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 Brunswick Canada, E3B 5H8 jim.cairns@luminultra.com

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, most studies have not used methods that distinguish between extracellular or dissolved ATP and the ATP contained only within microorganisms. While developing ATP assay reagents 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. A survey 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 the Biomass 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 be used to solve problems and enable continuous process improvement. For accurate estimation 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

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 activated sludge process. The continuing scientific interest in ATP monitoring of biological waste treatment processes is not surprising. As the keystone of metabolic activity (Lehninger, 1982), most of 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 the synthesis of new cells and biochemicals. Furthermore, ATP can be easily measured with high specificity by the firefly luciferase assay. The reaction is as follows:

ATP + O 2 + luciferin AMP + PPi + oxyluciferin + light

Where, ATP = Adenosine triphosphate AMP = Adenosine monophosphate PPi = pyrophosphate Mg++ = Magnesium ion The chemical energy produced from the breakdown of ATP is converted into light energy. 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 nonbiological solids that are poorly or non-biodegradable, which can accumulate in the floc of a biological waste treatment process. Doubtless, this occurs in other wastewater treatment systems. Therefore, conventional measurements such as mixed-liquor suspended 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 parameter in 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;

Mg + + luciferase

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) with no 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 with biocide 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 this experience, 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-liquor samples 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 an experiment. 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. Fullscale plant operations were monitored by collecting grab samples at various time intervals and locations throughout the plant. ATP analyses were conducted on subsamples 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 firefly luciferase 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 ATP analyses 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

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 activated sludge 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 that the 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 done by 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.
600.0 40.0

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(ng/mL)

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15.0 200.0

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Figure 1 tATP

TM

and dATP

TM

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 increases occurred 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).

BSI TM = dATP
Where,

TM

tATP TM

100%

dATP

tATP

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300.0

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(ng/mL)

25.0

BSITM = Biomass Stress Index tATPTM = Total ATP dATPTM = Dissolved ATP
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Percent Change from Neutral pH

BSI TM
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cATP TM
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0 7 7.5 8 8.5 9 9.5 10 10.5 11

pH

Figure 2 cATPTM and BSITM Response 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 pH 9.5, 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 ATP only). cATPTM is calculated as follows:

cATP TM = tATP TM dATP TM

Where, cATPTM = Cellular ATP tATPTM = Total ATP dATPTM = Dissolved ATP This is another advantage of accurate assessment of the dissolved ATP component its use 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, but cATPTM becomes more sensitive to changes in living biomass concentration.

Figure 3 shows the results of a similar experiment except that the pH adjustments were decreased to make the sample acidic.
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-200 4 4.5 5 5.5 6 6.5 7 7.5

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Figure 3 tATPTM and BSITM Response to Acid Stress

Through these results, it can be seen that the BSITM was considerably more sensitive to pH than the measurement of total ATP content. Food and nutrient deficiency and heat shock laboratory stress tests (data not shown) also demonstrated 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 container holding 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 the experiment). 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.

BSI TM
3

Relative Change from Initial Condition

-1

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tATP TM
-3 -4

-5 1 2 3 4 5 6

Time of Anoxic Conditions (hr)

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 more sensitive (Figure 5).
5

BSI TM
Relative Change from Initial Condition
0

-5

-10

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tATP TM

-20

-25 0 5 10 15 20 25 30

Time of Anoxic Conditions (hr)

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, in either 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.

3.5

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Relative Change from Initial Condition

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Figure 6 BSITM Relative 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.
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Figure 7 BSITM Change 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 controlled environment of the laboratory.

To determine if the measurement of dATPTM was typical in biological wastewater treatment processes a variety of plants were sampled and analyzed. Table 1 summarizes select 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

BSITM (%) Type of Plant Municipal Sewage Paper Mill Paper Mill Food Processing Plant Food Processing Plant Paper Mill Paper Mill Type of Bioreactor Minimum Activated Sludge Aerated Lagoon Series of Aerated Lagoons w/ Combined Air & Pure Oxygen Covered Anaerobic Lagoon Biological Nutrient Removal Reactor Upflow Anaerobic Sludge Blanket Activated Sludge 0.7 1.0 3.0 1.4 0.1 7.7 0.1 Maximum 36 44 73 14.5 4.7 40 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 quite suddenly. 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.

80%

Cell 2A
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(%)

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BSI
30% 20%

TM

Cell 2C
10%

0%
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

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 that measuring 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.
200

Cell 2A tATP TM
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Cell 2A cATP TM
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0
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

Figure 9 cATPTM Monitoring in Aerobic Lagoon Treating Pulp & Paper Effluent

Accounting for the dissolved ATP component allows a better assessment of the concentration 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.

The following figures show examples of how the BSITM can reveal stress in full scale operations.
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40 2

Reactor Dissolved Oxygen (mg/L)

BSI TM

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30 1.5 25

Dissolved Oxygen Concentration

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15

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0 22-Feb-04

29-Feb-04

7-Mar-04

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0 28-Mar-04

Figure 10 BSITM Response 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.
0.1 45

Period 1 R = -0.09 Reactor Influent Nutrient/COD Ratio

0.08

Nutrient-to-COD Ratio

Period 2 R = -0.38

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35

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20 0.04 15

BSI TM
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0
0 bFe 4 r-0 Ma 4 r-0 Ap 4 Ma 04 yJ -0 un 4 4 l -0 Ju 04 gAu 04 pSe

Figure 11 BSI

TM

Response to Macronutrient Deficiency

Reactor BSITM (%)

30

Reactor BSITM (%)

Figure 11 displays data collected during spring and summer at the same facility as described 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) then appeared 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 between nutrient-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 was experiencing 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.
12 900

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300 23-Apr-05

Figure 12 BSITM Response 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 aerated reactor 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

Aerated Tank Ammonia (mg/L NH3-N)

Aerated Tank BSI

TM

(%)

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 adenosine triphosphate 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.