Bioresource Technology 98 (2007) 11241127

Short Communication

Measurement of the degradation capacity of activated sludge for a xenobiotic organic

Nyuk-Min Chong , Tze-Yung Lin
Department of Environmental Engineering, DaYeh University, 112 Shan-Jiau Rd. Da-Tsuen, Changhua 515, Taiwan Received 20 December 2005; received in revised form 19 April 2006; accepted 20 April 2006 Available online 9 June 2006

Abstract Studies were conducted to evaluate the capacities of an activated sludge in degradation process for a xenobiotic. The results showed that during its acclimation to 2,4-D, the sludge sharply accumulated degradation capacities before degradation could proceed at a noticeable rate; moreover, during de-acclimation the sludge gradually lost some capacities in a manner resembling an exponential decay. 2006 Elsevier Ltd. All rights reserved.
Keywords: Xenobiotics; Degradation capacity; Acclimation; Degraders; Activated sludge; 2,4-D

1. Introduction When a persistent xenobiotic is Wrst placed as the sole carbon source in a degradation reaction with an indigenous microbial biomass, a prolonged lag period, in which the target organic compound remains intact, must pass before any utilization of the organic can be observed; neither can the biomass grow. After this lag period, the target is usually quickly degraded. The biomass is not initially able to degrade the target, but does so after a period of induction or biochemical adjustment, a procedure commonly known to environmental scientists and engineers as acclimation (Singleton, 1994; Buitron and Gonzalez, 1996; Mangat and Elefsiniotis, 1999). Degradation of a xenobiotic occurs after acclimation indicates the acquisition a degradation capacity by the biomass as time proceeds. This fact implies that the biomass contains null degradation power initially but has gradually acquired a certain amount over time with the presence of the target substrate. For a mixed culture, one explanation for acclimation can be the selection of specialized microorganisms (Wiggings et al., 1987; Hu et al., 1996;
* Corresponding author. Tel.: +886 4 851 1888x2354; fax: +886 4 851 1336. E-mail address: chong@mail.dyu.edu.tw (N.-M. Chong).

Hu et al., 1998), but the need for a pure culture to acclimate (Buitron and Gonzalez, 1996; Buitron et al., 1998) also indicates that a capacity value is harbored within the biomass. DiYculties in understanding the xenobiotic degradation process are experienced by environmental scientists and engineers because the most important quantity for the entity responsible for degradation has been neither deWned nor measured. While degradation of an ordinary substrate is explicitly reXected by a mass of microorganisms (growth), degradation of a xenobiotic substrate involves a capability factor within the biomass which has not always been reXected in growth. Only when a capability is gained can utilization of the target as a substrate occur, thereby resulting in growth of the then capable microorganisms. The diYculties begin with the deWnition of degradation capacity, followed by the method of its quantiWcation, then by the question concerning its kinetics to couple with substrate utilization in expressing the degradation process. The degradation capacity must be an entity that is practically accountable. It must be real and it must exist within the biomass. The researchers hypothesize that for a biomass such as an activated sludge, degradation capacities exist with and can be accounted for by the number of microorganisms that can degrade (degraders), while others are nondegraders. Even when the total number of microorganisms

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has not changed, degradation power can arise when the original non-degraders turn into degraders during acclimation, and vice versa during de-acclimation. The objective of this work was to perform detailed measurements of the degradation capacities of an activated sludge during its degradation of a persistent xenobiotic compound. 2. Methods The tool used for the measurement of the degradation capacity of a biomass for a xenobiotic organic was developed previously (Chong, 2005). BrieXy, the tool is made of an agar plate (referred to as the tool plate), which has the ability to diVerentiate degraders and non-degraders within a biomass. The diVerentiation is made possible by distinguishing degrader colonies by means of a red colorization; non-degraders are colored blue. The number of red and blue thus indicates the quantities of both degraders and non-degraders on the same plate. The number of degraders compared to total microbial counts (colony forming units, CFU) indicates the degradation potential or capacity. Degradation capacity shown using this tool can be expressed explicitly as degrader units or as the proportion of degraders within the bulk biomass (the latter was designated as fraction-of-red, FOR). The target xenobiotic substrate used to demonstrate the measurement function of the tool was the herbicide 2,4dichlorophenoxyaceticacid (2,4-D). The biomass used as a test subject was a mixed culture originally obtained from an undeveloped forest soil that had no record of 2,4-D contamination, nor it had metals (slag) contamination. The soil with its microorganisms was inoculated in nutrient broth (NB) for a number of subcultures from which a mixed culture was harvested each time a test was conducted. The mixed culture of microorganisms resembled the nature of an activated sludge and thus this name was used as the description of the biomass. Batches of such sludge were subjected to reactions with 2,4-D in shaker Xask (batch) reactors. The medium originally contained 50 mg/l of 2,4-D as the sole carbon source and minerals: FeCl3, 1.2 mg/l; CaCl2, 12 mg/l; MgSO4 7H2O, 65 mg/l; NH4Cl, 25 mg/l; K2HPO, 200.0 mg/l, and KH2PO4, 156.6 mg/l. During the degradation reaction 2,4-D concentrations remaining in the solution were measured at regular intervals from samples of reactor mixture that were Wltered through Wberglass Wlter (pore size 0.22 m); UV absorbance at 235 nm of a sample was compared to a calibration to obtain its 2,4-D concentration. Simultaneously a small amount of activated sludge suspension was withdrawn from the reactor and inoculated onto the tool plates after serial dilutions. Reference inoculations were made on nutrient agar (NA) and plates containing 2,4-D as the sole carbon source (B plates). Measurements were made for the rise and fall of degradation capacities of batches of activated sludge to 2,4-D during their acclimation and de-acclimation. Three sets of tests representing acclimation, de-acclimation, and re-acclimation of the activated sludge to 2,4-D were performed.

The acclimation tests were performed routinely during which a speciWc set of FORs was determined in duplicate concurrently with 2,4-D concentrations at the time. The de-acclimation was to subject the acclimated activated sludge in an idle condition to the old medium without 2,4-D. One de-acclimation test was for the activated sludge that was idle for 100 h following a complete 2,4-D degradation. At that point the FORs were determined. Another deacclimation test was for an activated sludge for 200 h after 2,4-D depletion. The FORs were measured during this time period. Re-acclimation of the acclimated activated sludge was performed after 2,4-D was depleted, by settling the sludge, decanting the clear supernatant of the old medium, and adding a new medium containing 2,4-D to the reactors. One re-acclimation test was for the activated sludge that had been idle for about 100 h (following the test for deacclimation). Another re-acclimation test was performed by reintroducing 2,4-D to an activated sludge three (3) consecutive times (full acclimation). The concentrations of 2,4-D and FORs were measured during the courses in which the re-acclimated activated sludge was set in reaction with 2,4-D. 3. Results 3.1. Acclimation 2,4-D degradation course by a mixed culture of soil origin (activated sludge) is shown in Fig. 1(a). The data shown are the average results of four acclimation tests (conducted at diVerent times). The curve was concurrent with the degradation capacity measurements. This 2,4-D course is typical of its acclimation and degradation by an activated
1.2 1.0 Indicated fraction 0.8 0.6 0.4 0.2 0.0
1.E+07

2,4-D, C/Co mean 2,4-D,C/Co FOR

2,4-D refed

CFU (unit/ml)

b
1.E+06 1.E+05

on NA degraders 0 50 100 150 200 250 300 350 400

0 1.E+04 Reaction time (hrs)

Fig. 1. (a) 2,4-D concentrations during acclimation and re-acclimation by a mixed culture of soil microorganisms cultivated as an activated sludge. The rise and fall of degradation capacities, measured by the tool plates as the fractions-of-red over the total number of colonies, are shown as FOR. (b) Total CFU and total number of degraders concurrent with and after 2,4-D degradation.

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sludge The fractions of degrader to total CFU (FOR) within the activated sludge determined simultaneously with the 2,4-D concentrations, are also shown along with the degradation curves in Fig. 1(a) (average of a duplicate). Degraders were not found during the lag period; however, beginning with the latest phase of the lag, degraders and thus degradation capacity evolved a little prior to substrate consumption, the rate of degrader build-up from zero to the maximum being faster than that for substrate disappearance. This trend of degrader accumulation indicated that the power-mediating degradation was built up before degradation could be driven at a noticeable rate. The total CFU could not show the trend of the substrate degradation (Fig. 1(b)). The CFU decreased slightly during the lag time (external substrate was not consumed but endogenous respiration occurred) and increased marginally during degradation. By comparison, number of degraders, coinciding with FORs, showed a dramatic increment. This phenomenon was the only way to detect how and when the persistent substrate was to be degraded. The number of degraders was indeed the intensity for degradation. The increase in total CFU could, rather, be explained by the growth of degraders. During the acclimation process, the degraders gained through conversion and made their degradation; growth took place simultaneously with the degradation. From the initial sharp evolution of degraders, conversion was considered the major mechanism; meanwhile, the growth yield was low, as is true for yield on a hard substrate. 3.2. De-acclimation The de-acclimation was examined for a decrease in degraders beginning with the latter part of degradation and also during and after a period when the target substrate 2,4D was depleted (Fig. 1(a) and Fig. 2). While the degraders increased quickly before degradation, they decreased considerably concurrently with the falling 2,4-D concentration. However, after the substrate was depleted, the number of degraders decreased only gradually. Fig 1(a) shows that the reduction in degraders was slight after about 100 h; Fig. 2 indicates that the degradation capacity during de-acclima-

tion also decreased at a slow rate. An exponential reduction with a half-life of about 120 h could be suitable for this deacclimation trend. Decreases in degradation capacity occurred quicker when the target was degrading than when it was completely degraded. During the period of 2,4-D break-down, a complex growth system, including the rise and fall of de-gradation intermediates that might be used by some or all microorganisms, occurred. On the other hand, in the absence of any organic substrate, extensive biochemical changes in the microbes would be unnecessary. 3.3. Re-acclimation When the same substrate was replenished to a onceacclimated activated sludge, the lag period before the substrate degradation depleted or shortened. This behavior is shown by the second degradation curve in Fig. 1(a). For the re-acclimating activated sludge that had experienced previous acclimation(s), the degraders were already present at the beginning, thereby resulting in an immediate degradation. The fraction of degraders (as well as their numbers) increased from what was remaining; substrate degradation was driven by the available degraders. Fig. 2 showed the FOR of an activated sludge after it was twice subjected to re-acclimation (i.e., complete degradation twice after a Wrsttime acclimation). After consecutive re-acclimations, a FOR approaching unity was found. A fast degradation rate was found as a result of this high capacity. 4. Discussion Measurements of the abstract quantity of the degradation capacity of activated sludge for a persistent xenobiotic organic compound were achieved by the use of a tool deWned and developed for such purpose. Some insights into acclimation and the actual trend of degradation potential have been made possible by these measurements. The results obtained in this research are useful in several ways: Wrst, the need to deWne a degradation capacity was very important for solving the previously reported diYculties in determining the causes of xenobiotic degradation. Our deWnition of this capacity has been proved correct and feasible. Second, the resulting quantity, pattern, and rate of increase and decrease in the degradation capacity demonstrate the kinetics of xenobiotic degradation. A mathematical description (modelling) of the acclimation/degradation mechanism is possible with this kinetics of degraders, rather than the kinetics of the total activated sludge. Third, a measurement of degrader quantity or its proportion is useful in gauging the degradation capacity of an activated sludge at any time. An important practical application can be generated basing on this measurement as it shows the quality of the biomass at the time of the test, whether or not the biomass possesses a capability resulting from its previous treatment history, or its origin that is conducive to degradation.

1.2 1.0 Indicated fraction 0.8 0.6 0.4 0.2 0.0 300 350 400 450 500 Reaction time (hrs) 550 600
2,4-D, C/Co FOR

Fig. 2. 2,4-D concentrations and FOR (average of a duplicate) at reintroduction of 2,4-D to a fully acclimated activated sludge. De-acclimation was examined during the periods of 2,4-D degradation and after 2,4-D depletion.

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5. Conclusions Following conclusions could be drawn from this study: 1. During activated sludge acclimation to a xenobiotic, degrader increased in amounts disproportional to bulk sludge growth; the increase occurred prior to substrate degradation, indicating the power-mediating degradation was acquired and built up before degradation could be driven at a noticeable rate. 2. At re-acclimation, immediate substrate degradation was driven by the available degraders left from previous acclimation. Further degrader increase continued during re-acclimation. 3. When the substrate was depleted, degraders decreased in a manner resembling the exponential decay with a halflife of about 120 h. Acknowledgements This research was supported in part through a grant from the National Science Council of Taiwan. The authors also wish to express appreciation to Dr. Cheryl Rutledge for her editorial assistance.

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