The biomass yielding process of xenobiotic degradation

Bioresource Technology 101 (2010) 4337–4342 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech The biomass yielding process of xenobiotic degradation Nyuk-Min Chong *, Shiu-Ching Tsai, Thanh Nga Le Department of Environmental Engineering, Da-Yeh University, No 168, University Rd., Dacun, Changhua 51591, Taiwan, ROC a r t i c l e i n f o Article history: Received 30 October 2009 Received in revised form 16 January 2010 Accepted 20 January 2010 Available online 11 February 2010 Keywords: Activated sludge Biomass yield 2,4-D Metabolic intermediates ATP a b s t r a c t Yields of activated sludge and an Arthrobacter sp biomass on organic xenobiotic 2,4-dichlorophenoxyacetic acid (2,4-D) and on the intermediates of selected 2,4-D metabolism pathways were measured Activated sludge yield on 2,4-D was lower by approximately 24–45% compared to the combined yields produced separately by the lower intermediates For activated sludge, cell synthesis only consumed 33% of the electrons generated from 2,4-D oxidation, while the other 67% were used for energy The high energy consumption, which was the primary cause of low activated sludge yield from 2,4-D degradation, occurred mainly in the catabolism of 2,4-D The degrader sludge supplied this catabolism energy demand with the ATP contained in the biomass As a result, the sludge’s ATP contents suffered a deficit that was not fully remunerated after 2,4-D was degraded Metabolism of the lower intermediates provided materials for further biomass growth and refilled part of the energy initially consumed Ó 2010 Elsevier Ltd All rights reserved Introduction Xenobiotic organics are foreign to most indigenous microorganisms, whether the microorganisms exist in mixed cultures (such as activated sludge) or in pure cultures The pathways employed by microorganisms for xenobiotic metabolism are mostly different from those commonly used for biogenic substrates Generally, indigenous microorganisms can acquire the pathways for xenobiotic degradation through a process called acclimation (Buitron et al., 1998; Chong and Lin, 2007; Singleton, 1994), in which enzymatic mechanisms evolve The acquired enzymatic elements are responsible for separate metabolism functions, particularly during the process of catabolism The catabolism (breaking down) of a xenobiotic goes through a series of intermediate steps until the lower intermediates are suitable for entering the central metabolism pathways of the cells The novel initial steps that are distant from the central pathways may hinder or detract from the contribution to overall growth-yield The yield disadvantages of a novel catabolic steps include two major aspects: (1) expending of energy or reducing power for breaking bonds that are oxidized (Harder, 1973; Kasberg et al., 1996), and (2) consuming carbon or organic molecules in oxidation that is necessary for the degradation (Muller and Babel, 2000) Yield of microbial biomass (typically activated sludge) from degrading a xenobiotic substrate is an important factor related to the degradability of the xenobiotic In the treatment of xenobiotic pollutants, the amount of activated sludge grown from utilizing a unit mass of the pollutant (the yield coefficient) is an important parameter for modeling and calculations of the xenobiotic treat* 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 2010 Elsevier Ltd All rights reserved doi:10.1016/j.biortech.2010.01.075 ment process (Chong, 2009) Because of its importance, yield is most frequently measured in the study of xenobiotic degradation In addition to the measurement of the overall yield after a xenobiotic is degraded, examination of the biomass yielding process during xenobiotic degradation is also necessary for the study of xenobiotic degradability The purpose of this study is to investigate the biomass yielding process during the microbial degradation of a xenobiotic To fulfill this purpose, yields of activated sludge and a pure culture from the acclimation and degradation of a xenobiotic were measured In addition, the yields of both biomasses on organic compounds, that are the intermediates at different downstream stages of the xenobiotic catabolism through pathways published in the literature, were also measured By comparing the overall biomass yields on the parent substrate with the sums of separate yields on its intermediates, the yields contributed by the stages of the metabolism process of the xenobiotic were revealed Using the activated sludge yield results, the material flow to the branches of cell synthesis and oxidation was calculated using stoichiometric equations Variation of activated sludge biomass energy contents during the course of the xenobiotic degradation was also measured to show how energy was spent when the biomass was going through the steps of the xenobiotic degradation process Methods 2.1 Growth substrates The growth substrates, each of which was subjected independently to degradation and growth-yield experiments, were 4338 N.-M Chong et al / Bioresource Technology 101 (2010) 4337–4342 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4-dichlorophenol (2,4DCP), glyoxylic acid, succinic acid, pyruvic acid, acetic acid, and formic acid 2,4-D was the parent xenobiotic The other growth substrates belong to the catabolism intermediates at different stages of the prominent 2,4-D metabolism pathways found in the literature The selection of 2,4-D catabolism intermediates is explained below: 2,4-D is proposed to be metabolized via a a-ketoglutarate dioxygenase dependent pathways or via a 2,4-D-dehalogenase pathways (Young, 2008), depending on the metabolizing microorganisms that exist mainly as pure cultures More microbial species employ the a-ketoglutarate dioxygenase than the 2,4-D-dehalogenase pathways, thus the former were considered more commonly suitable for mixed culture (activated sludge, described below) The two pathways differ in the (apparent) first step thus producing different first catabolism intermediates Both pathways form chlorocatecol (with mono- or di-chloride), which is subsequently cleaved by enzymes from one of two distinct classes: (1) intradiol (ortho), or (2) extradiol (meta) dioxygenases (Vaillancourt et al., 2006) Via the a-ketoglutarate dioxygenase pathways, 2,4-D is first turned into 2,4-DCP and glyoxylate (Fukumori and Hausinger, 1993; Loos et al., 1967; Tiedje and Alexander, 1969) When the dichlorocatechol formed is cleaved the ortho way (referred to as the ortho pathways), downstream intermediates at or near the central pathways are mol of acetate and mol of succinate When the dichlorocatechol formed is cleaved the meta way (referred to as the meta pathways), the lower intermediates are mol of pyruvate and mol of acetate while mol of formate is formed in an earlier step 2,4-D and 2,4-DCP are referred to as xenobiotics, while glyoxylic acid, succinic acid, pyruvic acid, acetic acid, and formic acid are referred to as the lower intermediates or biogenic (easy) substrates 2.2 Biomass The biomasses used for degradation and growth on each substrate included: (1) a mixed culture originated from soil (resembling and called activated sludge) and (2) a pure culture of Arthrobacter sp (DSM 20407) Activated sludge was the primary biomass whose yields on the growth substrates were measured Yields of the pure culture were also measured to investigate (1) whether or not the pathways known for this pure culture are suitable for activated sludge; and (2) whether yield is dependent on the substrate or on the degrading pathways Experiments with the activated sludge included: (1) measurements of yields on the early (first) 2,4-D catabolism product(s) through the a-ketoglutarate dioxygenase pathways; (2) measurements of yields on the downstream products of the ortho cleavage of dichlorocatechol; (3) measurements of yields on the downstream products of the meta cleavage of dichlorocatechol; and (4) measurements of the sludge’s energy (ATP) contents during its courses of acclimation and degradation of 2,4-D Experiments with the Arthrobacter sp were measurements of yields on the intermediates of 2,4-D catabolism through the a-ketoglutarate dioxygenase pathways and on the downstream products of the following modified-ortho ring cleavage Sufficient amount of each of the activated sludge and the pure culture biomass for use in a growth and degradation experiment was prepared in steps as follow: seeds of each culture were inoculated separately to Nutrient Broth (NB, Difco 234000) in shake flasks (150 ml); after growing for approximately 48 h in NB, the growth was sub-cultured (10 ml of seed) to new NB shake flasks; multiple (at least five) shake flasks were operated concurrently at the third sub-culture; the third sub-cultures were grown for 48 h (stationary phase); to avoid the carry-over of organic matters to the degradation reactions, each of the shake flask media was centrifuged (lightly at 5000 rpm, min), supernatant discarded, and the pellet was re-suspended in pH buffer; suspensions of cells grown from all (5) flasks were mixed from which a measured amount was used to inoculate into a reactor for a growth-yield experiment The activated sludge used for ATP measurements was cultivated long-term in a fed-batch reactor Feed to the fed-batch reactor consisted of sucrose (100 mg/l), peptone (18 mg/l), and mineral ingredients same as those for growth reactions shown below The same soil microorganisms as the NB culture were used as seeds The fedbatch reactor was operated to a (pseudo) steady-state at a mean cell residence time (hc) of days This activated sludge represented a biomass undergoing stable growing conditions and thus having a uniform ATP content, in contrast to a biomass grown in NB The effects of varying initial ATP contents in the biomass on the rate and yield of xenobiotic degradation, if any, are topics of further studies For inoculation into the 2,4-D degradation–ATP measurement experiments, the sludge from the fed-batch mixed liquor was settled for 30 before the supernatant was discarded (approximately 4/5 of the original liquid) The settled sludge was re-suspended in pH buffer The new mixture was settled again, supernatant was discarded, and a measured amount from the concentrated sludge was used to inoculate into a 2,4-D degradation reactor 2.3 Growth reactions Degradation and growth experiments were performed in batchtype shake flask reactors with each substrate as the sole carbon source Minerals FeCl3, 1.2 mg/l; CaCl2, 12 mg/l; MgSO4Á7H2O, 65 mg/l; NH4Cl, 25 mg/l; K2HPO, 200.0 mg/l; KH2PO4, 156.6 mg/l needed for microbial growth were provided Ammonium ion also acted as the nitrogen source Shake flasks provided oxygen which acted as the electron acceptor The shake flasks growth conditions included: agitation: 120 rpm orbital; temperature: room temperature at 25 ± °C; pH: buffered at approximately 7.2 Concentration of the biomass (X) was measured as suspended solid (SS); 2,4-D and 2,4-DCP concentrations were measured using UV spectrophotometry (wavelength 235 nm; Spectrophotometer: Shimadzu UV1800, its method detection limit for 2,4-D was determined in our laboratory as 0.065 mg/l) Concentrations of other substrates were measured as COD All samples for substrate measurement were filtered (Millipore Millex GS, pore size 0.22 lm) The initial concentrations of 2,4-D in the batch degradation experiments were 100 or 50 mg/l, and the initial concentration of 2,4-DCP was 50 mg/l Biomass (X) and substrate (S) concentrations were measured at regular intervals (typically day for the xenobiotics and h for the biogenic substrates) until complete degradation was found The ATP experiments were conducted in a similar way, with the addition of ATP measurements Experiments for the growth-yield and ATP were repeated three (3) and two (2) times, respectively 2.4 Yield calculation Activated sludge and Arthrobacter sp growth curves and substrate depletion curves were used to calculate the yield coefficients (y) of the biomass on 2,4-D, 2,4-DCP and glyoxylate A y was calculated using Eq (1): y¼ P P ÀX o Þ Â X i ðt i À t o ÞðSo À Si Þ ðX i À X o ÞðSo À Si Þ À ðXXÁt P P o ÀS1 Þ ðSo À Si Þ2 À ðSXÁt  X i ðt i À t o ÞðSo À Si Þ ð1Þ Eq (1) was formed from y ¼ P P X i X o ịSo Si ịỵkd P X i ðt i Àto ÞðSo ÀSi Þ ðSo ÀSi Þ2 1981), with the simplified equation for kd: (Kim et al., 4339 N.-M Chong et al / Bioresource Technology 101 (2010) 43374342 kd ẳ y So S1 ị À ðX À X o Þ X Á t1 where So and Xo are substrate and biomass (SS) concentrations (mg/ l), respectively, at initial time to; Si and Xi are substrate and biomass concentrations, respectively, at the ith time point ti; S1 and X1 are and substrate and biomass concentrations, respectively, at a substrate degradation end-point t1; X i is mean biomass concentration over the time interval to to ti; X is mean biomass concentration over the time interval to to t1; kd is endogenous decay coefficient (1/t) The yield coefficients for succinic acid, pyruvic acid, acetic acid, ÀX o Þ ; and formic acid were determined by the equation y ẳ DDXS ẳ X So S1 ị neglecting kd 2.5 Stoichiometric equations A stoichiometric equation for biochemical oxidation of an organic substance was formulated as the sum of three (3) oxidation half-reactions representing: (1) the organic substrate acting as the electron donor (Rd); (2) oxygen as an electron acceptor (Ra); and (3) cell synthesis (Rc) While Rd for an organic was written individually, the half equation for cell synthesis (Rc) using ammonium as a nitrogen source was written commonly as (Rittmann and McCarty, 2001) 1 CO2 ỵ HCO3 ỵ NHỵ4 ỵ Hỵ ỵ e 20 20 ! C5 H7 NO2 cellị ỵ H2 O 20 20 ð2Þ whereas the half equation for oxygen as an electron acceptor (Ra) was written as 1 O2 ỵ Hỵ ỵ e ! H2 O ð3Þ The overall equation, R, for the reaction coupling substrate utilization and biomass growth in the bio-oxidation of an organic was formed from R = feÁRa + fsÁRc À Rd (Rittmann and McCarty, 2001), where fs is the fraction of the donated electron used for the synthesis of new cells, and fe = À fs is the fraction of the donated electrons that generate energy used for maintenance An fs for the Requation of a substrate was calculated as follow: from the measured yield of a substrate, mole of cell mass (C5H7NO2) resulted from oxidizing mol of the substrate (mole-yield) was determined By adjusting fs (and fe = À fs), the coefficient of C5H7NO2 in equation R changed accordingly By trial-and-error, an appropriate fs was determined that fit the calculated coefficient of C5H7NO2 to the measured mole-yield (within ±2%) ATP concentration of the supernatant was measured using HPLC (Agilent Technologies 1200 Series; Column: Thermo Scientific ODS Hypersil 250 4.6 mm with particle size lm) Mobile phase A consisted of 150 mM KH2PO4 and 150 mM KCl (pH adjusted to 6.0 with 0.1 M KOH) Mobile phase B consisted of 85% mobile phase A and 15% acetonitrile The continuous gradient elution scheme was: at time 0, 0.28, 9.72, 13.89, and 19.44 min, 0%, 3%, 9%, 100%, and 100% of mobile phase B, respectively, was injected Column temperature was 17–19 °C Total flow rate was 0.54 ml/min Sample injection volume was 20 ll (total retention time was 25–30 min) ATP was detected by a UV detector at 254 nm ATP concentration (x lg-ATP/ml) was determined from the peak area compared with that of an external standard ATP contained in the sludge was callg-ATP/mg-SS as: (x lg-ATP/ml  6.0 ml)/ culated for (g  10À3 l  j mg-SS/l) Results and discussion 3.1 Measured yield Yields of activated sludge and Arthrobacter sp on 2,4-D, 2,4-DCP and selected 2,4-D catabolism downstream intermediates (lower intermediates) used as growth substrates are listed in Table Yield of activated sludge on 2,4-D was measured as 0.25 g-SS/g2,4-D (at kd determined as 0.019 1/d, similar to the value of previous studies (Chong, 2009)) This yield was converted to 55.3 g-SS/ mol-2,4-D, which is lower than the yields of activated sludge on most biogenic substrates (e.g 0.53 g-SS/g for glucose under the same experimental conditions) Fig shows the yields of activated sludge on 2,4-D, 2,4-DCP, glyoxylate, and selected 2,4-D lower intermediates of the ortho pathways Activated sludge yielded from mol of 2,4-D and from the sum of equivalent moles of lower intermediates that have been produced when 2,4-D catabolism has reached a particular stage, are presented in Fig The sum of separate yields of activated sludge on glyoxylate and 2,4-DCP was approximately equal to Table Experimental biomass yields on 2,4-D and its catabolism intermediates Substrate Acetate Pyruvate Succinate Formate Glyoxylate 2,4-DCP 2,4-D 2.6 ATP measurement a Adenosine triphosphate (ATP) contained in the cells of activated sludge microorganisms was measured along the courses of 2,4-D degradation by the sludge ATP was extracted from an aliquot of sludge (g ml, with j mg/l of SS) SS was first filtred (Advantec A045F047A, cellulose ester; pore size 0.45 lm) and the filterable was washed by passing through ml of 0.005% Sodium Dodecyl Sulfate (SDS) and then twice with deionized water (10 ml each time) The filter paper was placed in a beaker containing 6.0 ml of boiling 0.02 M Tris(hydroxymethyl) aminomethane (Tris buffer, pH 7.8) that contained 2% of trichloroacetic acid (TCA – for inactivation of ATP degrading enzymes) The beaker contents were heated (100 °C) with occasional shaking for 10 to 15 Cooled liquid volume was readjusted to exactly 6.0 ml using Tris, and was centrifuged 23,000g for The supernatant, containing ATP extracted from the cells, was decanted (and stored if necessary, at °C for less than h) for ATP measurement Biomass Activated sludge y (g-cella/mol substrate) Arthrobacter sp y (g-cella/mol substrate) 27.1 30.1 44.9 3.1 8.1 48.9 55.3 31.2 33.5 42.5 – 7.3 52.2 60.2 Microbial cells dry weight (as SS) 2,4-D, y = 55.3 glyoxylate y =8.1 glyoxylate y =8.1 10 2,4-DCP, y = 48.9 acetate y =27.1 20 1.0 1.03 succinate y =44.9 30 40 50 60 yield (g-SS/mol substrate) 1.45 70 80 Fig Yields of activated sludge growing on 2,4-D, 2,4-DCP and glyoxylate, and sum of 2,4-D’s catabolism downstream intermediates of the a-ketoglutarate dioxygenase-ortho pathways (values are averages of triplicate experiments) 4340 N.-M Chong et al / Bioresource Technology 101 (2010) 4337–4342 the sludge’s yield on 2,4-D, whereas the sum of separate yields of activated sludge on succinate, acetate and glyoxylate was 45% more than the yield produced by 2,4-D These results show that different yields were found for the same biomass grown on the same substrates (those of the lower intermediates) that came into the growth reaction from two (2) different sources: (1) those derived from breaking down the parent xenobiotic, and (2) those applied directly The yields on the intermediates derived from the parent are lower than the yields on the same organics applied directly The reason for this difference is because that breaking down the structure of the parent xenobiotic must have reduced cell mass productivity of the lower intermediates The yields of activated sludge on 2,4-D, 2,4-DCP, glyoxylate, and selected 2,4-D lower intermediates of the meta pathways are shown in Fig Combined yield of the meta pathway intermediates (pyruvate, acetate, and formate) was 24% more than the yield produced by 2,4-D By comparing the combined yield of the ortho pathway intermediates (Fig 1) with the combined yield of the meta pathway intermediates (Fig 2), it can be inferred that dichloro- 1.0 2,4-D, y = 55.3 3.2 Stoichiometric equations glyoxylate y=8.1 1.03 2,4-DCP, y = 48.9 gyoxylate gy y y=8.1 pyruvate y=30.1 10 20 acetate y=27.1 30 40 50 yield (g-SS/mol substrate) formate y = 3.1 1.24 60 70 Fig Yields of activated sludge growing on 2,4-D, 2,4-DCP and glyoxylate, and sum of 2,4-D’s catabolism downstream intermediates of the a-ketoglutarate dioxygenase-meta pathways (values are averages of triplicate experiments) 1.0 2,4-D, y = 60.2 glyoxylate y =7.3 glyoxylate y =7.3 10 chatecol catabolism through the ortho pathways is more biomass productive than through the meta pathways Based on yield economy, the ortho pathways should be more favorably employed by the majority of the mixed culture microorganisms Fig shows the yields of Arthrobacter sp on 2,4-D, 2,4-DCP, glyoxylate, and selected 2,4-D lower intermediates Arthrobacter sp harbors plasmid pJP4 (Chong and Chang, 2009) that leads 2,4-D degradation through the modified-ortho pathways (Liu et al., 2001; Schlomann, 2002) The modified-ortho pathways also have glyoxylate, succinate, and acetate as catabolic intermediates Fig shows that the pure culture produced slightly higher yields on 2,4-D and 2,4-DCP, but essentially similar yields on the biogenic intermediates, compared to activated sludge This comparison shows that: (1) the overall efficiency of activated sludge yielding process is lower than that of a pure culture, most probably because some species in the mixed culture not employ pathways that are uniform with the majority; (2) although it is generally true that the quantity of biomass yielded on most biogenic substrates is related more to the nature of the substrate than it is related to the kinds of microbial species, the yield on a xenobiotic substrate may be dependent on the kinds of pathways the microbial cells adopt for metabolizing the xenobiotic 0.99 2,4-DCP, y = 52.2 acetate y =31.2 20 succinate y =42.5 30 40 50 yield (g-SS/mol substrate) 60 1.34 70 80 Fig Yields of Arthrobacter sp growing on 2,4-D, 2,4-DCP and glyoxylate, and sum of 2,4-D’s catabolism downstream intermediates of the a-ketoglutarate dioxygenase – modified-ortho pathways (values are averages of triplicate experiments) Table lists the stoichiometry equations of the reactions in which activated sludge bio-oxidized 2,4-D, 2,4-DCP, glyoxylate, and selected 2,4-D lower intermediates Each equation has mixculture microbial cells (C5H7NO2) as a product The fs and fe that formed the equations are listed in Table For the intermediates having a biogenic nature, approximately 60% of the electrons generated from bio-oxidation of the donor were used in cell synthesis and 40% were used for energy (a 60/40 partition of the generated electrons used for synthesis/energy) Glucose as a benchmark gave a 70/30 partition The 60–70/40–30 partition for synthesis/energy is considered normal for most biogenic substrate (Hatzikioseyian and Tsezos, 2006) Much different from the synthesis/energy partition for biogenic substrates, bio-oxidation of xenobiotic substrate 2,4-D gave only 33% of its oxidation electrons for growth, but consumed the other 67% for energy Similar results were found for 2,4DCP (36% and 64% of the generated electrons were used for synthesis and energy, respectively) Formic and glyoxylic acids are nonxenobiotic exceptions for their low yields: formic acid has a low yield because of its one-carbon structure, and glyoxylic acid has a low yield because it is highly oxidized (Harder, 1973) A number of reasons may be the cause of the low yield, or high energy consumption of metabolizing a xenobiotic compared to metabolizing a biogenic substrate: (1) breaking down specific bonds, especially de-chlorination, consumes reducing power (NADH) which otherwise can be used for synthesis (Kasberg et al., 1996; Sander et al., 1991) (2) Transporting energy through Table Stoichiometric equations for 2,4-D and its catabolism intermediates oxidized by activated sludge for cell mass growth a b Substrate Stoichiometric equation Eq No Acetate Pyruvate Succinate Formate Glyoxylate 2,4-DCP 2,4-D Glucoseb a CH3 COO ỵ 0:8 O2 ỵ 0:24 NHỵ ! 0:24 C5 H7 NO2 ỵ 0:76 HCO3 ỵ 0:04 CO2 ỵ 0:76 H2 O C2 H3 O COO ỵ 1:15 O2 ỵ 0:27 NHỵ ! 0:27 C5 H7 NO2 ỵ 0:73 HCO3 ỵ 0:92 CO2 ỵ 0:73 H2 O ! 0:398 C H NO ỵ 1:602 HCO C3 H4 O2 COO þ 1:512 O2 þ 0:398 NHþ ỵ 0:409 CO2 ỵ 0:604 H2 O ỵ 0:112 CO ỵ 0:028 H O ! 0:028 C5 H7 NO2 þ 0:972 HCOÀ H COOÀ þ 0:36 O2 þ 0:028 NHỵ 2 CHO COO ỵ 0:64 O2 ỵ 0:072 NHỵ ỵ 0:072 H2 O ! 0:072 C5 H7 NO2 ỵ 0:712 CO2 ỵ 0:928 HCO3 ỵ 0:432 HCO ! 0:432 C H NO ỵ 4:268 CO ỵ 0:576 H O C6 H4 Cl2 O þ 3:836 O2 þ 0:432 NHþ 2 ỵ 2:0 HCl C8 H6 Cl2 O3 ỵ 5:055 O2 ỵ 0:489 NHỵ ỵ 0:489 HCO3 ! 0:489 C5 H7 NO2 ỵ 6:045 CO2 ỵ 1:511 H2 O ỵ 2:0 HCl C6 H12 O6 þ 1:79 O2 þ 0:84 NHþ þ 0:84 HCO3 ! 0:84 C5 H7 NO2 ỵ 2:64 CO2 ỵ 5:16 H2 O (4) (5) (6) (7) (8) (9) (10) (11) Activated sludge cell = C5H7NO2; mol wt = 113 Glucose for benchmarking 4341 N.-M Chong et al / Bioresource Technology 101 (2010) 4337–4342 Table Proportions of donated electrons spent on synthesis and energy fs and fe to form stoichiometric equations of Table Glucose for benchmarking the long and branching pathways magnifies energy losses caused by transport inefficiency (compared to 60% efficiency for energy transport from donor to synthesis of a biogenic substrate metabolism (Rittmann and McCarty, 2001, p 156)) (3) Carbon is oxidized to CO2, rather than to contribute to cell yield (Eq (10) shows the amount of substrate carbon spent in cell mass growth and CO2 production) Muller and Babel (2000) proposed that in the degradation of 2,4-D (via the a-ketoglutarate dependent dioxygenase), some intermediates must be oxidized to regenerate the organic components necessary for the continuation of the degradation process (4) Incomplete degradation produces lower cell yield Fradette et al (1994) proposed that residual extracellular intermediate metabolites (REIM) must be accounted for in 2,4-D degradation Excessive REIM of 2,4-D by activated sludge is likely ruled out in this study because numerous experiments had shown (both by UV scanning and HPLC detection; data not shown) that culture media contained little organics after 2,4-D was found depleted 3.3 Energy and yield Fig shows that during the course of activated sludge acclimation and degradation of 2,4-D, ATP contents of activated sludge microbial cells decreased corresponding to the disappearance of 2,4-D ATP contained originally in activated sludge cells had a maximum deficit of 0.23 M-ATP/M 2,4-D (Fig 4c) in this particular experiment case A regain of ATP, that started approximately at the time when 2,4-D was depleted, was most reasonably the result of the metabolism of the lower intermediates This regain of ATP did not reach the level initially contained in the activated sludge, so that a net loss of sludge’s ATP was found at the very end of 2,4-D acclimation and degradation process The low-yielding conditions discussed above can be translated into energy economy in the xenobiotic degradation process by activated sludge Fig indicates that an energy burden instead of an energy advantage was experienced by activated sludge during its degradation of 2,4-D The energy that was purposely input to make the xenobiotic bonds stable could not be retrieved for a gain, but must be overcome when the bonds are to be broken For the xenobiotic degrader sludge, the counteracting energy was shown to come from the sludge’s own energy reserve The free energy obtainable from 2,4-D oxidation to CO2 and H2O (this energy value was undetermined, and data are not found in the literature) was insufficient to reimburse the total deficit that occurred mainly during the early catabolism process Energy involvement in the cell growth process can be written into the stoichiometric equation (that describes material balances) by including energy carrying materials (most commonly ATP) in the equation For a thermodynamic description of growth, ATP is related to biomass in term of ATP-yield (yATP) yATP in the range of 2–30 g-cell/mol-ATP has been reported (Heijnen and van Dijken, 40 120 100 16 20 100 80 12 60 80 with 2,4-D no grow b 40 20 0 SS (mg/l) 2,4-D (mg/l 140 60 (mg/g-SS 0.40 0.46 0.43 0.72 0.64 0.64 0.67 0.30 ATP conc (mg/l) b 0.60 0.54 0.57 0.28 0.36 0.36 0.33 0.70 ATP g-SS) a For energy fea -5 Acetate Pyruvate Succinate Formate Glyoxylate 4-DCP 2,4-D Glucoseb For synthesis fsa 160 a 80 Proportion of electron used (10 Substrate 180 100 net loss c -0.023 M ATP M 2,4-D 0 Time (d) Fig ATP tracking during activated sludge acclimation and degradation of 2,4-D (a) Time courses of 2,4-D and SS; (b) ATP contents of activated sludge microorganisms in reaction with 2,4-D and in non-grow condition; and (c) ATP mass in unit reactor liquid volume (concentration) contained in reactor microorganisms Energy deficit (reference to initial ATP of activated sludge cells) and loss are indicated 1992) As an example of stoichiometric equation containing ATP and NADH, Muller and Babel’s (2000) theoretical 2,4-D degradation equation is: C8 H6 Cl2 O3 þ 0:75 NH3 þ 7:3 ATP ! 0:75 C4 H8 NO2 cellị ỵ CO2 ỵ 0:625 NADH ỵ FADH2 ð12Þ To examine how much extra energy compared to biogenic substrate is spent in activated sludge yielding process of 2,4-D degradation, Eq (10) was revised based on a 60/40 (synthesis/energy) partition instead of the 33/67 determined in this study The revised equation became C8 H6 Cl2 O3 ỵ 3:0 O2 ỵ 0:9 NHỵ4 ỵ 0:9 HCO3 ! 0:9 C5 H7 NO2 Cellị ỵ 4:4 CO2 ỵ 1:1 H2 O þ 2:0 HCl ð13Þ Activated sludge yield from Eq (13) is 101.9 g-cell/M 2,4-D (0.46 gcell/g 2,4-D), which gives an extra 46.6 g-cell/M 2,4-D compared to that measured in this study If a yATP of 14 g-cell/mol-ATP (median of 2–30) is applicable, the ATP that is to produce 46.6 g-cell would be 3.3 M ATP/M 2,4-D This 3.3 ATP, presumably the extra amount of energy that could make 2,4-D yield as much as a biogenic substrate is written into Eq (13) to make C8 H6 Cl2 O3 ỵ 3:0 O2 þ 0:9 NHþ4 þ 0:9 HCOÀ3 þ 3:3 ATP ! 0:9 C5 H7 NO2 Cellị ỵ 4:4 CO2 ỵ 1:1 H2 O ỵ 2:0 HCl 14ị Eqs (12) and (14) suggest that yield can be increased by inputting ATP into a xenobiotic degradation and growth reaction For example, by supplying 3–7 ATP/M 2,4-D from some sources, activated sludge yield on 2,4-D can be made comparable to the sludge’s yield on a biogenic substrate under similar conditions However, this conjecture can be true only if the amount of ATP could be input into the degrader cells, without altering the cells’ characteristics ATP supplied externally by substrates other than the xenobiotic would yield non-degrader microorganisms which offer unknown, if any, benefit 4342 N.-M Chong et al / Bioresource Technology 101 (2010) 4337–4342 to the degradation system The stoichiometric equation such as Eq (14) can no longer represent this mixed degrader and non-degrader system Expenditure of cells’ stored energy complicates, if not detrimental to, a biomass’ reaction with the xenobiotic in that (1), yield and degradation rate may be dependent on the initial energy contents of the cells; (2), the reacting cells must exercise a control mechanism which determines whether to yield a high amount of new cells with low per cell ATP contents or the other way around Conclusions The activated sludge yield on xenobiotic 2,4-D is approximately 0.25 g-SS/g-2,4-D Such a low yield is the consequence of the difficult 2,4-D catabolism pathways which consumes a large proportion of obtainable energy for non-cell-synthesizing purposes Biomass yield on 2,4-D is mainly contributed by catabolism downstream intermediates The yield restriction of 2,4-D indicates that in addition to the availability of capable microorganisms (with acquired novel pathways), degradability of a xenobiotic must also depend on how the degrader biomass can manage a balance, if not a profit, in metabolism energy during xenobiotic degradation References Buitron, G., Gonzalez, A., Lopez-Marin, L.M., 1998 Biodegradation of phenolic compounds by an acclimated activated sludge and isolated bacteria Water Science and Technology 37 (4–5), 371–378 Chong, N.-M., 2009 Modeling the acclimation of activated sludge to a xenobiotic Bioresource Technology 100 (23), 5750–5756 Chong, N.-M., Chang, H.W., 2009 Plasmid as a measure of microbial degradation capacity for 2,4-dichlorophenoxyacetic acid Bioresource Technology 100 (3), 1174–1179 Chong, N.M., Lin, T.Y., 2007 Measurement of the degradation capacity of activated sludge for a xenobiotic organic Bioresource Technology 98, 1124–1127 Fradette, S., Rho, D., Samson, R., Leduy, A., 1994 Biodegradation of 2,4dichlorophenoxyacetic acid (2,4-D) by Pseudomonas cepacia: stoichiometric study Canadian Journal of Chemical Engineering 72 (3), 497–503 Fukumori, F., Hausinger, R.P., 1993 Alcaligenes eutrophus JMP134 ‘‘2,4dichlorophenoxyacetate monooxygenase” is an a-ketoglutarate-dependent dioxygenase Journal of Bacteriology 175, 2083–2086 Harder, W., 1973 Microbial metabolism of organic C1 and C2 compounds Antonie van Leeuwenhoek 39, 650–652 Hatzikioseyian, A., Tsezos, M., 2006 Modelling of microbial metabolism stoichiometry: application in bioleaching processes Hydrometallurgy 83, 29– 34 Heijnen, J.J., van Dijken, J.P., 1992 In search of a thermodynamic description of biomass yields for the chemotrophic growth of microorganisms Biotechnology and Bioengineering 39, 833–858 Kasberg, T., Kaschabek, S., Müller, D., Reineke, W., 1996 Maleylacetate reductases functioning in the degradation of chloroaromatics International Biodeterioration and Biodegradation 37 (3–4), 247 Kim, J.W., Humenick, M.J., Armstrong, N.E., 1981 Analysis of bacterial growth and substrate removal kinetics by a statistical method Water Research 15, 1221– 1226 Liu, S., Ogawa, N., Miyashita, K., 2001 The chlorocatechol degradative genes, tfdTCDEF, of Burkholderia sp strain NK8 are involved in chlorobenzoate degradation and induced by chlorobenzoates and chlorocatechols Gene 268, 207–214 Loos, M.A., Roberts, R.N., Alexander, M., Dawson, J.E., 1967 Formation of 2,4dichlorophenol and 2,4-dichloroanisole from 2,4-dichlorophenoxyacetate by Arthrobacter sp Canadian Journal of Microbiology 13, 691–699 Muller, R.H., Babel, W., 2000 A theoretical study on the metabolic requirements resulting from a-ketoglutarate-dependent cleavage of phenoxyalkanoates Applied and Environmental Microbiology 66 (1), 339–344 Rittmann, B.E., McCarty, P.L., 2001 Environmental Biotechnology: Principles and Applications McGraw-Hill, Singapore Sander, P., Wittich, R.-M., Fortnagel, P., Wilkes, H., Francke, W.O., 1991 Degradation of 1,2,4-trichloro- and 1,2,4,5-tetrachlorobenzene by Pseudomonas strains Applied and Environmental Microbiology 57 (5), 1430–1440 Schlomann, M., 2002 Two chlorocatechol catabolic gene modules on plasmid pJP4 Journal of Bacteriology 184 (5), 4049–4053 Singleton, I., 1994 Microbial metabolism of xenobiotics: fundamental and applied research Journal of Chemical Technology and Biotechnology 59, 9–23 Tiedje, J.M., Alexander, M., 1969 Enzymatic cleavage of the ether bond of 2,4dichlorophenoxyacetate Journal of Agricultural and Food Chemistry 17, 1080– 1084 Vaillancourt, F., Bolin, J., Eltis, L., 2006 The ins and outs of ring-cleaving dioxygenases Critical Reviews in Biochemistry and Molecular Biology 41, 241–267 Young, E., April, 2008 UM-BBD The University of Minnesota Biocatalysis/ Biodegradation Database 2,4-D Pathways (retrieved 18.12.09)  ... a biomass grown in NB The effects of varying initial ATP contents in the biomass on the rate and yield of xenobiotic degradation, if any, are topics of further studies For inoculation into the. .. the parent xenobiotic The other growth substrates belong to the catabolism intermediates at different stages of the prominent 2,4-D metabolism pathways found in the literature The selection of. .. fs is the fraction of the donated electron used for the synthesis of new cells, and fe = À fs is the fraction of the donated electrons that generate energy used for maintenance An fs for the Requation