Biochemical applications of surface-enhanced infrared absorption spectroscopy An overview is presented on the application of surface-enhanced infrared absorption -LRB- SEIRA -RRB- spectroscopy to biochemical problems . Use of SEIRA results in high surface sensitivity by enhancing the signal of the adsorbed molecule by approximately two orders of magnitude and has the potential to enable new studies , from fundamental aspects to applied sciences . This report surveys studies of DNA and nucleic acid adsorption to gold surfaces , development of immunoassays , electron transfer between metal electrodes and proteins , and protein -- protein interactions . Because signal enhancement in SEIRA uses surface properties of the nano-structured metal , the biomaterial must be tethered to the metal without hampering its functionality . Because many biochemical reactions proceed vectorially , their functionality depends on proper orientation of the biomaterial . Thus , surface-modification techniques are addressed that enable control of the proper orientation of proteins on the metal surface . Introduction The seminal discovery of surface-enhanced Raman scattering -LRB- SERS -RRB- in the early 70s opened the field of surface-enhanced spectroscopy -LSB- 1 , 2 -RSB- . The phenomenon has subsequently also been observed at longer wavelengths and , ultimately , led to the realization of surface-enhanced infrared absorption spectroscopy -LRB- SEIRAS -RRB- -LSB- 3 -RSB- . Several reports appeared in the 90s on both practical and theoretical aspects of the phenomenon -LSB- 4 -- 6 -RSB- . SERS and SEIRAS have lately received attention in the field of biochemistry and biophysics , because of growing interest in bio-nanotechnology -LSB- 7 -RSB- . Typical approaches of bio-nanotechnology are constructions of hybrid devices in which bio-molecules , e.g. DNA or proteins , are combined with a solid sensing and/or actuating substrate , for example as an electrode . With this architecture the whole bandwidth of biological functions can be addressed by exchange of signals with the sensor/actuator . The concept of the hybrid bio-device is key to the development of biosensors for DNA or proteins , or for immunoassays on a chip , etc. . This concept is , moreover , valuable not only for technological progress but also for fundamental studies on proteins and other biologically active materials . Triggering the properties of the adsorbate on the substrate enables functional studies of biomolecules . A critical issue in the design of such a device is assessment of the interface between biomaterial and substrate in which the essential signal relay between the two different materials occurs . This signal relay comprises only small amounts of monolayer molecules at the interfaces which are difficult to detect by conventional Raman and IR techniques . Spectroscopic distinction from the strong background of the bulk is also difficult . This obstacle is overcome by exploiting the `` optical near-field effect '' of surface-enhanced spectroscopy in which the signal enhancement is restricted to the interface . Characteristic of vibrational techniques , SERS and SEIRA provide a wealth of molecular information on the level of a single chemical bond . SERS and SEIRA are complementary techniques , and each has its own advantages and disadvantages . SERS takes advantage of its enormous enhancement factor -LRB- of the order of 106 -- 1012 -RRB- . The strongest enhancement occurs as a result of the resonance condition if the biomolecule carries a chromophoric co-factor . The fluorescence which often accompanies this may render detection of the Raman spectrum difficult , however . Although the latter is not a problem with SEIRA , the surface-enhancement is only modest -LRB- ∼ 101 -- 103 -RRB- . SEIRAS probes almost all bands of the adsorbed species as long as the vibrational mode includes a dipole component perpendicular to the surface -LRB- surface-selection rule -RRB- -LSB- 5 -RSB- . Although the enhancement factor of SEIRAS is smaller than that of SERS , the cross-section for IR absorption is several orders of magnitude higher than the corresponding Raman cross-section . Thus , the modest enhancement of SEIRAS may be sufficient for many applications . To study the functionality of proteins by IR spectroscopy , the difference technique has provided an unprecedented amount of molecular information -LSB- 8 -- 10 -RSB- . The IR spectrum of a protein is recorded in one state -- often the resting state -- and subtracted from the IR spectrum of another state -- an active state or reaction intermediate . The difference spectrum then contains only the vibrational bands associated with the transition from one state to the other . All of the other vibrational bands are cancelled which drastically simplifies interpretation of the vibrational changes . As a consequence , the amplitudes of the difference bands are much smaller than the absorption bands of the entire protein -LRB- difference bands may be smaller by a factor of 10 − 4 , depending on the size of the protein -RRB- . Resolving the small difference bands requires acute spectroscopic sensitivity , in particular when surface-enhanced infrared difference spectroscopy -LRB- SEIDAS -RRB- is performed on a protein monolayer . In this report , we review applications of SEIRA in which biochemical processes were studied . Because SEIRAS was introduced to the field of biomolecules only recently , we consider it worthwhile to start with practical aspects of the method . Experimental considerations Preparation of thin metal-film substrates Preparation of the thin metal film is the critical part of a successful SEIRA experiment . Enhancement by SEIRA is very dependent on the size , shape , and particle density of the selected metal-island film . These properties are easily affected by the experimental conditions during film fabrication , e.g. rate of film deposition , type of the substrate , the substrate temperature , etc. -LSB- 11 -RSB- . SEIRA-active metal islands are usually prepared by high-vacuum evaporation of the metal on to a supporting substrate . Metals such as Au , Ag , Cu , and Pt are vapor-deposited by Ar sputtering , electron-beam heating , or resistive thermal heating of a tungsten basket . The thickness and the rate of deposition are monitored by use of a quartz crystal microbalance -LRB- QCM -RRB- . Controlling of the deposition rate is essential for optimum enhancement . Slow deposition -LRB- 0.1 nm s − 1 or less for deposition of Au or Ag on Si or CaF2 -RRB- generally results in greater enhancement -LSB- 11 -RSB- . This condition also depends on the type of metal , the type of substrate , and the metal film thickness , however , and must therefore be optimized for the system being used . As the morphology of the metal film affects the extent of surface enhancement , templates such as a periodic particle-array film prepared by nanosphere lithography have been used -LSB- 12 , 13 -RSB- . This approach not only increases the enhancement factor but the reproducibility of signal enhancement will enable quantitative SEIRAS . Although vacuum evaporation is routinely used , the equipment is costly and not readily available . An alternative means of forming a metal thin film is by chemical -LRB- electroless -RRB- deposition . Stable SEIRA-active thin films of Au , Pt , Cu , and Ag have been reported on Si or Ge substrates -LSB- 14 -- 17 -RSB- . The procedure for preparing a thin Au film on a silicon surface is described below : The surface of the Si is covered with 40 % w/v NH4F for a few minutes -LRB- typically 1 -- 3 min -RRB- to remove the oxide layer and to terminate the surface with hydrogen.After rinsing with water , a freshly prepared 1:1:1 mixture of : 0.03 mol L − 1 NaAuCl40 .3 mol L − 1 Na2SO3 +0.1 mol L − 1 Na2S2O3 +0.1 mol L − 1 NH4Cl , and2 % w/v HFis put on the Si surface for 60 -- 90 s.Although a shiny Au film is formed , the Au surface may still be contaminated with thio compounds from the plating chemicals . These are removed by electrochemical cycling of the potential between 0.1 and 1.4 V in 0.1 mol L − 1 H2SO4 until the cyclic voltammogram of polycrystalline gold appears -LRB- broad oxidation peak above 1.1 V and a sharp reduction peak at 0.9 V relative to the SCE -RRB- . One can , instead , apply a dc voltage of +1.5 V between the Au film and the counter electrode -LRB- e.g. a Pt wire -RRB- for ca. 1 min . Atomic force microscope -LRB- AFM -RRB- images of the chemically deposited Au film reveals an island structure similar to that of the vacuum-evaporated Au films , albeit with somewhat larger average diameter of the metal islands -LSB- 16 , 17 -RSB- . Another advantage of the chemical method is the stronger adhesion of the deposited metal layer to the substrate . This property helps significantly when long-term stability of the metal film is required , as is typical for preparation of biomimetic devices -LRB- vide infra -RRB- . An interesting option is the use of colloidal gold nano particles -LSB- 18 , 19 -RSB- . Colloidal gold is prepared by reducing tetrachloroauric -LRB- III -RRB- acid with sodium citrate . It is also commercially available in different particle sizes . Typically , 10 -- 50 nm colloidal gold is chosen . A major difference from the metal thin-film method is that the sample is attached to the colloidal gold suspension before measurement . The colloidal gold is then collected on an optical substrate by filtration or by centrifugation . The sample/colloid gold is measured either in the transmission configuration -LRB- with an IR card -RRB- or dried on an ATR prism . Removal of the metal film from the supporting substrate The metal film prepared by vacuum-evaporation is usually poorly adhesive . It is easily wiped off with ethanol or acetone . A metal film prepared by the chemical deposition method adheres much more strongly , and hence may not be removed by wiping or even by polishing with aluminium powder . Such metal films can be dissolved by immersion in a boiling solution of a 1:1:1 mixture of HCl -LRB- 32 % -RRB- , H2O2 -LRB- 30 % -RRB- , and H2O . Geometry and optical configuration The most widespread optical arrangement employs the so-called metal underlayer configuration -LRB- sample/metal film/supporting substrate ; Fig. 1a -RRB- . IR-transparent materials are usually used as supporting substrates . Highly refractive materials , for example Si , Ge , and ZnSe , are suitable for internal reflection optical geometry -LRB- Fig. 1a , -LRB- ii -RRB- -RRB- and CaF2 and BaF2 are more suitable for transmission geometry -LRB- Fig. 1a -LRB- i -RRB- -RRB- . For the former , relatively thick metal films -LRB- in the range of ten to several hundred nanometers -RRB- can be used whereas for the latter geometry the thickness of metal film should be kept to less than 10 nm , because the island structure starts to merge at higher thickness . As a result , the metal film is no longer transmissive -LSB- 11 , 20 -RSB- . Fig. 1Schematic diagram of the optical configuration of SEIRAS . -LRB- a -RRB- Metal underlayer configuration . -LRB- b -RRB- Metal overlayer configuration . The arrows denote the optical pathway of the IR beam in -LRB- i -RRB- transmission , -LRB- ii -RRB- attenuated total reflection , and -LRB- iii -RRB- external reflection geometry . -LRB- c -RRB- Spectro-electrochemical cell for SEIRA spectroscopy For internal reflection geometry -LRB- Kretschmann attenuated total-reflection geometry -RRB- , half-cylindrical , half-spherical , or trapezoidal prisms are commonly used . The former two types are advantageous for optical reasons , because the position of the focus on the metal film is not affected by changes in the angle of incidence of the IR beam . Si is commonly used as reflection element , because of its high chemical stability . One disadvantage is that the available spectral range is then limited to > 1000 cm − 1 . Although use of Ge enables use of a wider spectral window -LRB- > 700 cm − 1 -RRB- , it is not suitable for electrochemical experiments at potentials above 0.0 V -LRB- relative to the SCE -RRB- in acidic solution -LRB- pH < 4.0 -RRB- , in which it starts to dissolve . Although Ge matches well with Ag or Cu , combination with Au may cause a decrease in the enhancement factor , because of the formation of a Ge -- Au alloy at the boundary which strongly affects the morphology of the island structure of the film . Non-IR-transparent material , for example glass , polymers , carbon , and metal , can be used as a substrate only with external reflection which uses the metal overlayer configuration -LRB- metal particle/sample/supporting substrate , Fig. 1b -RRB- . In this configuration the thin metal layer -LRB- ca. 4 -- 7 nm -RRB- is directly evaporated on the sample/support surface . It should be noted that poorly reflective material is optically favorable , because of reduced distortion of band shape in the corresponding SEIRA spectrum -LSB- 21 , 22 -RSB- . This configuration is advantageous for detection in trace analysis of a sample adsorbed on glass or a polymer -LSB- 22 -RSB- . It is pointed out , however , that external reflection IR spectroscopy is not very useful for functional studies of biomaterial , because of the required presence of bulk water that strongly absorbs the IR probe beam . Chemical modification of the metal film surface It is crucial to chemically modify the bare metal if direct contact with the metal surface hampers the structure and function of the biomaterial . Strong interaction forces may even induce denaturation of proteins , because their secondary structure is very dependent on weak hydrogen-bonds between amino acids . The use of a chemically modified electrode -LRB- CME -RRB- is an approach used to tackle these obstacles -LSB- 23 -RSB- . The surface of the metal electrode is modified by heterobifunctional crosslinkers that comprise : a thiol group that is spontaneously adsorbed by the metal electrode ; a spacer group with different lengths of alkyl chain ; anda functional headgroup pointing toward the bulk solution . The CME surface can be conveniently prepared by the so-called `` SAM -LRB- self-assembled monolayer -RRB- method '' in which the SEIRA-active metal substrate is immersed in a solution containing the crosslinker molecule -LRB- normally for between 10 min and 2 h , depending on size of the molecule -RRB- . The SAM of the crosslinker is formed spontaneously by quasi-covalent bonding between the sulfur and the metal surface . The substrate is then thoroughly rinsed with the solvent so that a SAM of the crosslinker remains on the metal substrate . Adsorption of the protein by the chemically modified electrode -LRB- CME -RRB- is a suitable model for protein -- protein interaction . By varying the properties of the functional headgroup it is possible to mimic the specific conditions of the substrate during the protein -- protein or protein -- ligand interaction process . For example , use of a carboxyl headgroup mimics the side-chain terminus of Asp or Glu . The negatively charged carboxyl headgroup can specifically interact with positively charged amino acids , for example Lys , His , and Arg residues , on the protein surface . Another approach is to exploit the specific interaction of Ni2 + - chelated nitrilotriacetic acid -LRB- NTA -RRB- with a sequence of histidine residues genetically introduced into the target protein -LRB- His-tag -RRB- -LSB- 24 , 25 -RSB- . Details of this approach are given below . Applications Studies of nucleic acids and DNA SEIRAS studies on nucleic acid bases have provided information about the adsorption and orientation of thymine on silver island film -LSB- 26 -RSB- , and of cytosine -LSB- 27 -RSB- and uracil -LSB- 28 -RSB- on gold electrodes . These experiments have been expanded to studies of the hydrogen-bonding interaction between complementary bases -LRB- adenine -LRB- A -RRB- -- thiamine -LRB- T -RRB- and guanine -LRB- G -RRB- -- cytosine -LRB- C -RRB- -RRB- as model systems of DNA and RNA . Sato et al. used SEIRAS to study base-pairing between thymidine and 6-amino-8-purinethiol , a thio-derivatized adenine , immobilized on a gold electrode surface -LSB- 29 -RSB- . They observed a characteristic thymidine band at 1571 cm − 1 adsorbed on the adenine-modified surface by hydrogen-bonding . This base-pairing occurs at potentials higher than 0.1 V -LRB- relative to the SCE -RRB- , at which the N1 of adenine is deprotonated and orientated toward the solution . This result implies that hydrogen-bonding between the base pairs can be controlled by the applied electrode potential . As an interesting extension of the method , SEIRAS has been used as a diagnostic criterion in cancer research -LSB- 30 , 31 -RSB- . Although the reported enhancement factor of 3 -- 5 only modest -LSB- 31 -RSB- , the enhanced bands are impossible to observe with conventional IR spectroscopy but are crucial for determining the properties of nucleic acids of tumor tissues and their interaction with anticancer drugs . Immunoassays based on SEIRAS There is substantial interest in the development of biosensors in which the biological component is associated with a transducer to provide a means of detecting changes in the biological component . Most current biosensors consist of an enzyme or an antibody which interacts with the substrate or the respective antigen . The enzymes or antibodies are usually immobilized on a platform and the interaction between the enzyme and the substrate -LRB- or the antibody and the antigen -RRB- is monitored . Biosensors based on surface plasmon resonance -LRB- SPR -RRB- have received considerable attention in recent years . In this method , the biomaterial is immobilized on the surface of a metal-film-covered optical prism -LRB- ATR -- Kretschmann configuration -RRB- , i.e. the same optical principle is applied as with SEIRAS . The advantage of SEIRAS compared with SPR is that the chemical information derived from the vibrational spectrum identifies the nature of the adsorbed species yet the sensitivity of SEIRAS is comparable with that of SPR . The sensitivity of SPR is poor when the refractive index of the adsorbed species is close to that of the solvent , as it is for small organic molecules dissolved in an organic solvent . SEIRAS detects the individual vibrational modes of functional groups of the adsorbate , and the S/N ratio does not depend on the type of solvent if the vibrational bands of the solvent do not overlap with those of the adsorbate . SEIRAS can , moreover , be used to detect even minute structural changes that occur in the adsorbed layer during the adsorption process . Brown et al. have applied SEIRAS to biosensor analysis for determination of antibody -- antigen interactions -LSB- 32 -RSB- . In this experiment , antibodies for Salmonella -LRB- anti-SAL -RRB- were immobilized on a gold surface deposited on a silicon wafer . After taking the spectrum of anti-SAL , this `` dipstick '' sensor was immersed in a solution containing the antigen -LRB- SAL -RRB- . The SEIRA spectra of anti-SAL without SAL has characteristic antibody peaks at 1085 and 990 cm − 1 . Addition of SAL results in an additional band at 1045 cm − 1 which was assigned to the P -- O stretching vibration of the phospholipid in the cell wall , which indicates the presence of SAL in the solution . Initially , the analyte adsorbed by the `` dipstick '' sensor was probed by external reflection SEIRA spectroscopy . Later , the authors developed a new method for SEIRAS in which they used a colloidal gold surface as adsorption platform -LSB- 19 -RSB- . The colloidal gold -- antibody -- antigen complexes are left to assemble in solution , taking advantage of the tight binding of the antibodies to colloidal gold which stabilizes the colloid solution . When the sample has been assembled the complexes are collected , either by filtration on a porous polyethylene membrane -LSB- 19 -RSB- or by centrifugation on an ATR substrate -LSB- 18 -RSB- , and are then readily mounted in the FTIR spectrometer . Both `` dipstick '' and `` colloidal gold '' furnish similar spectral quality , although the enhancement of the latter approach is not mentioned . The colloidal gold system is more convenient and less expensive means of preparation than coating by sputtering . Filtration of the colloidal particles on disposable IR cards is also a rapid , easy , and cost-effective means of producing a SEIRA substrate which operates in transmission mode and does not require complicated optical arrangements . SEIRAS to probe protein functionality The metal used for surface enhancement , can also be used as an electrode . The enzymatic reactions of many biological systems , especially those of membrane proteins , are driven by an electrochemical gradient across the cell membrane . Such a system can be artificially reproduced on an electrode surface to mimic the physiological properties of a biological membrane . Electrochemically induced oxidation and reduction of a monolayer of cytochrome c -LRB- Cc -RRB- , a protein that mediates single-electron transfer between the integral membrane protein complexes of the respiratory chain , is the model system for electron transfer within and between proteins -LSB- 33 -RSB- . Electrons are directly injected to and/or withdrawn from Cc after proper contact has been established with the electrode by means of a suitable surface modifier . Proteins can be attached to such a chemically modified electrode -LRB- CME -RRB- by electrostatic attraction or covalent interaction . Figure 2 depicts potential-induced difference spectra of Cc that has been electrostatically adsorbed by different types of CME . The positive and negative peaks are indicative of conformational changes associated with the transition from the fully oxidized to the fully reduced state . Details of band assignment have been published -LSB- 14 -RSB- . The difference spectra reveal subtle changes of the secondary structure induced by rearrangement of the hydrogen-bonded network among the internal amino acid side-chains surrounding the heme chromophore . Note that the peak positions of the observed Cc bands do not depend on the type of CME layer whereas the relative intensities are strongly dependent on the CME . The former suggests that the internal conformational changes of Cc are not affected by interaction with the modifiers . The latter observation is attributed to the different surface structure , i.e. the orientation or position of the Cc relative to the CME underlayer -LSB- 14 -RSB- . Fig. 2Potential-induced redox difference spectra of a cytochrome c monolayer adsorbed on a variety of chemically modified electrodes : -LRB- a -RRB- mercaptopropionic acid , -LRB- b -RRB- mercaptoethanol , -LRB- c -RRB- cysteine , -LRB- d -RRB- dithiodipyridine . Minor spectral contributions from the surface modifier have been subtracted to selectively reveal the vibrational spectra of the adsorbed cytochrome c -LSB- 14 -RSB- Studies of molecular and protein recognition The acute sensitivity of SEIRAS is particularly useful when the technique is applied to studies of membrane proteins . Handling of the membrane proteins usually requires great care , because of their sensitivity to degeneration as soon as they are separated from the native lipid bilayer . The large size of the proteins also makes it difficult to control their orientation . A successful strategy for immobilization of membrane proteins is to attach the purified protein , by using the selective affinity of a genetically introduced histidine tag -LRB- His-tag -RRB- , to a nickel-chelating nitrilotriacetic acid -LRB- Ni-NTA -RRB- monolayer -LRB- SAM -RRB- self-assembled on a chemically modified gold surface . The lipid environment of the surface-anchored membrane protein is subsequently restored by in-situ dialysis of the detergent around the amphiphilic membrane protein . This approach results in orientated immobilization and reconstitution of the native matrix , which enhances the stability of the membrane protein and restores full functionality . The success of the method has been demonstrated by use of cytochrome c oxidase -LRB- CcO -RRB- -LSB- 24 , 25 -RSB- . Recombinant CcO -LRB- solublized by detergent -RRB- from Rhodobacter sphaeroides was immobilized on the stepwise-formed Ni-NTA SAM bound to the gold surface . It is advantageous to follow each surface-modification step by in-situ SEIRAS , which ensures qualitative and quantitative control of each reaction step . The protein is subsequently embedded in the lipid layer by removing the detergent by addition of Bio-Beads . SEIRA spectra reveal the increase of several lipid bands during the reconstitution without any dissociation of protein from the surface . Atomic-force microscopy -LRB- AFM -RRB- provides direct evidence of the formation of the lipid bilayer . Spots of size ∼ 7 nm appear in the AFM image ; this is in accordance with the diameter of a single CcO molecule -LSB- 34 -RSB- . It is remarked that the same approach can be used to bind CcO to a rough Ag surface . Integrity of the native structures of the heme cofactors -LRB- heme a and heme a3 -RRB- was shown by surface-enhanced resonance Raman spectroscopy -LRB- SERRS -RRB- -LSB- 35 , 36 -RSB- . Full functionality of the lipid-reconstituted CcO electrode is demonstrated by the fact that a catalytic current resulting from reduction of oxygen , the native property of CcO , is observed when an electron is donated by the adsorbed Cc . In such an experiment orientation of the CcO is controlled by the position of the His-tag on either side of the membrane surface CcO . With molecular genetic techniques , the His-tag is introduced either in the C-terminal tail of subunit I or in the C-terminus of subunit II . By binding CcO through the respective His-tag , the former orients CcO such that the binding site of Cc is exposed to the bulk solution . The latter orientation obstructs the binding site for Cc , because of the barrier imposed by the lipid bilayer . Indeed , SEIRAS , in combination with electrochemistry , reveals that Cc binds and initiates the catalytic reaction of CcO only when the Cc binding site faces the bulk aqueous phase and that Cc does not interact with the oppositely oriented CcO -LSB- 24 , 25 -RSB- . Interestingly , the electrochemically induced redox difference surface-enhanced infrared difference absorption -LRB- SEIDA -RRB- spectrum of the Cc -- CcO complex is remarkably similar to that of Cc adsorbed on a carboxy-terminated SAM -LRB- mercaptoundecanoic acid , MUA -RRB- -LRB- Fig. 3c and d -RRB- . It is noted that SEIDA spectra of Cc are sensitive to the properties of the terminal headgroup of the SAM that it interacts with -LSB- 14 -RSB- . The similarity of the spectra of Cc -- CcO and Cc -- MUA suggests a preponderance of carboxylate residues at the physiological docking site of Cc , i.e. , those of the side-chains of aspartate or glutamate units . Ferguson-Miller et al. have , indeed , identified residues Glu148 , Glu157 , Asp195 , and Asp214 -LRB- all in subunit II of CcO from R. sphaeroides -RRB- as the major interaction partners of Cc -LSB- 37 -RSB- . Fig. 3Potential-induced IR difference spectra of : -LRB- a -RRB- cytochrome c bound to a monolayer of cytochrome c oxidase tethered to a gold surface via His-tag/Ni-NTA interaction ; -LRB- b -RRB- tethered monolayer of cytochrome c oxidase alone ; -LRB- c -RRB- difference between -LRB- a -RRB- and -LRB- b -RRB- which recovers the vibrational contribution from cytochrome c only ; -LRB- d -RRB- cytochrome c adsorbed to a monolayer of mercaptoundecanoic acid The same strategy has recently been applied to immobilization of photosystem 2 -LRB- PS2 -RRB- on an Au surface with the purpose of developing a semi-artificial device for production of hydrogen by photosynthetic oxidation of water -LSB- 38 -RSB- . In this report , SEIRA was used to determine the adsorption kinetics of PS2 on the Ni-NTA-modified Au surface . On illumination of the surface-bound PS2 a photocurrent was generated . The action spectrum corresponds to the absorption spectrum of PS2 , indicating that the observed photocurrent is caused by photoreaction of PS2 . This methodology is a general approach for immobilization of proteins , because the introduction of affinity tags is routine with modern genetic techniques . Other tags beyond the His-tag may also be an option . Thus , orientational control of protein adsorption on a solid surface can be conveniently achieved . An oriented sample is mandatory when the vectorial function of membrane proteins is addressed . Many membrane proteins are asymmetric in their functionality , because they translocate ions or solutes preferentially in one direction or because their stimulant , e.g. ligand , binding partner protein , membrane potential , etc. , affects the protein from one side only . Summary and outlook Despite the accomplishments reported in this article , studies of biomaterials by SEIRA is still in its infancy . Potential-induced difference spectroscopy using SEIRA is a promising possibility for study of the functionality of proteins . The stimulus in such studies is not limited to the electric trigger , as reviewed here , but can also be light illumination , temperature jump , or chemical induction . Many protein functions can be addressed by these means and the mechanism of action of these molecular machines may be resolved down to the level of a single bond . The sensitivity may not be sufficient for functional difference spectroscopy on any protein with SEIDAS , however . As the enhancement factor for SEIRA is modest compared with that for SERS , optimization of the enhancement by proper design of the metal surface is crucial . Although enhancement factors are usually in the range 10 -- 100 , factors as high as 1000 have sometimes been reported -LSB- 16 -RSB- . To obtain the latter enhancement reproducibly , however , development of homogeneous island metal film preparation under strict topological control will be mandatory .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O B O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O B B O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O


Analysis

of

pharmaceuticals

in

wastewater

and

removal

using

a

membrane

bioreactor

Much

attention

has

recently

been

devoted

to

the

life

and

behaviour

of

pharmaceuticals

in

the

water

cycle

In

this

study

the

behaviour

of

several

pharmaceutical

products

in

different

therapeutic

categories

-LRB-

analgesics

and

anti-inflammatory

drugs

lipid

regulators

antibiotics

etc.

-RRB-

was

monitored

during

treatment

of

wastewater

in

a

laboratory-scale

membrane

bioreactor

-LRB-

MBR

-RRB-

The

results

were

compared

with

removal

in

a

conventional

activated-sludge

-LRB-

CAS

-RRB-

process

in

a

wastewater-treatment

facility

The

performance

of

an

MBR

was

monitored

for

approximately

two

months

to

investigate

the

long-term

operational

stability

of

the

system

and

possible

effects

of

solids

retention

time

on

the

efficiency

of

removal

of

target

compounds

Pharmaceuticals

were

in

general

removed

to

a

greater

extent

by

the

MBR

integrated

system

than

during

the

CAS

process

For

most

of

the

compounds

investigated

the

performance

of

MBR

treatment

was

better

-LRB-

removal

rates

-RRB-

and

effluent

concentrations

of

e.g.

diclofenac

ketoprofen

ranitidine

gemfibrozil

bezafibrate

pravastatin

and

ofloxacin

were

steadier

than

for

the

conventional

system

Occasionally

removal

efficiency

was

very

similar

and

high

for

both

treatments

-LRB-

e.g.

for

ibuprofen

naproxen

acetaminophen

paroxetine

and

hydrochlorothiazide

-RRB-

The

antiepileptic

drug

carbamazepine

was

the

most

persistent

pharmaceutical

and

it

passed

through

both

the

MBR

and

CAS

systems

untransformed

Because

there

was

no

washout

of

biomass

from

the

reactor

high-quality

effluent

in

terms

of

chemical

oxygen

demand

-LRB-

COD

-RRB-

ammonium

content

-LRB-

-RRB-

total

suspended

solids

-LRB-

TSS

-RRB-

and

total

organic

carbon

-LRB-

TOC

-RRB-

was

obtained

Introduction

Most

pharmaceutical

substances

are

by

nature

biologically

active

and

hydrophilic

in

order

that

the

human

body

can

take

them

up

easily

and

persistent

to

avoid

degradation

before

they

have

a

curing

effect

Depending

on

the

pharmacology

of

a

medical

substance

it

will

be

excreted

as

a

mixture

of

metabolites

as

unchanged

substance

or

conjugated

with

an

inactivating

compound

attached

to

the

molecule

-LSB-

-RSB-

When

they

enter

a

wastewater-treatment

plant

xenobiotics

are

not

usually

completely

mineralized

They

are

either

partially

retained

in

the

sludge

or

metabolized

to

a

more

hydrophilic

but

still

persistent

form

and

therefore

pass

through

the

wastewater-treatment

plant

-LRB-

WWTP

-RRB-

and

end

up

in

the

receiving

waters

Their

removal

in

WWTPs

is

variable

and

depends

on

the

properties

of

the

substance

and

process

conditions

-LRB-

e.g.

sludge

retention

time

-LRB-

SRT

-RRB-

hydraulic

retention

time

-LRB-

HRT

-RRB-

temperature

-RRB-

-LSB-

-RSB-

Levels

of

many

pharmaceutically

active

compounds

-LRB-

PhACs

-RRB-

are

barely

reduced

and

they

are

therefore

detected

in

WWTP

effluents

The

presence

of

PhACs

in

surface

drinking

and

wastewaters

is

well

documented

in

the

literature

-LSB-

-RSB-

Although

present

at

low

concentrations

in

the

environment

drugs

can

have

adverse

effects

on

aquatic

organisms

These

effects

are

chronic

rather

than

acutely

toxic

and

depend

on

exposure

-LRB-

bioavailability

-RRB-

susceptibility

to

the

compound

in

question

and

the

degradability

of

the

compound

-LSB-

-RSB-

To

ensure

compliance

with

future

discharge

requirements

upgrading

of

existing

wastewater-treatment

facilities

and

implementation

of

new

technologies

is

envisaged

as

the

next

step

in

improvement

of

wastewater

treatment

In

the

last

ten

to

fifteen

years

the

use

of

membranes

in

wastewater

reclamation

has

attracted

much

interest

Membrane

technology

has

become

a

technically

and

economically

feasible

alternative

for

water

and

wastewater

treatment

especially

because

of

high

SRTs

achieved

within

compact

reactor

volumes

In

the

MBR

the

concentration

of

microorganisms

can

be

increased

to

up

to

mg

L

Because

of

this

high

biomass

concentration

the

rate

of

degradation

is

higher

and

specialists

are

grown

for

problematic

compounds

Another

advantage

of

membrane

treatment

is

separation

of

suspended

solids

by

membranes

so

they

are

not

limited

by

the

settling

characteristics

of

the

sludge

The

performance

in

terms

of

effluent

quality

is

believed

to

be

better

but

there

is

a

striking

lack

of

knowledge

about

the

behaviour

of

trace-pollutants

Literature

data

on

this

subject

is

still

very

limited

and

contradictory

-LSB-

-RSB-

Ultrafiltration

membranes

do

not

enable

greater

retention

of

the

drugs

investigated

in

this

study

as

a

result

of

filtration

effects

the

molecular

sizes

of

the

compounds

selected

are

at

least

a

factor

of

smaller

than

the

pore

size

of

the

membranes

Additional

removal

of

hydrophobic

compounds

by

membranes

can

nevertheless

be

expected

because

they

are

adsorbed

by

particles

deposited

as

a

layer

on

the

membrane

surface

this

effect

for

hydrophilic

compounds

is

not

yet

very

well

defined

however

-LSB-

-RSB-

The

objectives

of

this

work

were

detection

of

target

pharmaceuticals

in

wastewater

influents

and

effluents

observation

of

their

elimination

in

the

CAS

process

and

comparison

with

results

obtained

for

a

laboratory-scale

MBR

provided

with

a

plate-and-frame

submerged

membrane

module

The

pharmaceutical

products

investigated

were

analgesics

and

anti-inflammatory

drugs

-LRB-

ibuprofen

ketoprofen

naproxen

diclofenac

indomethacin

acetaminophen

mefenamic

acid

and

propyphenazone

-RRB-

lipid

regulators

and

cholesterol-lowering

statin

drugs

-LRB-

clofibric

acid

gemfibrozil

bezafibrate

pravastatin

and

mevastatin

-RRB-

antibiotics

-LRB-

erythromycin

azithromycin

sulfamethoxazole

trimethoprim

and

ofloxacin

-RRB-

psychiatric

drugs

-LRB-

fluoxetine

and

paroxetine

-RRB-

an

antiepileptic

drug

-LRB-

carbamazepine

-RRB-

β-blockers

-LRB-

atenolol

sotalol

metoprolol

and

propranolol

-RRB-

anti-histaminics

-LRB-

famotidine

and

loratidine

-RRB-

anti-ulcer

agents

-LRB-

lansoprazole

and

ranitidine

-RRB-

an

anti-diabetic

-LRB-

glibenclamide

-RRB-

and

a

diuretic

-LRB-

hydrochlorothiazide

-RRB-

These

compounds

had

different

physicochemical

properties

-LRB-

i.e.

neutral

ionic

hydrophilic

hydrophobic

-RRB-

Their

chemical

structures

and

CAS

numbers

are

listed

in

the

Appendix

If

their

behaviour

during

wastewater

treatment

could

be

more

reliably

related

to

process

design

and

operating

conditions

process

performance

could

possibly

be

improved

by

alteration

of

these

conditions

in

accordance

with

the

types

of

molecule

that

are

difficult

to

eliminate

Experimental

Materials

and

standards

Chemical

standards

of

carbamazepine

lansoprazole

loratidine

famotidine

trimethoprim

ofloxacin

atenolol

metoprolol

azithromycin

dihydrate

erythromycin

hydrate

fluoxetine

hydrochloride

ranitidine

hydrochloride

sulfamethoxazole

propranolol

hydrochloride

indomethacin

acetaminophen

mefenamic

acid

clofibric

acid

bezafibrate

mevastatin

and

sotalol

hydrochloride

were

purchased

from

Sigma

Aldrich

-LRB-

Steinheim

Germany

-RRB-

propyphenazone

pravastatin

and

paroxetine

hydrochloride

from

LGC

Promochem

-LRB-

London

UK

-RRB-

ketoprofen

diclofenac

gemfibrozil

ibuprofen

and

naproxen

from

Jescuder

-LRB-

Rubí

Spain

-RRB-

glibenclamide

from

SIFA

Chemicals

-LRB-

Liestal

Switzerland

-RRB-

and

hydrochlorothiazide

from

Pliva

-LRB-

Zagreb

Croatia

-RRB-

All

pharmaceutical

standards

were

of

high-purity

grade

-LRB-

-RRB-

Isotopically

labelled

compounds

used

as

internal

standards

were

from

Sigma

Aldrich

from

Dr

Ehrenstorfer

-LRB-

Augsburg

Germany

-RRB-

and

and

from

CDN

Isotopes

-LRB-

Quebec

Canada

-RRB-

All

solvents

-LRB-

methanol

acetonitrile

and

water

-RRB-

were

HPLC-grade

and

were

purchased

from

Merck

-LRB-

Darmstadt

Germany

-RRB-

as

also

was

hydrochloric

acid

-LRB-

HCl

-RRB-

ammonium

acetate

-LRB-

-RRB-

and

acetic

acid

-LRB-

HAc

-RRB-

Nitrogen

for

drying

purity

was

from

Air

Liquide

-LRB-

Spain

-RRB-

Stock

solutions

of

individual

standards

-LRB-

g

L

-RRB-

and

internal

standards

were

prepared

in

methanol

and

stored

at

°C

Stock

solutions

of

ofloxacin

pravastatin

and

sulfamethoxazole

were

renewed

monthly

because

of

their

limited

stability

A

standard

mixture

in

which

the

compounds

were

at

a

concentration

of

approx.

mg

L

was

prepared

from

the

stock

solutions

Further

dilutions

of

this

mixture

were

prepared

in

-LRB-

v/v

-RRB-

methanol

water

and

were

used

as

working

standard

solutions

A

mixture

of

internal

standards

prepared

by

dilution

of

individual

stock

solutions

in

methanol

was

used

for

internal

standard

calibration

Membrane

bioreactor

-LRB-

MBR

-RRB-

A

submerged

MBR

of

approximately

L

active

volume

equipped

with

two

flat

sheet

membranes

-LRB-

size

area

pore

size

μm

-RRB-

purchased

from

Kubota

-LRB-

Osaka

Japan

-RRB-

was

installed

in

a

municipal

WWTP

in

Rubí

-LRB-

Barcelona

Spain

-RRB-

Although

the

nominal

porosity

of

the

membranes

was

μm

-LRB-

microfiltration

-RRB-

a

fouling

layer

of

proteins

and

microorganisms

formed

on

the

surface

of

the

membranes

reduced

the

effective

porosity

to

μm

which

brought

the

type

of

filtration

into

the

ultrafiltration

range

-LSB-

-RSB-

The

MBR

was

operated

in

parallel

with

the

CAS

process

-LRB-

aeration

tank

and

secondary

settling

tank

-RRB-

The

biocenosis

of

the

MBR

was

grown

from

inoculated

sludge

from

the

municipal

WWTP

-LRB-

aeration

basin

-RRB-

and

cultivated

over

a

period

of

approximately

month

to

reach

steady-state

conditions

The

hydraulic

retention

time

was

set

to

h

by

regulating

the

effluent

flow

and

the

SRT

was

infinite

because

no

sludge

was

discharged

from

the

reactor

The

laboratory-scale

MBR

was

operated

dynamically

in

intermittent

permeation

mode

cycles

of

permeation

for

min

interrupted

with

min

of

halt

Influent

and

permeate

flows

were

controlled

by

use

of

flow

meters

and

computer-controlled

pumps

Continuous

aeration

was

provided

by

means

of

a

sparger

pipe

situated

at

the

bottom

of

the

reaction

vessel

the

oxygen

concentration

was

kept

between

and

mg

L

The

temperature

inside

the

reactor

was

°C

throughout

sampling

Wastewater-treatment

plant

-LRB-

WWTP

-RRB-

Rubí

WWTP

was

designed

for

inhabitant

equivalents

During

the

sampling

programme

the

WWTP

was

operating

with

an

average

daily

flow

of

day

A

mixture

of

municipal

hospital

and

industrial

wastewater

is

treated

Treatment

consists

of

pretreatment

preliminary

treatment

primary

sedimentation

and

secondary

-LRB-

biological

-RRB-

treatment

Pretreated

wastewater

goes

through

a

physical

process

of

settling

in

a

primary

clarifier

Secondary

treatment

occurs

in

pre-denitrification

-LRB-

anaerobic

-RRB-

and

nitrification

-LRB-

aerobic

-RRB-

tanks

and

two

secondary

clarifiers

Secondary

sludge

is

recirculated

to

a

primary

clarifier

which

improves

the

settling

characteristics

of

the

primary

sludge

and

increases

sludge

age

A

mixture

of

primary

and

secondary

-LRB-

activated

-RRB-

sludge

is

processed

-LRB-

thickening

dewatering

-RRB-

and

anaerobically

digested

and

biogas

produced

is

used

to

heat

a

digester

The

hydraulic

retention

time

of

CAS

treatment

in

WWTP

Rubí

calculated

for

the

average

daily

flow

is

approximately

h

During

the

sampling

programme

the

plant

was

operating

with

an

SRT

of

approximately

days

WWTP

effluent

is

discharged

into

the

river

Riera

de

Rubí

which

flows

into

the

Mediterranean

sea

Sampling

and

sample

preparation

Sampling

was

conducted

during

May

and

June

Twenty-eight

samples

were

analyzed

All

wastewater

samples

were

taken

as

time-proportional

grab-samples

bearing

in

mind

the

HRT

of

the

MBR

and

CAS

processes

The

sampling

points

were

primary

sedimentation

tank

effluent

as

the

inflow

to

the

conventional

treatment

plant

and

membrane

bioreactor

CAS

effluent

andMBR

effluent

Wastewater

samples

were

collected

in

amber

glass

bottles

pre-rinsed

with

ultra-pure

water

as

composite

samples

the

volume

depended

on

the

type

of

sample

-LRB-

mL

for

influent

wastewater

and

mL

for

effluent

-RRB-

Immediately

on

arrival

at

the

laboratory

they

were

filtered

through

glass

fibre

filters

and

then

through

μm

Nylon

membrane

filters

from

Whatman

-LRB-

UK

-RRB-

The

target

compounds

were

extracted

in

one

step

by

a

method

described

elsewhere

-LSB-

-RSB-

using

a

Baker

vacuum

system

-LRB-

J.T.

Baker

The

Netherlands

-RRB-

and

Waters

-LRB-

Milford

MA

USA

-RRB-

Oasis

HLB

cartridges

-LRB-

mg

mL

-RRB-

previously

conditioned

at

neutral

pH

with

mL

methanol

then

mL

deionised

water

-LRB-

HPLC

grade

-RRB-

Elution

was

performed

twice

with

mL

methanol

at

a

flow

of

mL

min

The

extracts

were

then

evaporated

under

a

nitrogen

stream

and

reconstituted

with

mL

-LRB-

v/v

-RRB-

methanol

water

Chemical

analysis

LC

analysis

was

performed

with

a

Waters

-LRB-

Milford

MA

USA

-RRB-

HPLC

system

coupled

to

a

Micromass

Quattro

-LRB-

Manchester

UK

-RRB-

triple

quadrupole

mass

spectrometer

equipped

with

a

Z-spray

electrospray

interface

Chromatographic

separation

was

achieved

on

a

Purospher

Star

endcapped

column

-LRB-

mm

mm

particle

size

μm

-RRB-

and

a

guard

column

both

from

Merck

A

specific

multi-residue

analytical

method

was

set

up

for

measurement

of

the

concentrations

of

the

pharmaceutical

compounds

in

wastewaters

-LSB-

-RSB-

Analysis

was

performed

in

multiple-reaction-monitoring

-LRB-

MRM

-RRB-

mode

in

both

positive

and

negative

electrospray-ionization

mode

This

method

was

refined

for

analysis

of

hydrochlorothiazide

and

glibenclamide

MRM

transitions

selected

for

each

compound

are

summarized

in

Table

In

accordance

with

the

performance

characteristics

defined

in

EU

Commission

Decision

for

confirmation

and

identification

of

pharmaceuticals

when

using

LC

tandem

MS

as

the

instrumental

technique

a

minimum

of

three

identification

points

are

required

When

using

LC

MS

MS

-LRB-

QqQ

-RRB-

analysis

two

MRM

transitions

are

sufficient

to

confirm

the

identity

of

a

compound

The

MRM

ratio

calculated

as

the

relationship

between

the

abundances

of

both

transitions

and

the

LC

retention

time

are

also

criteria

used

to

confirm

the

presence

of

an

analyte

in

the

samples

In

this

study

therefore

transitions

between

a

precursor

ion

and

the

two

most

abundant

fragment

ions

were

chosen

for

each

analyte

when

working

in

MRM

mode

resulting

in

four

identification

points

enough

to

conform

with

the

aforementioned

EU

directive

When

poor

fragmentation

was

observed

for

the

compounds

only

one

transition

could

be

monitored

Confirmation

of

the

identities

of

these

was

achieved

by

matching

their

LC

retention

times

with

those

of

standards

Shifts

in

retention

times

were

less

than

so

the

confirmation

was

regarded

as

sufficiently

reliable

For

internal

standards

only

one

transition

was

selected

because

they

were

isotopically

labelled

compounds

unlikely

to

be

found

in

environmental

samples

Table

transitions

of

the

compounds

analyzedCompoundMRM

To

compensate

for

matrix

effects

from

sample

matrices

internal

standard

calibration

and

adequate

dilution

of

sample

extracts

were

used

on

the

basis

of

the

previously

published

method

-LSB-

-RSB-

Recoveries

of

the

method

were

determined

by

spiking

Influent

samples

and

CAS

and

MBR

effluents

were

spiked

in

triplicate

with

a

standard

mixture

of

selected

compounds

to

a

final

concentration

of

μg

L

Spiked

samples

and

a

blank

sample

were

analysed

by

the

above

mentioned

method

Recoveries

of

the

target

pharmaceuticals

are

listed

in

Table

with

method

detection

limits

-LRB-

MDL

-RRB-

for

influent

and

effluent

samples

MDLs

and

method

quantification

limits

-LRB-

MQL

-RRB-

were

calculated

on

the

basis

of

signal-to-noise

ratios

-LRB-

S/N

-RRB-

of

and

respectively

Table

recoveries

of

the

selected

compounds

and

method

detection

limits

-LRB-

MDL

-RRB-

in

ng

L

-LRB-

-RRB-

MDL

-LRB-

ng

L

-RRB-

InfluentMBR

effluentCAS

effluentInfluentMBR

and

CAS

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Mefenamic

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Clofibric

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

aThe

relative

standard

deviation

-LRB-

-RRB-

of

the

recoveries

is

given

in

parentheses

-LRB-

n

-RRB-

Results

and

discussion

It

is

well

documented

that

WWTPs

are

major

contributors

of

pharmaceuticals

in

the

environment

WWTP

Rubí

mainly

receives

domestic

hospital

and

industrial

wastewater

and

effluent

concentrations

of

several

monitored

compounds

exceed

μg

L

levels

Ranges

of

output

loads

for

WWTP

Rubi

for

each

pharmaceutical

and

mean

values

-LRB-

g

day

-RRB-

are

reported

in

Table

The

quantities

of

pharmaceuticals

discharged

into

the

environment

are

calculated

by

multiplying

the

detected

effluent

concentrations

by

an

average

daily

flow

rate

of

day

The

total

amount

of

pharmaceutical

compounds

discharged

by

WWTP

Rubi

into

the

environment

exceeded

g

day

-LRB-

an

average

value

-RRB-

The

most

abundant

compounds

with

average

individual

loads

of

g

day

were

the

anti-inflammatory

drugs

ibuprofen

naproxen

and

diclofenac

the

lipid

regulators

gemfibrozil

and

bezafibrate

the

diuretic

hydrochlorothiazide

and

the

β-blocker

atenolol

Table

daily

output

loads

of

the

investigated

pharmaceuticals

for

Rubí

WWTPPharmaceuticalEffluent

load

-LRB-

g

day

-RRB-

MeanRangeAnalgesics

and

anti-inflammatory

nd

Mefenamic

Psychiatric

nda

Antiepileptic

Hypoglycaemic

nd

Lipid

regulator

and

cholesterol

lowering

statin

Clofibric

PravastatinndndaNot

detectable

-LRB-

below

the

LOQ

-RRB-

To

assess

the

efficiency

of

elimination

by

the

MBR

substance-specific

analysis

must

be

performed

and

the

bulk

properties

DOC

and

COD

of

wastewater

influents

and

effluents

must

also

be

determined

The

performance

of

the

MBR

system

is

summarized

in

Table

The

data

are

presented

for

the

sampling

period

Removal

efficiencies

of

for

TSS

and

for

total

COD

were

achieved

during

the

membrane

process

Average

effluent

ammonia

concentration

was

μg

L

in

the

MBR

effluent

compared

with

μg

L

in

the

CAS

effluent

It

is

known

that

membrane

processes

are

quite

efficient

at

removing

COD

and

TOC

from

wastewater

-LSB-

-RSB-

Improved

COD

removal

is

attributed

to

the

combination

of

complete

retention

of

particulate

material

by

the

membrane

including

suspended

COD

and

high

molecular

weight

organisms

and

to

avoidance

of

biomass

washout

problems

common

in

activated

sludge

systems

Consequently

stable

conditions

are

provided

for

growth

of

specialized

microorganisms

which

are

the

able

to

remove

poorly

biodegradable

components

Table

of

the

performance

of

the

MBR

systemPropertyInfluentMBR

effluentCAS

effluentTSS

-LRB-

mg

L

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

CODtotal

-LRB-

mg

L

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

TOC

-LRB-

mg

L

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

mg

L

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

aValues

are

averages

from

n

samples

with

standard

deviations

in

parentheses

Of

pharmaceutical

products

included

in

the

analytical

method

were

detected

in

the

wastewater

entering

WWTP

Rubí

Box

plots

of

measured

concentrations

of

each

pharmaceutical

are

showed

in

Figs.

and

Ten

measured

values

are

given

for

influent

and

MBR

effluent

concentrations

and

eight

for

CAS

effluent

-LRB-

data

are

missing

for

two

sampling

programmes

-RRB-

For

each

variable

the

box

has

lines

at

the

lower

quartile

-LRB-

-RRB-

median

-LRB-

-RRB-

and

upper

quartile

-LRB-

-RRB-

values

The

whiskers

are

the

lines

extending

from

each

end

of

the

box

to

show

the

extent

of

the

data

up

to

times

the

interquartile

range

-LRB-

IQR

-RRB-

Outliers

are

marked

with

symbols

Fig.

during

MBR

and

CAS

treatment

of

the

analgesics

and

anti-inflammatory

drugs

naproxen

-LRB-

a

-RRB-

ketoprofen

-LRB-

b

-RRB-

ibuprofen

-LRB-

c

-RRB-

mefenamic

acid

-LRB-

d

-RRB-

diclofenac

-LRB-

e

-RRB-

indomethacin

-LRB-

f

-RRB-

acetaminophen

-LRB-

g

-RRB-

and

propyphenazone

-LRB-

h

-RRB-

Fig.

during

MBR

and

CAS

treatment

of

the

antibiotics

ofloxacin

-LRB-

a

-RRB-

sulfamethoxazole

-LRB-

b

-RRB-

and

erythromycin

-LRB-

c

-RRB-

the

β-blockers

atenolol

-LRB-

d

-RRB-

and

metoprolol

-LRB-

e

-RRB-

the

anti-ulcer

agent

ranitidine

-LRB-

f

-RRB-

the

antiepileptic

drug

carbamazepine

-LRB-

g

-RRB-

and

the

psychiatric

drug

paroxetine

-LRB-

h

-RRB-

Fig.

during

MBR

and

CAS

treatment

of

the

lipid

regulator

and

cholesterol-lowering

statin

drugs

gemfibrozil

-LRB-

a

-RRB-

bezafibrate

-LRB-

b

-RRB-

clofibric

acid

-LRB-

c

-RRB-

and

pravastatin

-LRB-

d

-RRB-

the

diuretic

hydrochlorothiazide

-LRB-

e

-RRB-

and

the

hypoglycaemic

agent

glibenclamide

-LRB-

f

-RRB-

The

highest

influent

concentrations

-LRB-

μg

L

-RRB-

were

measured

for

the

analgesics

and

anti-inflammatory

drugs

naproxen

ibuprofen

ketoprofen

diclofenac

and

acetaminophen

the

antihyperlipoproteinaemic

drugs

gemfibrozil

and

bezafibrate

the

β-blocker

atenolol

and

the

diuretic

hydrochlorothiazide

For

other

compounds

input

concentrations

were

usually

in

the

range

ng

L

Because

the

low

concentrations

measured

were

sometimes

close

to

the

limits

of

quantification

any

hypothesis

about

the

efficiency

of

their

elimination

is

questionable

Mean

removal

was

nevertheless

calculated

for

each

of

the

pharmaceutical

compounds

the

results

are

presented

in

Table

The

most

important

removal

pathways

of

organic

compounds

during

wastewater

treatment

are

biotransformation/biodegradation

adsorption

by

the

sludge

-LRB-

excess

sludge

removal

-RRB-

andstripping

by

aeration

-LRB-

volatilization

-RRB-

Table

removal

of

selected

pharmaceuticals

by

the

MBR

and

CAS

processesCompoundElimination

-LRB-

-RRB-

in

MBRaCASbAnalgesics

and

anti-inflammatory

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Mefenamic

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Anti-ulcer

-LRB-

-RRB-

-LRB-

-RRB-

Psychiatric

-LRB-

-RRB-

-LRB-

-RRB-

Antiepileptic

drugsCarbamazepineNo

eliminationcNo

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

No

-LRB-

-RRB-

No

-LRB-

-RRB-

-LRB-

-RRB-

Hypoglycaemic

-LRB-

-RRB-

-LRB-

-RRB-

Lipid

regulator

and

cholesterol

lowering

statin

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Clofibric

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

a

bValues

are

averages

with

relative

standard

deviations

-LRB-

-RRB-

in

parentheses

for

n

or

n

samplescCompounds

were

classified

as

no

elimination

if

elimination

was

less

than

Because

of

the

low

values

of

the

Henry

coefficients

-LRB-

KH

-RRB-

of

the

compounds

investigated

-LSB-

-RSB-

the

fraction

removed

by

volatilization

can

be

neglected

-LSB-

-RSB-

The

two

processes

abiotic

-LRB-

adsorption

-RRB-

and

biotic

degradation

-LRB-

transformation

by

microorganisms

-RRB-

could

not

be

distinguished

because

no

method

was

developed

for

analysis

of

the

target

compounds

in

sludge

The

term

removal

is

therefore

used

here

for

conversion

of

a

micropollutant

to

compounds

other

than

the

parent

compound

Elimination

efficiency

of

the

laboratory-scale

MBR

and

the

full-scale

CAS

process

was

comparable

for

naproxen

ibuprofen

acetaminophen

hydrochlorothiazide

and

paroxetine

All

were

removed

to

a

large

extent

by

both

systems

-LRB-

removal

was

greater

than

except

for

hydrochlorothiazide

for

which

it

was

between

and

-RRB-

Hydrochlorothiazide

and

paroxetine

were

eliminated

slightly

better

by

conventional

treatment

Similar

results

for

the

behaviour

of

these

drugs

during

conventional

treatment

have

been

reported

by

several

authors

-LSB-

-RSB-

For

ketoprofen

diclofenac

bezafibrate

and

gemfibrozil

removal

by

the

MBR

system

was

very

high

and

uniform

-LRB-

-RRB-

with

the

exception

of

two

sampling

programme

It

is

assumed

this

variation

could

have

been

a

result

of

reduced

microbial

activity

or

altered

sorption

and

flocculation

conditions

No

plausible

explanation

can

be

given

for

the

drastically

reduced

efficiency

of

removal

of

clofibric

and

mefenamic

acid

by

MBR

in

two

sampling

programmes

otherwise

these

were

eliminated

with

efficiencies

between

and

High

and

steady

removal

-LRB-

-RRB-

in

the

MBR

was

also

observed

for

ranitidine

and

ofloxacin

In

conventional

treatment

all

these

pharmaceuticals

were

eliminated

with

a

wide

range

of

efficiencies

always

lower

than

those

obtained

by

the

MBR

Better

removal

of

readily

biodegradable

micropollutants

by

the

MBR

could

be

because

of

the

smaller

flock

size

of

the

sludge

which

enhances

mass

transfer

by

diffusion

and

therefore

increases

elimination

Taking

into

consideration

the

composition

of

sludge

originating

from

a

membrane

bioreactor

-LRB-

specialized

microorganisms

large

amount

of

active

biomass

in

suspended

solids

-RRB-

improved

removal

is

to

be

expected

this

was

confirmed

by

our

experiments

A

possible

explanation

of

substantially

greater

attenuation

of

diclofenac

by

the

MBR

-LRB-

average

removal

efficiency

compared

with

in

CAS

-RRB-

could

be

the

greater

age

of

the

MBR

sludge

Improved

removal

is

observed

with

increasing

solids

retention

time

-LSB-

-RSB-

Another

explanation

could

be

greater

adsorption

potential

of

the

MBR

sludge

because

the

organic

matter

content

is

greater

than

for

CAS

sludge

According

to

results

from

the

EU

project

Poseidon

-LSB-

-RSB-

adsorption

processes

affect

elimination

of

diclofenac

Literature

data

on

this

matter

is

still

very

contradictory

Clara

et

al.

reported

poor

removal

of

diclofenac

in

laboratory-scale

WWTPs

whereas

in

full-scale

plants

removal

varied

from

less

than

to

between

and

for

some

of

the

facilities

investigated

-LSB-

-RSB-

Heberer

et

al.

-LSB-

-RSB-

reported

low

removal

efficiencies

in

a

WWTP

whereas

Ternes

et

al.

documented

significant

-LRB-

-RRB-

elimination

of

diclofenac

-LSB-

-RSB-

Removal

of

carbamazepine

was

in

contrast

very

poor

-LRB-

-RRB-

and

effluent

concentrations

for

both

MBR

and

CAS

were

frequently

greater

than

influent

levels

Poor

elimination

of

this

neutral

drug

has

been

reported

by

many

authors

-LSB-

-RSB-

Glucuronide

conjugates

of

carbamazepine

can

presumably

be

cleaved

in

sewage

thus

increasing

environmental

concentrations

-LSB-

-RSB-

Rates

of

removal

of

the

antibiotic

sulfamethoxazole

were

very

variable

in

both

treatments

investigated

According

to

Drillia

et

al.

its

microbial

degradation

will

depend

on

the

presence

of

readily

biodegradable

organic

matter

in

wastewater

this

varies

during

both

MBR

and

CAS

treatment

-LSB-

-RSB-

Also

a

substantial

amount

of

sulfamethoxazole

enters

WWTPs

as

its

human

metabolite

which

can

possibly

be

converted

back

to

the

original

compound

during

treatment

-LSB-

-RSB-

Efficiency

of

removal

of

atenolol

metoprolol

pravastatin

erythromycin

and

indomethacin

varied

in

both

MBR

and

CAS

treatment

This

could

not

be

explained

Fluctuation

of

elimination

efficiency

was

also

observed

for

propyphenazone

-LRB-

for

MBR

and

for

CAS

-RRB-

and

glibenclamide

-LRB-

for

MBR

and

for

CAS

-RRB-

Effluent

concentrations

greater

than

those

recorded

for

the

influent

could

be

explained

by

the

presence

of

input

conjugate

compounds

that

are

transformed

into

the

original

compounds

during

treatment

Because

these

conjugates

were

not

included

in

the

analysis

no

firm

conclusion

can

be

made

about

their

biotransformation

especially

because

sampling

inaccuracy

can

also

lead

to

errors

Conclusion

Several

pharmaceutical

products

-LRB-

e.g.

ibuprofen

naproxen

acetaminophen

ketoprofen

diclofenac

bezafibrate

gemfibrozil

ranitidine

ofloxacin

hydrochlorothiazide

and

paroxetine

-RRB-

with

high

rates

of

attenuation

can

be

expected

to

be

completely

removed

from

wastewater

by

adsorption

or

degradation

or

a

combination

of

both

during

membrane

treatment

For

most

of

the

compounds

investigated

MBR

effluent

concentrations

were

significantly

lower

than

in

the

effluent

from

conventional

treatment

Elimination

of

hydrochlorothiazide

and

paroxetine

was

slightly

better

in

CAS

treatment

Some

substances

-LRB-

e.g.

carbamazepine

-RRB-

were

not

removed

by

either

MBR

or

CAS

treatment

No

relationship

was

found

between

the

structures

of

target

compounds

and

their

removal

during

wastewater

treatment

however

The

range

of

variation

of

the

efficiency

of

removal

by

the

MBR

system

was

small

for

most

of

the

compounds

in

conventional

treatment

greater

fluctuations

were

observed

and

removal

efficiency

was

found

to

be

much

more

sensitive

to

changes

in

operating

conditions

-LRB-

temperature

flow

rate

etc

-RRB-

Although

membrane

technology

seems

a

promising

means

of

removal

of

pharmaceutical

compounds

the

MBR

process

investigated

would

not

completely

halt

discharge

of

micropollutants

Membrane

treatment

processes

should

be

optimized

by

modification

of

the

membranes

-LRB-

variation

of

the

materials

and

reduction

of

molecular

mass

cut-off

limits

-RRB-

and/or

by

modification

of

the

treatment

process

-LRB-

inoculation

of

special

microorganisms

-RRB-

The

efficiencies

of

diverse

microbial

populations

in

elimination

of

selected

pharmaceuticals

and

optimization

of

design

and

operating

conditions

of

a

laboratory-scale

MBR

will

be

the

main

objectives

of

our

future

investigations

That

would

provide

guidelines

for

scale-up

of

a

biological

pilot

plant

and

its

evaluation

by

integration

into

an

industrial

process

water-recycling

system

Because

of

the

current

lack

of

information

on

the

behaviour

of

pharmaceuticals

in

surface

and

wastewaters

however

further

studies

are

required

on

the

occurrence

fate

and

effects

of

these

substances

in

the

environment

O O B O O O O O O B I O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O

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