Bioresource Technology 104 (2012) 181–186 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech Biogenic substrate benefits activated sludge in acclimation to a xenobiotic Nyuk-Min Chong a,⇑, MaiLy Luong b, Ching-Shyung Hwu c a Department of Environmental Engineering, DaYeh University, No 168, University Road, Dacun, Changhua 51591, Taiwan, ROC Department of Environmental Science, Hanoi University of Science, Vietnam National University, 334 Nguyen Trai Street, Thanh Xuan, Hanoi, Vietnam c Department of Safety, Health and Environmental Engineering, Hungkuang University, 34, Chungchie Road, Shalu Township, Taichung 43302, Taiwan, ROC b a r t i c l e i n f o Article history: Received 18 August 2011 Received in revised form 27 October 2011 Accepted November 2011 Available online 11 November 2011 Keywords: Acclimation Xenobiotic Activated sludge Biogenic substrate Diauxic growth a b s t r a c t Activated sludge that originated from a biogenic fed-batch reactor under steady-state was re-cultivated with the same biogenic substrates to test the changes in the sludge’s performance in acclimation and degradation of a xenobiotic Re-cultivations with varying biogenic concentrations were conducted at time points ranging from 16 d before to d after the acclimation reactions Biogenic re-cultivation energizes sludge cells thereby benefiting the re-cultivated biomass by shortening its acclimation lag time Lag time increases on both sides of the re-cultivation time where lag has been shortened the most: (1) in short recultivation times before and after acclimation reactions, high concentrations of new or unfinished biogenic substrates cause diauxic growth that delays acclimation; (2) in long re-cultivation times, the re-cultivated biomass loses its energy-rich advantage Both these lag lengthening situations have their worst cases in which acclimation lag times become longer than that of the original sludge, thus counterbalancing the benefits Ó 2011 Elsevier Ltd All rights reserved Introduction Xenobiotic organic chemicals, including phenoxy acid herbicides, are so defined because they are foreign to natural (indigenous) microorganisms Although xenobiotics containing wastewaters can be suitably treated using biological methods (Chin et al., 2005; Ettala et al., 1992; Hill et al., 1986; Meric et al., 2003), the xenobiotic nature of the pollutant requires the treatment plant microorganisms (typically activated sludge) to go through an acclimation phase before the microorganisms evolve the degradation capability for treating the influent xenobiotics The doubt and concern about successful biological treatment of xenobiotics is twofold: a prolonged acclimation phase (length of lag time) before the start of degradation, and slow degradation kinetics after the lag Between these two concerns, the more important one is often the acclimation lag time, rather than the treatment kinetics, which may be met with the adjustment of operating conditions within a reasonable range, especially for those mildly persistent xenobiotics Activated sludge biomass grown on the feed of biogenic substrate must be in a healthy physiological condition These healthy conditions should be favorable to the microorganisms when they must go through the energy-expensive xenobiotic acclimation process However, inconsistent results are found in literature about the effects of biogenic organics on degradation of man-made ⇑ Corresponding author Tel.: +886 851 1340; fax: +886 851 1336 E-mail address: chong@mail.dyu.edu.tw (N.-M Chong) 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd All rights reserved doi:10.1016/j.biortech.2011.11.004 xenobiotics or hydrocarbons There have been some study cases of both beneficial and adverse effects of biogenic organics on xenobiotic degradation The beneficial cases include: citrate on toluene (Harrison and Barker, 1987); natural amino acids on mono-substituted phenol (Shimp and Pfeander, 1985); natural organics such as manure on two chloro- and a nitro-herbicides (Moorman et al., 2001); fatty acids on soil hydrocarbon (Nelson et al., 1996); pyruvate on naphthalene (Lee, 2003) Conversely, adverse cases are also found: glucose or amino acids on xylene and toluene (Swindoll et al., 1988); ethanol on benzene, toluene and xylene (Corseuil et al., 1998); glycolic acid and glucose on p-cresol (Lewis et al., 1986); yeast extract and milk on 3-nitrobenzoate, 4-chlorobenzoate, 4-chlorophenol (Hu et al., 2005) The microbiological aspects of xenobiotic degradation are examined here in order to diagnose the causes of the inconsistent results listed above about the effects of biogenic on xenobiotic degradation: (1) the involvement of symbiotic functions or cometabolisms needed for the microbial communities to succeed in degrading the target xenobiotics For the degradation of those xenobiotics in the examples listed above, the requirement of ecological cooperation of the microbial communities should not be overly demanding; the microbial diversity that activated sludge contains can well satisfy the ecological needs for degradation of a xenobiotic solely (Sanapareddy et al., 2009; Wagner et al., 2002) This ecological factor can slightly affect xenobiotic acclimation in either the advantageous or the disadvantageous ways, if there is any effect at all Therefore, the ecological factor can have a neutral or unimportant effect on the efficiency of activated sludge in its 182 N.-M Chong et al / Bioresource Technology 104 (2012) 181–186 acclimation to a xenobiotic (2) The effect that activated sludge use a biogenic substrate preferentially to xenobiotic substrate (diauxic growth) (Basu et al., 2006; Chong and Chiou, 2010; Harder and Dijkhuizen, 1982) This factor can slow down xenobiotics acclimation and thus affect xenobiotic degradation disadvantageously (3) Biogenic substrate enhancement of the elements needed for mediating xenobiotic degradation Such elements include enzyme activities (Margesin et al., 2000; Taylor et al., 2002) and energy (ATP) contents in the microbial cells (Wilson et al., 1986) The production of catabolic enzymes means that the microorganisms are successful in evolution of their degradation capability To drive these chemical reactions, including the production of enzymes and the subsequent catabolism of the xenobiotics, the microbial cells must possess a rich energy reserve Consequently, enhancing the cells’ energy-richness can be advantageous for the cells to break down the stable structure of the target xenobiotic In summary, diauxic growth and energy richness are the major factors requiring systematic examination to resolve the inconsistencies noted above The purpose of this study, therefore, was to investigate the effects of nourishing activated sludge with biogenic substrates on the sludge’s performance or efficacy in acclimation and degradation of a xenobiotic In addition to seeking a unified explanation for the inconsistent results mentioned above, this study also intended to seek indications about the xenobiotic treatment potency of activated sludge when a xenobiotic appears periodically in the influent of the activated sludge treatment plant To fulfill the purpose of this study, experiments were conducted with which a biomass of steady-state growing activated sludge was separately re-cultivated with a feed of biogenic nature (containing sucrose and peptone) The times at which re-cultivation of the sludge started were before, after and concurrently with the sludge’s acclimation and degradation of a model xenobiotic 2,4-dichlorophenoxyacetic acid (2,4-D) The test variables included (1) the points of time when re-cultivations started; and (2) the biogenic substrate concentrations in the re-cultivation feed The lengths of lag time during acclimation and the kinetics of the succeeding degradation were quantified using a mathematical acclimation model developed by Chong (2009) The advantages or disadvantages of biogenic substrates for the sludge biomass’ xenobiotic acclimation and degradation were examined using the model parameters that describe the efficiency of an acclimation process The major factors studied were the sludge cells’ energy contents and biogenic interference on the sludge biomass’ performance in acclimation and degradation of the xenobiotic Methods 2.1 Target xenobiotic and activated sludge common activated sludge biomass produced from a continuous activated sludge treatment plant 2.2 Re-cultivations of activated sludge This study employed a major experimental design referred to as the re-cultivation of the activated sludge biomass The details of re-cultivation are as follow: (1) the biomass re-cultivated was harvested the fed-batch reactor; (2) the re-cultivating feed was biogenic in nature, consisting of sucrose and peptone at the proportion of 4:1 on the weight to weight basis (w/w) This ratio was kept constant when the biogenic feed concentration was changed; (3) the re -cultivation times (the times at which the feed and biomass started reaction) were divided into three time segments relative to the starttime of a 2,4-D acclimation and degradation reaction: before (precultivation), concurrent, and after (post-cultivation) The biogenic re-cultivation scheme, with variations of re-cultivation time and biogenic substrate feed concentration, is listed in Table (with designations of the tests) The re-cultivation media contained minerals listed above, different concentrations of biogenic substrates (Table 1), and activated sludge thickened from the fed-batch reactor suspension with minimum carry-over of supernatant (sludge suspension settled for longer than 30 and approximately 70% of supernatant discarded) The re-cultivation reactors were operated in shake-flasks (300 ml conical flasks containing 150 ml liquid) The activated sludge concentration (measured as suspended solids, SS) initially added to the re-cultivation reactors was approximately 100 mg-SS/l For the pre-cultivation reactors, biogenic feeds were administered once and the reactors were then let idle (shaking was maintained) until it was time the sludge biomass was re-harvested and used in new reactors for the 2,4-D acclimation and degradation tests For the concurrent (0 h) and post-cultivation tests, biogenic substrates were added, at the intended times, directly to the 2,4-D acclimation reactors Concentrated biogenic substrates were spiked-fed to the postcultivation reactors to avoid excessive change in liquid volume 2.3 Acclimation and degradation experiments The activated sludge biomass used for acclimation and degradation of 2,4-D was harvested from the composite of multiple pre-cultivation reactors Sludge concentrations (SS) were measured to calculate the amounts of sludge to be transferred to the Table Biogenic substrate concentration and culture time applied to the re-cultivation of activated sludge biomass used for 2,4-D acclimation and degradation.a Culture timec The target xenobiotic was the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) The initial activated sludge seeds were obtained from a soil that did not have any record of 2,4-D nor metal (slag) contamination The mixed culture from soil was grown to a suitable amount on Nutrient Broth (NB Difco 234000) for a number of subcultures in shake-flasks before the culture was seeded to a long-term cultivation reactor that was operated in a fed-batch mode The fed-batch reactor was fed once everyday with biogenic substrates (100 mg/l sucrose and 25 mg/l peptone) The feed also contained minerals: FeCl3 1.0 mg/l, NH4Cl 30.0 mg/l, K2HPO4 200.0 mg/l, KH2PO4 156.0 mg/l, MgSO4 31.0 mg/l Reactor suspension, one-tenth (1/10) of the liquid volume in the reactor tank, was wasted everyday so that a mean cell resident time (hc) of 10 d was achieved The fed-batch reactor was operated uninterrupted for a prolonged period to reach a pseudo-steady state The sludge obtained from this fed-batch reactor was referred to as the original sludge, which resembled the Biogenic conc.b (mg/l) 20 50 100 200 Designationsc Post-cultivation Concurrent Precultivation À1 d À2 d À4vd 0h 1h 8h 2d 3d h20 S h20 S h50 S h50 S d20 S d20 S d50 S d50 S À1 d100 S À2 d100 S À4 d100 S h100 S h100 S h100 S d100 S d100 S h200 S h200 S d200 S d200 S a Initial sludge concentration was 50 mg/l (SS) in all 2,4-D acclimation and degradation tests b Biogenic substrates consisted of sugar (sucrose) and peptone at the ratio of 4:1 w/w The concentrations listed denoted sugar concentration only c Designation rules: n1 T n2S n1 stands for re-cultivation time relative to the start of acclimation test; T stands for time (in hour or day); n2S stands for concentration of biogenic feed (sugar) n1-T and n2S are used independently when re-cultivation time (row of the table), and re-cultivation feed concentration (column), respectively, are described in the text and figures N.-M Chong et al / Bioresource Technology 104 (2012) 181–186 acclimation reactors The liquid in an acclimation reactor was made up with approximately 95% of the supernatant of the precultivation reactors In the concurrent and the post-cultivation tests, the biomass was harvested directly from the fed-batch reactor, as a thickened sludge with minimum carry-over of supernatant The starting sludge concentrations were all 50 mg-SS/l Media in acclimation reactors contained 100 mg/l of 2,4-D and minerals listed above, while in the cases of concurrent and postbiogenic feeds the media also contained sucrose and peptone as the biogenic substrates 2,4-D and minerals were added to the acclimation reactors from concentrated stock solutions to avoid excessive changes in the concentrations of all soluble constituents in the acclimation reactors Acclimation and degradation of 2,4-D were conducted in batch shake-flasks (250 ml conical flasks containing 100 ml liquid, shaken at 100 rpm orbital, at room temperature (25 ± °C)) Samples were withdrawn at regular intervals (typically day) from the flasks for the measurements of 2,4-D concentrations remaining in solution and sludge concentrations that indicated sludge growth COD was also measured for the samples of selected concurrent and post-cultivation tests Majority of the acclimation and degradation experiments were conducted in duplicate 2.4 Measurements 2,4-D concentrations in the bulk reactor suspensions were measured The absorption/adsorption of 2,4-D onto biomass was negligible (evidences not shown) The reactor suspensions were filtered (through Millipore Millex GS, pore size 0.22 lm) 2,4-D concentrations in the filtrates were measured using HPLC (Agilent 1200 Series) The column used was reverse phase C-18, length 250 mm and / 4.6 mm, with particle size lm (Phenomenex Luna 00G-4041E0) The mobile phase consisted of acetonitrile (CH3CN) in H2O (80% v/v), pH adjusted to 4.0 with concentrated H3PO3 Flow rate was ml/min Sample injection volume was 20 ll Retention time was 3.5–3.8 2,4-D was detected by a UV detector at 283 nm The concentration of activated sludge was measured as dried weight of suspended solids (SS) Activated sludge samples were filtered through fibre-glass filters (Whatman GF/C), from which SS was determined from the filterable portion after drying Biogenic substrate was measured using COD for selected tests SS and COD measurements were performed following the standard methods (APHA, 1998) of SM2540-D (filtered and dried at 103–105 °C) and SM5220-C (closed reflux, titrimetric), respectively Adenosine triphosphate (ATP) concentrations (mass-ATP/massSS) were measured for activated sludge cells from the h and precultivation reactors of the 100S test series (re-cultivation feed containing 100 mg/l sucrose and 25 mg/l peptone) Detailed ATP extraction, measurement, and calculation methods are reported in Chong et al (2010) 2.5 Acclimation kinetics The kinetics of acclimation was described with a mathematical acclimation model established by Chong (2009) The 2,4-D data points obtained from an acclimation and degradation test were fitted with the output of the model This model fitting gave quantitative values of the length of lag time and the kinetics of degradation for an acclimation and degradation course The values of all model parameters (see Chong (2009)) were set equal to those of the studies reported in Chong (2009), except Xmax, a, and s which describe the degrader conversion rate in the model equation shown below (Eq (2) of Ref Chong (2009)): X a tịconversion ẳ X max tị ỵ eatsị 1ị 183 where Xa concentration of degraders (mg/l), Xmax maximum amount of degrader converted from non-degrader (mg/l), t is time (d), s time at which maximum conversion rate occurs (t), and a maximum Xa conversion rate (1/t) Xmax, a, and s of a 2,4-D course were determined by non-linear regression in which a model output was fitted to a set experimental data points (average of duplicate tests) The regression program used was DNRLIN (double precision) of the IMSL library (Visual Numerics, Inc.) A model output used for fitting to the data was computed by simultaneous integration of the differential equations of the model using the Runge–Kutta algorithm DNRLIN and integration were performed on a desktop computer (Compaq Visual Fortran compiler) From the results a and s, the length of lag time, k, was calculated from k ¼ s À a2 (Zwietering et al., 1990) Results and discussion 3.1 Modeling of acclimation performance Fig shows the results of 2,4-D acclimation and degradation tests by the biomass of the re-cultivation scheme 100S (100 mg/l sucrose and 25 mg/l peptone) (Table 1) (Results of the complete test scheme are shown in Fig A.1) The data show the 2,4-D degradation courses and the curves are best fits to the experimental data points with the model outputs Model parameters that not concern degrader development or degradation kinetic were held constant at the values reported by Chong (2009) Values for lm, Ks and Ki (the Haldane kinetic parameters specific to Xa), which were slightly adjusted in the efforts of achieving the best fits, were practically constant: lm, Ks and Ki were 3.11 1/d (standard deviation = 0.06), 138.15 mg/l (s.d = 8.8), and 48.15 mg/l (s.d = 6.22), respectively Modeling results indicate that Xmax, a, and s of the degrader conversion rate function (see definition of parameters in Eq (1)) are the parameters that determine the shape of a model output that fits an acclimation and degradation course Still, Xmax was relatively constant (48.18 mg-SS/l, s.d = 0.81), a and s remain the major parameters that express the change of the activated sludge biomass’ performance in acclimation and degradation of the xenobiotic The modeling technique used in this study leads to a method that is useful in describing the performances of different activated sludge biomass In this study, the sludge biomass’ performance is changed by the different times at which the sludge was re-cultivated on biogenic substrates, and at different concentrations of the biogenic substrates in the re-cultivation feeds The differences among the performances are usually small, yet a and s are the instrumental parameters precise enough in describing small changes in the performance of the sludge biomass s mainly dictates the length of lag time, and a, the maximum rate of degrader conversion, reflects the rate at which the sludge biomass is to acclimate to the target xenobiotic Because the parameters for specific growth rate (lm, Ks and Ki) and also Xmax are essentially constant, a determines the overall time required for the complete degradation for the xenobiotic The biogenic effects on xenobiotic acclimation are examined based on a and s (lag equivalence) in the following discussion, especially on the effect related to the sludge’s energy richness 3.2 Effects of diauxic growth Fig shows that both re-cultivation times and biogenic feed concentrations change the length of lag time With respect to biogenic feed concentration, all feed concentrations help in shortening lag times compared to the original sludge The 100S biogenic 184 N.-M Chong et al / Bioresource Technology 104 (2012) 181–186 100.0 -1d 2,4-D (mg/l) 80.0 0h 1h 60.0 16d -2d 2d original -1d -2d -4d 16d 12d 40.0 20.0 3d 5d 2d 8h 1h 0h 8h 5d 3d original -4d 12d 0.0 Time (d) Fig Acclimation and degradation of 2,4-D by activated sludge biomass re-cultivated with biogenic substrates 100S (100 mg/l sucrose and 25 mg/l peptone) at different times before, concurrent, or after the sludge’s acclimation reaction with 2,4-D Experimental data (data points) were fitted with the acclimation model (lines), which calculated lag time and acclimation and degradation kinetics substrate produced sludge biomass that had the shortest lag time, followed closely by 200S, 50S, and 20S (see Table for concentrations of biogenic substrate in the re-cultivation feeds) This order of lag advantage suggests that there is an optimum feed concentration that is most effective in shortening acclimation lag time With respect to the time of re-cultivation, sludge re-fed d previously showed the shortest lag time for all biogenic feed concentrations, most noticeably 100S and 200S Lag times on both sides of 3-d re-cultivation time were longer than the shortest lag The reasons behind the trend of the length of lag with respect to re-cultivation time are discussed below: (1) For short pre-cultivation times, including concurrent, 1-h, 8-h and even the 2-d re-cultivations, lag times are longer than the shortest lag at 3-d re-cultivation In the 2,4-D acclimation reactors of these short re-cultivation times, biogenic substrates are present simultaneously with 2,4-D for some period of time (see biogenic COD of the 0h100S, 0h200S, and À1d100S tests in Fig A.2) The lengthening of lag time by the presence of biogenic substrate with 2,4-D simultaneously is best explained with the effect that the activated sludge biomass uses the biogenic substrates for growth in preference to 2,4-D (more commonly known as diauxic growth) Diauxic growth means that the sludge biomass has a chance to utilize the easy substrates, and thus the biomass temporarily ignores or escapes from the xenobiotic acclimation stress This pause of the acclimation process lets the xenobiotic intact for additional time, thereby causing the elongation of the acclimation lag time Acclimation resumes, or in the worst case, the microorganisms must restart a new acclimation process (Brandt et al., Lag (d) 5.5 4.5 original sludge 20S 50S 100S 200S 3.5 2.5 -4 -2 10 68 Re-cultivation time (d) 12 14 16 Fig Length of lag time as the result of re-cultivation time and biogenic feed concentration Lag of original sludge as reference 2004), after the biogenic substrates are consumed to an extent near completion Evidences of diauxic growth are found: (1) Early and extra SS growth shown for the 0-h case (Fig A.3) In the 0-h tests, SS growth accompanied biogenic substrate dissipation but not degradation of 2,4-D Such a trend in SS growth was not successfully matched by the model output because the model does not contain terms that describe non-degrader growth from the biogenic substrates extra to those lower intermediates of 2,4-D catabolism For those tests where biogenic substrates were absent (consumed), e.g., 3-d and 16-d re-cultivations, model outputs closely match experimental SS growths, which growths are typical of the sludge growth from the sludge’s acclimation and degradation of 2,4-D solely (2) Prominent delay of acclimation in post-cultivation tests (Fig 1) Acclimation function of the sludge biomass that had started but not completed its acclimation with 2,4-D experienced severe interference by the addition of biogenic substrates The diauxic growth effect was most severe for the À1 d post-cultivation tests, followed by the À2 d tests Biogenic substrates added d afterward had little effect because the acclimation process therein had essentially completed (2) For pre-cultivation times that are longer than the time the sludge biomass requires to consume all the biogenic substrates (e.g., d), the re-cultivated biomass is well nourished, negative effect of diauxic growth has subsided and the biogenic benefit (energy enrichment, Section 3.3) dominates (3) For long pre-cultivation times, the re-cultivated sludge biomass gradually loses the nourishment supplied earlier by the biogenic substrates In the cases of 100S feed, for example, lag time increased from the shortest lag at 3-d pre-cultivation Lag time increased in a trend that can be approximated with an exponential function, which will reach an ultimate in a long pre-cultivation time However, the ultimate lag time may not have any practical significance because sludge that has lasted such a long pre-cultivation time (non-growing sludge) may have suffered serious decay The discussion about lag advantage is based on a reference to the lag of the original sludge, which was the sludge of a 10-d hc However, the xenobiotic acclimation quality of the original sludge is found in this study to be slightly different from that projected for the sludge re-cultivated 10 d previously: a longer lag time (Fig 2), and a faster degrader conversion rate (Fig 3a) These results indicate the difference in xenobiotic acclimation quality between sludge harvested from a continuously fed rector operated under a hc and the sludge biomass of re-cultivation time equals to hc It is true that the hc value may not precisely represent the biological quality of the original sludge because hc is only a nominal value N.-M Chong et al / Bioresource Technology 104 (2012) 181–186 2.5 a α (1/d) original sludge 2.0 1.5 5.5 b Lag (d) original sludge 4.5 0h 1h 8h 3.5 2.5 10 30 50 ATP (mg/g-SS) 70 Fig ATP contents of sludge cells related to (a) maximum rate of degrader conversion (a) Line is an arbitrary fit of a with exponential function; and (b) lag time Lag times of h, h and h followed different trend than those of other precultivation tests due to diauxic growth effect Adjustment of bioreactor operation, by strict adherence to the ideal definition of a mean-cell-residence-time, will change either the hc value or the intrinsic biological quality of the sludge The sludge’s biological quality thus obtained can be more truly represented by a hc that is most accurately operated Under the present conditions, hc of the original sludge (10 d in this case) can be considered equivalent to the re-cultivation time in expressing the sludge’s xenobiotic acclimation quality Furthermore, the results of this study can extend the interpretation of hc, from its conventional one such as growth phase (or sludge age), to one including the sludge’s potency in acclimation to a xenobiotic This new interpretation of hc will provide a guideline for treatment plant operations coping with periodic xenobiotic influent Overall, both the strength of biogenic substrates and the time point of biogenic presence relative to the biomass’ reaction with the xenobiotic are important in affecting biomass’ acclimation lag time to the xenobiotic A biogenic feed is inherently beneficial for xenobiotic degradation, except where the biogenic substrates exist concurrently with the xenobiotics and at strength high enough to cause a pause of the acclimation process Detailed measurements of biogenic substrate concentrations that start or end the unfavorable diauxic growth are valuable for the precise modeling of the diauxic growth effects 3.3 Effects of energy richness Despite that biogenic substrates, when present, inevitably interfere with the sludge biomass’ acclimation to the xenobiotic, sludge re-grown on the biogenic helps the sludge biomass in shortening lag when the biomass is to acclimate to 2,4-D as the sole substrate This benefit is mainly conferred by the sludge cells’ energy contents that were enriched from metabolizing the biogenic substrates The well nourished sludge cells contain a high energy level to sustain the energy consuming xenobiotic acclimation process To relate the biogenic effect on 2,4-D acclimation, the ATP contents 185 of the sludge cells at times after the one-time feed of biogenic substrates (the 100S) were measured (see results in Fig A.4) Fig 3a shows that a, the maximum rate of degrader conversion, is positively proportional to the energy states (ATP levels) of the sludge cells A mathematical function relating a with respect to ATP can be readily established Because degradation rate is dependent on a, the overall 2,4-D degradation rate is also proportional to ATP Fig 3b shows the length of lag time with respect to ATP contents of the cells Length of lag time decreases with the increase of ATP, only that the trend was broken by diauxic growth when the biogenic substrates were present during the acclimation phases Diauxic growth counteracts the sludge’s lag shortening advantage brought by the enriched energy in the sludge cells But only severe diauxic growth counterbalanced the benefits of the energy richness Low biogenic re-cultivation (20S and 50S) of sludge all had beneficial effect on decreasing lag times Low amounts of biogenic substrates are able to enrich ATP of the sludge cells, but at low concentrations, the biogenic substrates are quickly consumed so that the biogenic substrates not cause any significant delay of the acclimation process During acclimation, degraders are formed from cells that can successfully evolve the required agents (mostly enzymes) that are capable of catabolism of the xenobiotic This degrader conversion process involves chemical reactions that are energy consuming (Chong et al., 2010) This study determines the positive relation of degrader conversion rate (a) with ATP The activated sludge biomass’ overall performance in acclimation and degradation of a xenobiotic is characterized by the length of lag time and the rate of degradation The relationships of lag time and a with ATP indicate that the biomass’ acclimation and degradation performance is most predominantly dependent on the energy contents of the sludge cells Such a relationship provides new insight into the mechanisms of the xenobiotic acclimation process The advantage of energy enrichment of activated sludge biomass by biogenic substrate, when taken into consideration together with diauxic growth, will explain why there are inconsistent results in previous studies about the effect of biogenic substrates on the acclimation and degradation of a xenobiotic Conclusions Biogenic substrates are beneficial to activated sludge’s acclimation and degradation of a xenobiotic by enriching the energy contents of the sludge cells This beneficial factor always occurs which shortens acclimation lag time and enhances the overall rate of xenobiotic degradation The counteracting factor is diauxic growth of activated sludge on the biogenic substrates High biogenic concentration present during the acclimation process brings a diauxic growth severe enough to counterbalance the energy-rich benefit When these interacting factors are considered together, definite results about the effect of biogenic substrate on xenobiotic degradation are obtained, unifying those inconsistencies reported in the literature Acknowledgement This study was supported partially by research grants provided by the National Science Council of the Republic of China The major support was from Grant No NSC97-2221-E-212 -040 Appendix A Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2011.11.004 186 N.-M Chong et al / Bioresource Technology 104 (2012) 181–186 References APHA (American Public Health Association), 1998 Standard Methods for the Examination of Water and Wastewater, 20th ed Washington, DC Basu, A., Apte, S.K., Phale, P.S., 2006 Preferential utilization of aromatic compounds over glucose by Pseudomonas putida CSV86 Appl Environ Microbiol 72 (3), 2226–2230 Brandt, B.W., Kelpin, F.D.L., van Leeuwen, I.M.M., Kooijman, S.A.L.M., 2004 Modelling microbial adaptation to changing availability of substrates Water Res 38 (4), 1003–1013 Chin, H., Elefsiniotis, P., Singhal, N., 2005 Biodegradation of 2,4dichlorophenoxyacetic acid using an acidogenic anaerobic sequencing batch reactor J Environ Eng Sci (1), 57–63 Chong, N.-M., 2009 Modeling the acclimation of activated sludge to a xenobiotic Bioresour Technol 100 (23), 5750–5756 Chong, N.-M., Chiou, J.-N., 2010 Biodegradation of BTE in soil by indigenous microbial populations with and without biogenic substrates World J Microbiol Biotechnol 26 (9), 1587–1594 Chong, N.-M., Tsai, S.-C., Le, T.N., 2010 The biomass yielding process of xenobiotic degradation Bioresour Technol 101 (12), 4337–4342 Corseuil, H.X., Hunt, C.S., Ferreira, R.D.S., Alvarez, P.J.J., 1998 The influence of the gasoline oxygenate ethanol on aerobic and anaerobic BTX biodegradation Water Res 32, 2065–2072 Ettala, M., Koskela, J., Kiesila, A., 1992 Removal of chlorophenols in a municipal sewage treatment plant using activated sludge Water Res 26, 797–804 Harder, W., Dijkhuizen, L., 1982 Strategies of mixed substrate utilization in microorganisms Philos Trans R Soc B-Biol Sci 297, 459–480 Harrison, E.M., Barker, J.F., 1987 Sorption and enhanced biodegradation of trace organics in a groundwater reclamation Scheme-Gloucester site, Ottawa, Canada J Contam Hydrol 1, 349–373 Hill, N.P., MacIntyre, A.E., Perry, R., Lester, J.N., 1986 Behaviour of chlorophenoxy herbicides during activated sludge treatment of municipal wastewater Water Res 20, 45–52 Hu, Z., Ferrainab, R.A., Ericsonb, J.F., Smetsa, B.F., 2005 Effect of long-term exposure, biogenic substrate presence, and electron acceptor conditions on the biodegradation of multiple substituted benzoates phenolates Water Res 39, 3501–3510 Lee, K., 2003 Effect of additional carbon source on naphthalene biodegradation by Pseudomonas putida G7 J Hazard Mater 105 (1–3), 157–167 Lewis, D.L., Kollig, H.P., Hodson, R.E., 1986 Nutrient limitation and adaptation of microbial populations to chemical transformations Appl Environ Microbiol 51, 598–603 Margesin, R., Zimmerbauer, A., Schinner, F., 2000 Monitoring of bioremediation by soil biological activities Chemosphere 40, 339–346 Meric, S., Eremektar, G., Ciner, F., Tünay, O., 2003 An OUR-based approach to determine the toxic effects of 2,4-dichlorophenoxyacetic acid in activated sludge J Hazard Mater 101 (2), 147–155 Moorman, T.B., Cowan, J.K., Arthur, E.L., Coats, J.R., 2001 Organic amendments to enhance herbicide biodegradation in contaminated soils Biol Fertil Soils 33, 541–545 Nelson, E.C., Walter, M.V., Bossert, I.D., Martin, D.G., 1996 Enhancing biodegradation of petroleum hydrocarbons with Guanidinium fatty acids Environ Sci Technol 30, 2406–2411 Sanapareddy, N., Hamp, T.J., Gonzalez, L.C., Hilger, H.A., Fodor, A.A., Clinton, S.M., 2009 Molecular diversity of a North Carolina wastewater treatment plant as revealed by pyrosequencing Appl Environ Microbiol 75 (6), 1688–1696 Shimp, R.J., Pfeander, F.K., 1985 Influence of easily degradable naturally occurring carbon substrates on biodegradation of monosubstituted phenols by aquatic bacteria Appl Environ Microbiol 49, 394–401 Swindoll, C.M., Aelion, C.M., Pfaender, F.K., 1988 Influence of inorganic and organic nutrients on aerobic biodegradation and on the adaptation response of subsurface microbial communities Appl Environ Microbiol 54, 212–217 Taylor, L.P., Wilson, B., Mills, M.S., Burns, R.G., 2002 Comparison of microbial numbers and enzymatic activities in surface soils and subsoils using various techniques Soil Biol Biochem 34, 387–401 Wagner, M., Loy, A., Nogueira, R., Purkhold, U., Lee, N., Daims, H., 2002 Microbial community composition and function in wastewater treatment plants Antonie Van Leeuwenhoek 81 (1), 665–680 Wilson, J.T., Miller, G.D., Giorse, W.C., Leach, F.R., 1986 Relationship between the ATP contents of subsurface material and the rate of biodegradation of alkylbenzenes and chlorobenzene J Contam Hydrol 1, 163–170 Zwietering, M.H., Jongenburger, I., Rombouts, F.M., van’t Riet, K., 1990 Modeling of the Bacterial Growth Curve Appl Environ Microbiol 56 (6), 1875–1881 ... effect of biogenic substrates on the acclimation and degradation of a xenobiotic Conclusions Biogenic substrates are beneficial to activated sludge s acclimation and degradation of a xenobiotic. .. performance in acclimation and degradation of the xenobiotic Methods 2.1 Target xenobiotic and activated sludge common activated sludge biomass produced from a continuous activated sludge treatment... using a mathematical acclimation model developed by Chong (2009) The advantages or disadvantages of biogenic substrates for the sludge biomass’ xenobiotic acclimation and degradation were examined