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Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering
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Respirometric assessment of substrate binding by antibiotics in peptone biodegradation
Ilke Pala Ozkok , Tugce Katipoglu Yazan , Emine Ubay Cokgor , Guclu Insel , Ilhan Talinli & Derin Orhon a a a b a a a a
Environmental Engineering Department, Faculty of Civil Engineering, Istanbul Technical University, Maslak, Istanbul, Turkey b Turkish Academy of Sciences, Piyade Sokak, Cankaya, Ankara, Turkey
Available online: 26 Oct 2011
To cite this article: Ilke Pala Ozkok, Tugce Katipoglu Yazan, Emine Ubay Cokgor, Guclu Insel, Ilhan Talinli & Derin Orhon (2011): Respirometric assessment of substrate binding by antibiotics in peptone biodegradation, Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 46:13, 1588-1597 To link to this article: http://dx.doi.org/10.1080/10934529.2011.609442
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Journal of Environmental Science and Health, Part A (2011) 46, 1588–1597 Copyright C Taylor & Francis Group, LLC ISSN: 1093-4529 (Print); 1532-4117 (Online) DOI: 10.1080/10934529.2011.609442
Respirometric assessment of substrate binding by antibiotics in peptone biodegradation
ILKE PALA OZKOK1, TUGCE KATIPOGLU YAZAN1, EMINE UBAY COKGOR1, GUCLU INSEL1, ILHAN TALINLI1 and DERIN ORHON1,2
1 2
Environmental Engineering Department, Faculty of Civil Engineering, Istanbul Technical University, Maslak, Istanbul, Turkey Turkish Academy of Sciences, Piyade Sokak, Cankaya, Ankara, Turkey
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The study evaluated the inhibitory impact of antibiotics on the biodegradation of peptone mixture by an acclimated microbial culture under aerobic conditions. A fill and draw reactor fed with the peptone mixture defined in the ISO 8192 procedure and sustained at steady state at a sludge age of 10 days was used as the biomass pool with a well-defined culture history. Acute inhibition experiments involved running six parallel batch reactors seeded with biomass from the fill and draw reactor and the same peptone mixture together with pulse feeding of 50 mg/L and 200 mg/L of Sulfamethoxazole, Erythromycin and Tetracycline. Substrate utilization was evaluated by observing the respective oxygen uptake rate profiles and compared with a control reactor, which was started with no antibiotic addition. While all available external substrate was removed from solution, addition of antibiotics induced a significant decrease in the amount of oxygen consumed, indicating that a varying fraction of peptone mixture was blocked by the antibiotic and did not participate to the on-going microbial growth mechanism. This observation was also compatible with the concept of the uncompetitive inhibition mechanism, which defines a similar substrate blockage through formation of an enzyme- inhibitor complex. Keywords: Antibiotics, biodegradation, oxygen uptake rate, process stoichiometry, respirometry, uncompetitive inhibition.
Introduction
Antibiotics are among xenobiotic compounds that are persistent to biodegradation and have a tendency to accumulate in the environment.[1] These substances are now major products of the pharmaceutical industry. They are widely used in human and veterinary medicine, aquaculture for preventing and/or treating microbial infections. [2] Possible irresponsible usage of these substances leads to resistant pathogenic microorganisms living in the surface waters and soil, which causes a large threat to human and environmental health. Antibiotics which are released to aquatic environment may contaminate raw, treated, recycled, irrigation and recreation water. Moreover, negative effects such as toxicity and inhibition may be observed on ecosystem bacteria.[3] The fate of antibiotics in wastewater treatment systems is of particular concern, because they are likely to exert adverse effects on treatment process efficiency and bypass treatment systems. This study focused on the fate and effect of three major antibiotics, namely Sulfamethoxazole, Erythromycin and Tetracycline.
Address correspondence to Derin Orhon, Turkish Academy of Sciences, Ankara, Turkey; E-mail: orhon@itu.edu.tr Received January 31, 2011.
Sulfamethoxazole (SMX) is a sulfanilamide bacteriostatic antibiotic, which is a structural analog of paraaminobenzoic acid (PABA). Its mode of action involves the inhibition of normal utilization of PABA for the synthesis of folic acid, which is important for DNA production. Removal of SMX was observed in biological treatment systems with a long hydraulic detention time, such as extended aeration, where there is an absence of a readily biodegradable substrate.[4] However, Erythromycin (ERY ) classified among macrolide bacteriostatic anitibiotics - inhibitors of bacterial protein biosynthesis -, was reported to be resistant to biodegradation.[5] Giger et al.[6] reported that complete removal of macrolide antibiotics was not possible in wastewater treatment plants and therefore the residual antibiotics accumulate in the receiving water bodies. Tetracycline (TET) is classified among the Tetracyclines group, which are broadspectrum bactreiostatic antibiotics, that reversibly bind to the 30S ribosomal subunit and inhibit translation; causing adverse effects on protein biosynthesis. It was reported that TET was removed by sorption onto sludge in activated sludge systems[7,8] and this removal mechanism was attributed to the tendency of TET to form very low solubility complexes by binding with ions like calcium, magnesium and iron.[9] Moreover, the selected inhibitors are bacteriostatic antibiotics, which limit the growth of bacteria
Substrate binding by antibiotics in peptone biodegradation by interfering with any aspect of bacterial metabolism unlike bactericidal antibiotics. In other words, the selected antibiotics are not expected to kill or inactivate bacteria, but they slow down their metabolic activities towards growth or reproduction. In the literature, the studies conducted with antibiotics only focus on either measurement of the substrate or the antibiotic compound. Experimental substrate or antibiotic profiles may at times be misleading for an accurate evaluation as observed levels of substrate reduction does not necessarily relate to utilization associated with the metabolic activities of biomass; they may also be triggered by a number of auxiliary physical/chemical processes such as entrapment, adsorption, binding, etc. Recognition of dissolved oxygen as a model component in current activated sludge models provided a new dimension for the understanding of biodegradation characteristics and substrate removal. The rate of oxygen utilization, commonly defined as the oxygen uptake rate (OUR), can now be continuously monitored throughout a controlled experiment in a batch reactor as the overall output of all related energy/oxygen consuming reactions. The resulting OUR profile can be interpreted for assessing chemical oxygen demand (COD) fractions with different characteristics and the stoichiometry and kinetics of major biochemical reactions involved.[10–13] OUR is now prescribed as a standard procedure for inhibition assessment.[14] It is successfully used for the evaluation of the inhibitory impact induced by heavy metals[15] and various organic compounds including antibiotics.[16–18] A similar respirometric approach was also used as part of a comprehensive modeling study, to verify and validate the behavior and the inhibitory impact of antibiotics in municipal wastewater treatment.[19] The majority of the studies in the literature examined effects of lower concentrations of antibiotics characterizing wastewater generated after usage of these compounds. However, control of antibiotics at source, in the effluent of the pharmaceutical industry is equally important. In the literature, antibiotic concentrations in pharmaceutical wastewaters are reported to increase up to 200 mg/L.[4, 9] Therefore, in this study, higher antibiotic concentrations were chosen in order to represent antibiotic production wastewaters. In this context, the objective of this part of the study was the assessment of the inhibitory impact of three different antibiotics, namely, SMX, ERY and TET on the biodegradation of peptone-meat extract mixture prescribed as the standard organic substrate in the ISO 8192 inhibition test procedure. The study utilized respirometric analysis and evaluated the acute effects of the selected antibiotics at two concentrations of 50 and 200 mg/L at first exposure to the microbial community using the oxygen uptake rate profiles generated in batch reactors. The impact of inhibitors has generally been evaluated in terms of process kinetics negatively affecting substrate utilization.[13, 20, 21] The respirometric ap-
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proach adopted in the study enables to focus primarily on inhibition stoichiometry – i.e. the inhibitory effect of antibiotics resulting in the entrapment and reduction in the level of available substrate, a significant aspect that is totally overlooked in previous studies. This step is estimated to be an indispensable prerequisite because a kinetic interpretation is bound to be erroneous without an accurate account of the biodegradable substrate.
Materials and methods
Experimental approach The experimental approach first involved using a microbial culture with a well-defined culture history. For this purpose, a main reactor was continuously operated at steady-state at a sludge age of 10 days; the reactor was fed with the peptone- meat extract mixture prescribed in the ISO 8192 test[14] on a fill a draw basis once a day and sustained a biomass acclimated to the selected organic substrate. Then the acclimated microbial community taken from the main fill and draw reactor was used as the biomass seed for two different sets of batch experiments: (i) the ISO tests, and (ii) respirometric (OUR) tests. Both series of experiments involved a number of batch reactors initially inoculated with the same microbial culture from the main reactor and started with and without antibiotic additions (control). The impact of SMX, ERY and TET – the selected antibiotics – at two different concentrations of 50 mg/L and 200 mg/L was evaluated both on the ISO tests and on the respirometric tests for generating the OUR profiles corresponding to selected initial conditions. Experimental setup The main fill and draw reactor was designed to have a volume of 14 L (VT ); it was started using the seed sludge taken from the aeration tank of a domestic wastewater treatment plant and fed with the selected peptone-meat extract mixture in a way to secure an initial COD concentration of 600 mg COD/L upon feeding.[14] The selected organic substrate will be called peptone mixture thereafter for simplicity. The stock substrate solution was prepared to contain in one liter; 16 g of peptone, 11 g of meat extract, 3 g of urea, 0.7 g of NaCl, 0.4 g of CaCl2 . 2H2 O, 0.2 g of MgSO4 . 7H2 O and 2.8 g of K2 HPO4 . Besides the organic carbon source (peptone-meat extract mixture), macro nutrients (K2 HPO4 : 320 g/L, and KH2 PO4 : 160 g/L) and micro nutrients (MgSO4 .7H2 O: 15 g/L, FeSO4 .7H2 O: 0.5 g/L, ZnSO4 .7H2 O: 0.5 g/L, MnSO4 .H2 O: 0.41 g/L, CaCl2 .2H2 O: 2.65 g/L) were added to the reactors. pH in the reactor was kept at neutral level. During each daily feeding period, reactor was settled for 1 h (ts ) and decanted until 2 L (V0 ). Aeration was continuously provided and the oxygen concentration in the
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reactor was kept above 3 mg/L to maintain aerobic conditions. Reactor was operated until it reached steady state conditions, which was monitored by suspended solids (SS), volatile suspended solids (VSS) and COD parameters. At steady state, biomass concentration of the reactor was stabilized around 2000 mg VSS/L. In order to prevent any possible interference induced by nitrification, a nitrification inhibitor (Formula 2533, Hach Company) was added to the reactors. ISO 8192 test These tests were conducted to determine the impact of the selected antibiotics at doses of 50 mg/L and 200 mg/L, as prescribed in the ISO 8192 procedure. The test involved adding the peptone mixture solution to the test vessels in a way to sustain an initial COD concentration of 600 mg/L and this solution was neutralized prior to the assay. Similarly, the biomass inoculum corresponding to an initial VSS concentration of 2000 mg/L was added to the vessels to secure an initial S0 /X0 ratio of 0.30 mg COD/mg VSS. Then, the vessels were filled up to a final volume of 250 mL with aerated tab water. The same procedure was employed for the other test vessels testing 5 to 200 mg/L antibiotics concentrations. The vessels were continuously aerated and samples were taken for dissolved oxygen measurements at 30 minutes and 180 minutes, defined as the incubation or contact times according to the ISO 8192 procedure.[14] Dissolved oxygen measurements were performed by a WTW Inolab Oxi Level 2 oxygen meter in 50 mL air-tight vessels. The stirring rate was kept low, so that an oxygen supply due to stirring was negligible even in open test vessels. The initial oxygen concentration in the test vessels was maintained in the range of 7–8 mg/L. Oxygen uptake rate tests Additional respirometric tests were conducted to generate the OUR profiles of the acclimated biomass under non-inhibiting (control) and inhibiting conditions. OUR measurements were performed with an Applitek RA-
Combo-1000 continuous respirometer. Respirometric tests were conducted in seven parallel batch reactors, each started with the necessary amount of acclimated biomass seeding alone to obtain endogenous oxygen uptake rate (OUR) level of biomass. One of the batch reactors served as the control reactor and started with the peptone mixture alone. The acute impact of each antibiotic was tested with two batch reactors; one started with a 50 mg/L and the other with 200 mg/L dose of the selected antibiotic. Peptone mixture alone or together with the corresponding dose of antibiotic was added to the reactor to obtain desired S0 /X0 ratio and the OUR data were monitored. Detailed information related to the batch OUR experiments are given in Table 1. Analytical procedures COD was measured using the procedure defined by ISO 6060.[22] For soluble COD determination, samples were subjected to filtration by means of Millipore membrane filters with a pore size of 0.45 µm. The Millipore AP40 glass fiber filters were used for SS and VSS measurements that were performed as defined in Standard Methods.[23]
Conceptual framework
Inhibitory actions in substrate biodegradation are conveniently evaluated using the analogy of enzyme-catalyzed reactions. In fact, the same approach was adopted to provide conceptual support to the empirical Monod-type expression now commonly utilized in defining microbial growth in activated sludge systems. As described in detail in the literature,[24, 25] the enzyme analogy was mostly introduced to differentiate two major types of inhibitory effects, both with retardation effects on microbial growth: competitive inhibition and non-competitive inhibition. Recent studies have also indicated that the inhibitory impacts of chemicals should be visualized, not only in the utilization of the readily biodegradable substrate for microbial growth, but also in the hydrolysis of the slowly biodegradable substrate.[13] The common feature of both types of inhibition
Substrate binding by antibiotics in peptone biodegradation is that the inhibitory action only affects process kinetics, so that the available biodegradable substrate is fully utilized. In uncompetitive inhibition however, the inhibitor (I) attacks the enzyme substrate complex, [ES], and forms an enzyme substrate inhibitor [ESI] complex, which does not undergo further biochemical reactions. This way, it blocks a part of the available substrate for biodegradation, as indicated by the following reactions and kinetic expressions with a dissociation constant, KI . E+S k1 1591
Table 2. Impact of antibiotics on oxygen uptake rate based on the ISO test. OUR values (mg O2 /L.h) Antibiotic concentration (mg/L) — 50 50 50 190 200 200
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The significant aspect that differentiates uncompettitive inhibition from the other types is that the induced effect is primarily stoichiometric, i.e. the fraction of substrate bound by the inhibitor does not become available for microbial growth as indicated by the following mass balance equation: [ES] = [S] − [ESI] (4)
The basic stoichiometry and mass balance for available substrate is of capital importance for evaluating the impact of inhibitors, mainly because without any consideration of substrate blockage, a kinetic interpretation is bound to be distorted and misleading. Almost all similar studies reported in the literature overlooked substrate blockage as they only relied on measurements of substrate profiles which cannot differentiate the bound fraction not utilized by biochemical reactions. The introduction of the OUR profiles for inhibitory impact constitutes the basis of the original approach in this study in determining substrate binding potential of the selected antibiotics in a way defined by uncompetitive inhibition.
sistency of results with respirometric experiments yielding the entire OUR curves. As mentioned before, the OUR tests were conducted with two antibiotic concentrations of 50 mg/L and 200 mg/L, mainly because they are representative of the range commonly encountered in the individual effluent streams at related pharmaceutical plants. [4, 8] The results are expected to be meaningful because removal at the source is always advocated as the best means of controlling these chemicals.
ISO 8192 test The ISO 8192 test prescribes the use of an oxygen meter, presumably as it is a more commonly available equipment. Obviously the same results can also be obtained with continuous respirometry, as carried out in the part of the study assessing continuous OUR profiles. Table 2 outlines results derived both with the O2 meter and respirometry. The OUR levels indirectly calculated in the ISO 8192 test was consistent with the OUR profiles only for the control test (Fig. 1a), they yielded no matching results for the inhibitory action of antibiotics. Therefore, the O2 meter measurements were only conducted for the set of antibiotics at 50 mg/L. The following observations may be underlined from the experimental results: (i) Although not consistent, both approaches indicated significant impact of the antibiotics after 30 minutes, while the impact at 180 min remained almost negligible. The OUR reduction with respect to the control test was in the range of 12–37% with the ISO8192 test (Fig. 1b) and slightly higher (15–59%) for continuous respirometry depending on the type of the antibiotic; (ii) The adverse effect of antibiotics were not more pronounced as the initial dose was increased from 50 mg/L to 200 mg/L; it remained at the same level or even slightly reduced for some of the antibiotics. As expected, the nature of the test does not provide additional clues for explaining the results
Results and discussion
Respirometric tests yielding full OUR profiles served as the primary basis for evaluating acute impact of selected antibiotics on the biodegradation of the peptone mixture. The impact was interpreted by changes inflicted by antibiotics on the shape of the initial OUR profile obtained in the control test conducted without antibiotic addition. The ISO 8192 test involves inherent drawbacks as it only provides a response for prescribed time sections (30 and 180 minutes) along the ongoing biodegradation process without any clues on the meaning of results. [13, 15] It was also conducted to obtain preliminary index values as often done for most tested chemicals,[26, 27] primarily for testing the con-
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Fig. 1. (a) OUR profile of peptone in the control reactor; (b) Inhibitory impact of antibiotics according to ISO 8192 test.
obtained. Such clues will be derived when interpreting results with continuous OUR profiles in the following section. Experiments with OUR profiles The study involves assessment of acute effects of antibiotics, to which the microbial system is exposed for the first time. The evaluation assumes that antibiotics remain nonbiodegradable for the short term tests as indicated in the literature. On the other hand as a further study, biodegradability of antibiotics will also be investigated involving continuous exposure of acclimated biomass. The OUR curve obtained from the biodegradation of the peptone mixture is shown in Figure 1a. The maximum OUR of the biomass gives the first peak around 200 mg/L.h., which is due to readily biodegradable COD components in the peptone mixture. The profile continues to drop with different rates corresponding to degradation of different COD fractions present in the selected organic substrate. The area under the OUR curve gives the total oxygen consumption, which is calculated as 190 mg/L, excluding the part related to endogenous respiration. The acute effect of antibiotic addition at a dose of 50 mg/L on the OUR profiles associated with biomass accli-
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Fig. 2. OUR profiles obtained with 50 mg/L addition of (a) SMX–50 mg/L; (b) TET–50 mg/L and (c) ERY–50 mg/L on the OUR profile.
mated to peptone mixture was displayed in Figure 2. The initial S0 /X0 ratios of the batch reactors used in this experimental set were adjusted as 0.3 mg COD/mg VSS for SMX and ERY , and 0.32 mg COD/mg VSS for TET. Three major observations could to be underlined as significant results of this test: (i) The antibiotic addition affected the shape of the OUR profile and the maximum OUR level. In fact, each antibiotic yielded a different OUR profile. Addition of SMX did not affect the maximum oxygen uptake rate (Fig. 2a). However, in the case of TET addition, the maximum oxygen uptake rate of the biomass has dropped from 200 mg/L.h. to 150 mg/L.h., as shown
Substrate binding by antibiotics in peptone biodegradation
Table 3. Mass balance between oxygen consumption and COD utilization based on OUR profiles. Remaining soluble COD (mg/L) Initial antibiotic concentration (mg/L) — 50 50 50 190 200 200 Total oxygen consumed (mg/L) 190 166 97 61 177 137 138 COD utilized COD bound (mg/L) (mg/L) 600 488 285 179 521 403 406 — 112 315 421 79 197 194 Soluble metabolic product, SP (mg/L) 36 29 17 11 31 24 24
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Peptone + antibiotic — 122 43 149 350 60 395
Runs Control SMX TET ERY SMX TET ERY
Total 36 151 60 160 381 84 419
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in Figure 2b. Similarly, ERY dosing caused it to drop to around 160 mg/L. h. (Fig. 2c). (ii) In all experiments, the OUR profile was allowed to decrease to the level of endogenous respiration after an observation period of 300–400 min – similar or slightly shorter as compared with the control reactor. The final endogenous respiration level in the OUR also indicated the depletion of all external organic substrate taking part in metabolic reactions. (iii) Despite complete removal of the same amount of peptone mixture initially present in all test reactors, antibiotic dosing induced a significant decrease in oxygen consumption. In fact, the amount of oxygen consumed for the growth of microorganisms for additions of SMX, TET and ERY were calculated as 166, 97 and 61 mg/L, respectively (Table 3). The decrease in the total amount of oxygen associated with microbial metabolism appears to be the primary indication of the inhibitory impact of selected antibiotics. The study also investigated changes in the nature of inhibition induced at higher doses of antibiotics (Fig. 3). In this context, when an antibiotic dose of 200 mg/L was applied, the maximum oxygen uptake rate in the corresponding OUR curves has dropped from 200 mg/L.h. to 160 mg/L.h. for TET and SMX additions. Addition of ERY however caused it to drop down to around 125 mg/L.h. Detailed information on the experimental conditions is given in Table 1. The system performance is better observed in terms of the total oxygen consumed during the OUR test, which were evaluated as 177 mg O2 for SMX, 137 mg O2 for TET and 138 mg O2 for ERY. The interesting feature of the experimental results is that these levels are higher than the ones associated with 50 mg/L antibiotic additions (Table 3). The observed COD removals need careful evaluation as they also include and reflect the effect of soluble microbial product generation and unbound antibiotics remained in solution. This is explained by the dissociation of the substrate/inhibitor complex at higher concentrations, releasing additional substrate for microbial utilization.
Evaluation of the inhibitory impact
Two characteristics of the OUR profile should be considered for the evaluation of the results in this study: (i) The OUR area above the endogenous respiration level directly gives the amount of oxygen consumed, O2 , at the expense of all available organic substrate (biodegradable COD) utilized by means of the following mass balance expression: O2 = CS(1 − YH ) (5)
where, CS is the biodegradable COD concentration and YH is the heterotrophic yield coefficient (mg cell COD/mg COD). Consequently, with a known/predetermined amount of biodegradable substrate, the OUR curve may be used to determine YH and/or inert COD components.[28] (ii) The OUR experiment is started at the endogenous respiration level before the addition of substrate onto biomass in the reactor; the experiment ends when the OUR drops to the same level again, indicating that all available external substrate has been consumed. The organic substrate (peptone mixture) used in the experiments is by nature totally biodegradable; this is one of the main reasons for its selection and recommendation as the standard substrate for biodegradation experiments. Because the biodegradable COD in the control reactor was completely depleted after the OUR profile dropped to the initial endogenous respiration level, COD remaining in the control reactor represents the residual soluble microbial products, SP , generated in the course of biochemical reactions; [29,30] in the proposed decay associated models, SP is conveniently expressed as a fraction of the influent biodegradable COD, CS1 in terms of a yield coefficient, YSP : [31] SP = YSP CS1 (6)
Using the data of the control reactor, a YSP value of 0.06 mg COD/mg COD was calculated, since an SP value
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Ozkok et al. place as part of peptone-meat extract removal under batch conditions.[32] The significant feature of the impact of antibiotics on the biodegradation of peptone mixture is the reduction of oxygen consumption in the OUR experiments despite the fact that the OUR profiles drop down to the level of endogenous respiration within the observation period, indicating that all available biodegradable COD is utilized. This observation is against basic stoichiometry and cannot be explained by the conventional understanding of the inhibitory impact, which would retard biodegradation by either reducing the maximum specific growth rate, µHmax and/or increase the half saturation coefficient, KS . Both types of effects are kinetic in nature, slowing down the rate of substrate utilization. The observed change in the OUR profile inflicted by this type of inhibition would be a longer period to reach the endogenous respiration level but the same area under the OUR curve or the same level of oxygen consumption. Moreover, the decrease in oxygen utilization cannot be explained with the inactivation and/or decrease of the biomass in the system either. The result of reduced active biomass concentration in the system would simply cause the system to continue substrate degradation at a slower rate, which would prolong the period required for the substrate to be depleted. The corresponding OUR curve would eventually reach the endogenous decay level, thus keeping the area under the OUR curve same as the non-inhibited system, as the amount of substrate utilized remains the same. Additional clarification and support for the arguments outlined above may be provided by model simulation. The peptone mixture used in the experiments is a well-studied organic substrate; major COD fractions it embodies with different biodegradation characteristics and corresponding biodegradation kinetics have already been ascertained by model calibration. The model structured for this purpose has the basic template of ASM1 modified for endogenous respiration.[25, 32] Detailed description of the mechanistic model used for this purpose is given in previous similar studies. [13, 32, 33] Basically, model evaluation defines the organic content of the peptone mixture in terms of three major COD fractions, namely the readily biodegradable COD, SS together with two hydrolysable COD fractions SH1 and SH2 . The same model can also be successfully calibrated with the OUR profile obtained for the control reactor including only the peptone mixture, as shown in Figure 4. The process kinetics and the corresponding values of model coefficients, listed in Table 4, closely approximate the kinetics defined in a similar studys,[32,33] in view of the fact that the peptone mixture used is a commercial product and its composition slightly changes from one batch to the other. It should be noted that the adopted model defines OUR in terms of the well known Monod-type growth rate expression, if endogenous respiration is excluded – i.e., if the
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Fig. 3. Impact of higher antibiotic dozes on the OUR profile (a) SMX–190 mg/L; (b) TET–200 mg/L (c) ERY–200 mg/L.
36 mg/L was generated at the expense of 600 mg/L of peptone mixture COD initially supplied in the reactor. Furthermore, 190 mg/L of oxygen consumed during the experiment corresponded to a yield coefficient, YH , of 0.66 mg cell COD/mg COD using the basic mass balance expression (Eq. 1). This value is slightly higher than the range of 0.60–0.64 mg cell COD/mg COD commonly associated with heterotrophic microbial growth because it reflects the effect of the substrate storage that takes
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Fig. 4. Effect of growth inhibition/biomass inactivation on the OUR profile.
OUR curve is considered above the endogenous respiration level: OUR = d SO µH ˆ SS = (1 − YH ) XH dt YH K S + SS (7)
of 20–90%. The results illustrated in Figure 4 indicate the following: (i) The shape of the OUR profile was affected, the initial peak gradually being converted to extended plateaus; (ii) the substrate utilization/oxygen consumption period was extended beyond 400 minutes (around 0.3 days) where all substrate was depleted in the control reactor; the simulation showed that with inhibitor addition, 100 mg/L of biodegradable COD remained unutilized after 0.3 days. (iii) the most important, growth inhibition and/or biomass inactivation did not affect oxygen consumption which remained the same - with less than 1% deviation due to model evaluation - when all substrate was removed. A similar model evaluation was also carried out to simulate the effect of the inhibitor on the half-saturation coefficient, KS (competitive inhibition) and on both µHmax and KS . In the first case the OUR profile was simulated when KS was gradually increased from 10 mg/L in the control reactor to 80; 100 and 300 mg/L (Fig. 5a); in the second part of the simulation the same KS increase was applied when the µHmax XH level in the control reactor was decreased by 40% (Fig. 5b). The results remained the same: while the
Based on process stoichiometry, this expression is identical to Eq. 5 and sets the same equivalence between substrate utilized and oxygen consumed. It also enables to visualize the impact of inhibition: According to the traditional understanding of inhibition, the inhibitor – i.e., the antibiotic in this study – would affect the µHmax XH couple, either by lowering the maximum growth rate, µHmax (non-competitive inhibition) or by reducing the viability of biomass, that is reducing XH . It may be argued that observed changes in the OUR profiles could be the result of a decrease in biomass in view of the bactericidal effect of antibiotics. This stipulation has been checked by model simulation: The results of the model calibration mentioned above (Table 4) yielded 5.1/day for µHmax; 1600 mg/L for XH and therefore 8160 mg/L.day for the µHmax XH couple. Five different model simulations were performed imposing a growth inhibition and/or biomass inactivation by reducing the µHmax XH level in the range
Table 4. Calibration results of biodegradation characteristics of the peptone mixture. Model parameter & state Maximum Specific Growth Rate for X H, µ H ˆ Half Saturation constant for growth of X H, K S Maximum Hydrolysis Rate for SH1 , khs Hydrolysis Half Saturation Constant for SH1 , K X Maximum Hydrolysis Rate for SH2 , khx Hydrolysis Half Saturation Constant for SH2 , K XX Endogenous Decay Rate for X H , bH Heterotrophic Yield Coefficient, Y H Unit 1/day mg COD/L 1/day g COD/g cell COD 1/day g COD/g cell COD 1/day g cell COD/g COD This study 5.1 10 4.0 0.08 1.22 0.08 0.14 0.66 Orhon et al.,[32] 4.6 4 4.30 0.03 2.10 0.08 0.20 0.60
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Ozkok et al. tone mixture utilized dropped from 600 mg/L in the unaffected control reactor to 488 mg/L with SMX; to 285 mg/L with TET and to 179 mg/L with ERY , which exerted the strongest effect. A parallel decrease could be calculated for the generation of the residual soluble metabolic products, SP as shown in Table 3. Interestingly, the remaining soluble COD in the reactor at the completion of the OUR test (endogenous respiration level) did not show the same trend: for SMX, the total COD associated with the [ESI] complex was calculated as 171 mg/L and the remaining COD contained around 70% of the antibiotic/substrate complex, the remaining 30% presumably being entrapped/attached to the biomass. The strongest biomass entrapment was attributed to TET, which yielded the lowest remaining COD level of 60 mg/L including SP (Table 3). When the antibiotic dosage was increased to 190–200 mg/L more oxygen was consumed for all selected antibiotics and more substrate utilized as compared with the lower dose of 50 mg/L. This observation is against the basic inhibition theory which predicts increased adverse effects with higher doses; but, it may be explained with the concept of dissociation of the substrate/inhibitor complex releasing higher substrate levels for microbial utilization. The remaining COD concentrations were substantially higher at this dosage, indicating that not all available antibiotic was bound with substrate and the remaining COD included aside the [ESI] complex, the unattached/free antibiotic fraction. Complex formation potential of the selected antibiotics maintained the same character so that TET yielded again the lowest level of remaining COD. The results provide supporting experimental evidence for the work of Plosz et al.,[19] defining sorption and desorption of antibiotics onto biomass under aerobic conditions, with the additional information that these processes also involve substrate entrapment and dissociation of the entrapped substrate as defined by uncompetitive inhibition. Unlike the respirometric analysis of the inhibitory action at antibiotic concentration of 50 mg/L, higher oxygen consumption and the corresponding substrate release mechanism observed at higher antibiotic doses cannot be explained by the same uncompetitive inhibition analogy and requires further investigation.
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Fig. 5. Model simulation of the OUR profile for the effect of (a) competitive inhibition (KS ) and (b) mixed inhibition (KS and µHmax XH ).
shape of the OUR profile was affected, the amount of oxygen consumption remained the same when substrate was totally removed. However, changes in the experimental OUR profiles observed in this study are totally different in nature and are shown in Figures 2 and 3, as compared with the simulated profiles displayed above: After antibiotic addition, the biodegradation time remains basically the same but the amount of oxygen consumed is significantly decreased depending on the selected antibiotic and applied dosage: The inhibitory effect becomes essentially stoichiometric, indicating that a varying fraction of peptone mixture is blocked by the antibiotic and does not participate to the on-going microbial growth mechanism. This observation is also compatible with the concept of the uncompetitive inhibition mechanism. Using the enzyme analogy commonly adopted for the interpretation of inhibition reactions, uncompetitive inhibition involves partial blockage of available substrate by the inhibitor. In this context, all observations could be explained by uncompetitive inhibition but the extent of the inhibitory impact greatly varied as a function of dosage and type of antibiotics. At 50 mg/L dosage, the amount of pep-
Conclusions
Inhibitory impact of selected antibiotics was observed as a decrease in the amount of oxygen consumed in the OUR test. Although the organic substrate was completely removed from solution and the final value of the OUR curve reached the endogenous respiration plateau, the corresponding oxygen consumption always remained below the level defined by basic stoichiometry and mass balance, leading to conclude that organic substrate was
Substrate binding by antibiotics in peptone biodegradation partially blocked by the antibiotic and could not be utilized in microbial metabolism. At 50 mg/L, the substrate ratio blocked was 20% for SMX, 52% for TET and 70% for ERY. Model simulation showed that the results could not be explained by conventional growth inhibition of biomass inactivation. However, they were compatible with uncompetitive inhibition, where substrate is partially bound as a substrate/inhibitor complex and becomes unavailable for utilization. Higher oxygen consumption observed at increased antibiotic dosage (200 mg/L) was attributed to partial dissociation of the substrate/inhibitor complex, releasing substrate, which provided higher substrate levels for microbial utilization. Additional studies are now underway to elucidate the fate of unutilized organic substrate within biomass and the inhibitory effects of the same antibiotics under continuous/chronic exposure.
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References
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