected during spring and summer at the same facility asdescribed in Figure 10. Oxygen level was found to be limiting in the period to the left of

the red vertical line. However, after additional aeration was installed in mid-June, there

was no longer a dependence on oxygen after this upgrade (i.e. the DO constraint was

effectively eliminated). Rather, nutrient delivery (i.e. Nitrogen and Phosphorous) thenappeared to become the limiting influence on biomass health and activity. For example,

prior to the period in which nutrient feed decreased, there was little correlation betweennutrient-to-COD ratio and BSITM (R = -0.09). However, following the reduction in

nutrient supply, the correlation increased significantly (R= -0.38). The observation that

the relationship was not completely correlated suggests that the population wasexperiencing additional types of stress. Long term statistical comparisons of BSITM with

other parameters have the potential to identify other stress factors allowing for continuous

process improvement.

0

2

4

6

8

10

12

5-Mar-05 12-Mar-05 19-Mar-05 26-Mar-05 2-Apr-05 9-Apr-05 16-Apr-05 23-Apr-05

AeratedTankBSITM(%)

300

400

500

600

700

800

900

AeratedTankAmmonia(mg/LNH3-N)

BSITM

Ammonia

Figure 12 BSI

TMResponse to Ammonia Toxicity

In contrast with Figure 11, Figure 12 shows the effect on BSITM

when ammonia,

normally a beneficial nutrient, is present at toxic levels. In this example, the aeratedreactor accepted the effluent from an upstream continuously-stirred anaerobic digester

treating starch mill wastewater. The chart shows that there is a corresponding rise in the

BSITM

as ammonia reached critical levels.

CONCLUSIONS

Dissolved ATP appears to be ubiquitous in biological wastewater treatment processes and

often represents a significant proportion of the total sample ATP. For accurate estimation

of viable biomass, its measurement is essential. After correction for dissolved ATP


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content, intracellular ATP measurements can be used more effectively for process control

such as adjusting food to microorganism ratio and sludge age.

A stress index can be calculated based on the ratio of dissolved ATP to total ATP. For

most types of stress, this index can be more responsive to stress than simply measuring

total ATP. It can rapidly differentiate between biomass reduction and stress. For example,if total ATP decreases, it could mean a loss in biomass or it could mean a stressful

condition has decreased the ATP content of the cells. Based on our tests, the use of BSITM

and cATPTM can distinguish between these two possibilities. Statistical process analyses

on the data generated from such a technology can be used to solve problems and enable

continual process improvement

REFERENCES

Patterson, J.W.; Brezonik, P.L.; Putnam, H.D. (1970) Measurement and significance of

adenosine triphosphate in activated sludge.Environ. Sci. Technol. 4(7) 569-575.Levin, G. V.; Schrot, J. R.; Hess, W. C. (1975) Methodology for application of adenosinetriphosphate determination in wastewater treatment. Environ. Sci. Technol. 9(10),

961965.

Lehninger A. L. (1982) Principles of Biochemistry, Part II,1011 pp. Worth Pubs. Inc., New York, USA.

Lefebrve, Y.; Coulture, P.; Couillard, D. (1988). An analytical procedure for the

measurement of ATP extracted from activated sludge. Can. J. Microbiol.34,

1275-1279.Archibald, F.; Methot, M.; Young, F.; Paice, M. G. (2001). A Simple System to Rapidly

Monitor Activated Sludge health and performance. Wat. Res.35 (10) 2543 2553.

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