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Treatment of opium alkaloid containing wastewater in sequencing batchreactor (SBR)—Effect of gamma irradiation

Cavit B. Bural a, Goksel N. Demirer a, Omer Kantoglu b, Filiz B. Dilek a,n

a Middle East Technical University, Department of Environmental Engineering, 06531 Ankara, Turkeyb Turkish Atomic Energy Authority, Saraykoy Nuclear Research and Training Center, 06982, Kazan, Ankara, Turkey

a r t i c l e i n f o

Article history:

Received 10 August 2009

Accepted 24 September 2009

Keywords:

Opium alkaloid containing wastewater

Biodegradability

SBR

Gamma irradiation

a b s t r a c t

Aerobic biological treatment of opium alkaloid containing wastewater as well as the effect of gamma

irradiation as pre-treatment was investigated. Biodegradability of raw wastewater was assessed in

aerobic batch reactors and was found highly biodegradable (83–90% degradation). The effect of

irradiation (40 and 140 kGy) on biodegradability was also evaluated in terms of BOD5/COD values and

results revealed that irradiation imparted no further enhancement in the biodegradability. Despite the

highly biodegradable nature of wastewater, further experiments in sequencing batch reactors (SBR)

revealed that the treatment operation was not possible due to sludge settleability problem observed

beyond an influent COD value of 2000 mg dm�3. Possible reasons for this problem were investigated,

and the high molecular weight, large size and aromatic structure of the organic pollutants present in

wastewater was thought to contribute to poor settleability. Initial efforts to solve this problem by

modifying the operational conditions, such as SRT reduction, failed. However, further operational

modifications including addition of phosphate buffer cured the settleability problem and influent COD

was increased up to 5000 mg dm�3. Significant COD removal efficiencies (470%) were obtained in both

SBRs fed with original and irradiated wastewaters (by 40 kGy). However, pre-irradiated wastewater

provided complete thebain removal and a better settling sludge, which was thought due to degradation

of complex structure by radiation application. Degradation of the structure was observed by GC/MS

analyses and enhancement in filterability tests.

& 2009 Elsevier Ltd. All rights reserved.

1. Introduction

A variety of industrial processes generate highly pollutedwastewater which constitute a major environmental concern andrequire adequate treatment prior to discharge into receivingwaters. Meeting the stringent discharge limits set by environ-mental regulations is a challenge for such industrial facilities.Among those, high strength effluents from opium alkaloidprocessing plants has been standing problematic for years dueto complex structural compounds contained in the wastewater.The pollutant constituents in opium alkaloid wastewater mainlyconsist of phytin, morphine, codein, thebain, aniline, toluol, vax-like substances and cellulose (Kac-ar et al., 2003; Aydın, 2002).

Opium poppy is a medicinally important plant, because it isthe only commercial source of the pharmaceutical alkaloids suchas the pain reliever morphine, the cough suppressant codeine andthe antitumor agent noscapine (narcotine) (Frick et al., 2007; Yeet al., 1998). Opium poppy is cultivated in a number of countriesamong which Turkey and India are accepted as traditional

producers. As India uses raw opium, Turkey uses poppy strawfor alkaloid extraction. As the global demand for alkaloids hasbeen increasing over the past two decades, the demand has beenmainly covered by poppy straw produced by Australia, France,Hungary, Spain and Turkey. According to International NarcoticsControl Board (INCB) reports, Turkey was the leading producer ofpoppy straw (in morphine equivalent) accounting for 34% of theworld production in the year 2006 (INCB, 2008).

Current literature on the treatment of opium alkaloid factoryeffluents is very limited because the cultivation and processing ofopium is not practiced in most of the developed countries (Sevimliet al., 1999). Several treatment options including chemical,physicochemical and biological methods investigated so far byother researchers (Kinli, 1994; Sevimli et al., 2000; Aydın et al.,2002; Koyuncu, 2003) could not present a whole solid solution tothe problem. For the treatment of such wastewaters, biologicaltreatment methods are generally prefered as they are cheap andeasy to apply. Considering the very high strength of thesewastewaters, anaerobic–aerobic biological treatment in sequenceis required. However, due to very complex nature of the organicmatters contained, even these sequence applications cannotreadily treat the alkaloid containing wastewater. Therefore, thetreatment of this industrial wastewater is still a challenging task.

ARTICLE IN PRESS

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/radphyschem

Radiation Physics and Chemistry

0969-806X/$ - see front matter & 2009 Elsevier Ltd. All rights reserved.

doi:10.1016/j.radphyschem.2009.09.013

n Corressponding author. Tel.: +90 312 2105877; fax: +90 312 210 2646.

E-mail address: [email protected] (F.B. Dilek).

Radiation Physics and Chemistry 79 (2010) 519–526

Author's personal copyARTICLE IN PRESS

Chemical pre-treatment prior to biological treatment can beused to improve the system performance for this kind of complexindustrial wastewaters. Recently, chemical treatment methods,based on the generation of strong oxidizing hydroxyl radicals,known as advanced oxidation processes (AOPs), have been widelyapplied for pollutant degradation (Perez et al., 2002). Among theAOPs, application of radiation technology—including 60Co-gammairradiation and e-beam accelerator—in wastewater treatment isdrawing considerable attention around the world (Bao et al.,2002; Duarte et al., 2002; Barrera-Diaz et al., 2003; Jo et al., 2006;Kim et al., 2007). Their application is based on interaction ofreactive oxidizing and reducing species,which are formed byradiolysis of water, with organic pollutants in both primary (directeffects) and secondary ionizations (indirect effects) (Drzewicz etal., 2003). For complex industrial wastewaters, radiation treat-ment alone may require high absorbed doses. Hence, thecombination of radiation technology with conventional treatmentprocesses, such as biological treatment (Jo et al., 2006), is morepromising.

Therefore, the aim of this study was to investigate the aerobicbiological treatability of high strength opium alkaloid industrywastewaters in a sequencing batch reactor (SBR) system inconjunction with gamma irradiation as means of pre-treatment.

2. Materials and methods

2.1. Wastewater characteristics

The wastewater used was obtained from an Opium AlkaloidsFactory located in Afyon Province, Turkey. In this plant, opiumpoppy harvested under the control and license of TurkishGovernment is processed to produce morphine and otherimportant alkaloids for medicinal purposes. The plant has acapacity to process 72 tons of opium per day and the specificwastewater production is about 6.7 m3 per ton of opium capsule.Thus, the plant generates around 480 m3 wastewater per day withhigh organic matter concentration, and intense dark brown color(Sevimli et al., 1999). The characteristics of wastewater werevariable throughout the study. The ranges for some importantparameters are presented in Table 1.

Considering the very high organic content of the raw waste-water, it would be impossible to treat it directly in an aerobictreatment system. Therefore, anaerobic pre-treatment was con-sidered to be essential, and the original wastewater was firstsubjected to an anaerobic treatment in a laboratory scale UASBreactor. The results of this part of the study showed that it wouldbe possible to decrease the COD value down to around 5000 mgdm�3 from around 30 000 mg dm�3 through anaerobic treatment(data not shown and to be published elsewhere). Therefore, in thisstudy, aerobic treatment of opium alkaloid containing wastewater

having COD of 5000 mg dm�3 was investigated, and accordingly,the raw wastewater was diluted to attain this value.

2.2. Inoculum

Inoculum used was collected from return sludge line of theactivated sludge unit of Ankara Municipal Wastewater TreatmentPlant. Prior to inoculation, activated sludge was aerated for oneday and washed two times with tap water to remove remainingorganics in the bulk liquid. When higher concentration of seedwas needed, the sludge was sedimented in Imhoff cone and thesettled part was used.

2.3. Nutrient addition

Although COD of the wastewater was quite high, nitrogen andphosphorus concentrations were not adequate for biologicaltreatment (COD:N:P=100:2.3:0.01). Hence, stock solutions ofthese nutrients were prepared and supplied during the experi-mental period to sustain microbial activity. A mixture of K2HPO4

and KH2PO4 was used as both phosphorus and buffer sourcewhereas NH4Cl was used as nitrogen source. COD:N:P ratiossupplied in each experiment were provided under relatedsections.

2.4. Aerobic biodegradability of raw alkaloid wastewater—Batch

experiments

Preliminary batch reactor experiments were performed inerlenmeyer flasks with 500 cm3 working liquid volume. InitialCOD concentration of around 5000 mg dm�3 was made up byappropriate dilution. Also a batch reactor supplemented withpeptone as carbon source with similar initial COD value was set inorder to compare the degradability of wastewater with a readilybiodegradable substrate. Reactors were inoculated to have seedconcentration around 55–70 mg dm�3. Nutrients were suppliedfrom stock solutions at a COD:N:P of 100:5:2 in order to satisfynutritional requirements of biomass. The daily volume of waterlost via evaporation was compensated by adding ordinary tapwater just before each sampling. 5–7 cm3 samples were taken atvarious time intervals and analyzed for COD concentration afterfiltering through 0.45 mm filters. Aeration supplied via air pumpswas enough to completely mix the reactor contents, therefore noadditional mixing was provided. Experiments were conductedunder ambient air conditions.

2.5. Radiation application

Radiation applications were carried out by Turkish AtomicEnergy Authority (TAEK) by using Issledovatel Px-g-30 Russian60Co gamma rays irradiator at a fixed dose rate of 2.23 kGy h�1 inair at ambient temperature.

2.6. Effect of radiation on the biodegradability of raw alkaloid

wastewater

Samples of wastewater were exposed to g-irradiation with 40and 140 kGy absorbed doses. BOD5/COD ratios were sought forboth raw and irradiated samples to explore the effect ofirradiation on the biodegradability of wastewater.

Table 1Characteristics of the opium alkaloid wastewater.

Parameter Unit Range

Chemical oxygen demand (COD) (total) mg dm�3 30000–43078

Soluble COD mg dmg dm�3 28500–40525

Biochemical oxygen demand (BOD5) mg dm�3 16625–23670

pH – 4.5–5.36

Total suspended solids mg dm�3 555–2193

Volatile suspended solids mg dm�3 382–1395

Total Kjheldahl nitrogen mg dmg dm�3 396–1001

NH4-N mg dm�3 61.6–259

Total P mg dm�3 4–5.21

Color (after coarse filtration) Pt–Co 4375–4750

C.B. Bural et al. / Radiation Physics and Chemistry 79 (2010) 519–526520


2.7. Sequencing batch reactor (SBR) setup and operation

Experiments were conducted in 2.5 dm3 glass vessels withtotal working volumes of 2 dm3 unless otherwise specified. Atstart-up, reactors were inoculated with mixed culture at avolumetric ratio of 1

4 i.e. 25% of working volume. Nutrientsrequired by biomass were supplied at COD:N:P of 100:5:2, unlessotherwise specified. Reactors were operated in fill-reaction-settle-draw modes following a 24 h cycle. The cycle periods weremanually controlled. The reaction period allocated was at least22 h as the settling time was varied from 20 min up to 2 haccording to operational needs. After sedimentation, the super-natant was drawn instantaneously as effluent and the sameamount of feeding was applied in pulse mode to start the nextcycle. The solid retention times (SRT) were varied between 6 and20 days throughout the study. Desired SRT values were main-tained by wasting a fraction of mixed liquor daily at the end ofeach reaction period. Target for dissolved oxygen concentration inreactors were set above 2 mg dm�3 by using air pumps. Theintensity of supplied aeration was enough to attain complete mixcondition in reactors without any further need for mixing. Unlessotherwise specified, the experiments were conducted at 25 1C,which was maintained by a temperature controlled waterbath.The attainment of steady-state condition was determined bymonitoring the mixed liquor suspended solids (MLSS), the mixedliquor volatile suspended solids (MLVSS) and the COD removalefficiencies. For MLSS and MLVSS analysis, completely mixedsamples were taken from the reactors at the end of reactionperiods. COD analyses were performed at the beginning and endof the reaction periods, on samples filtered through 0.45 mmfilters. Settling characteristics were monitored based on theSludge Volume Index (SVI).

In total, six sets of experiments, using reactors R1, R2, R3, R4,R5 and R6, were carried out in SBRs. Due to complex nature andhigh strength of opium alkaloid containing wastewater, gradualincrements in influent strength was attempted to allow microbialbiomass to acclimatize. Accordingly, the experiment was initiatedwith diluted wastewater having 500 mg dm�3 COD (R1). Theinfluent strength was increased to 2000 mg dm�3 over time (R2).Due to some operational problems (i.e. sludge settling) experi-enced during this experiment, some additional runs wereconducted in an attempt to overcome the problem (R3), e.g. SRTwas reduced from 20 days down to 6 days, reactor working liquidvolumes were reduced to 1.6 dm3 to avoid overflows fromreactors, fresh seed at a mass ratio of 1

2 was introduced to renewthe culture and allow a new microbial selection, and if possible,few drops of paraffin oil were added into reactors to suppressfoaming and accordingly associated biosolids loss, wheneverneeded (data not shown). But, no cure was achieved. Then,radiation treatment was considered as an attempt to cure thesettling problem experienced. Before initiating related SBR run,effect of radiation application to help degrade the complexstructure of alkaloid wastewater was tested through filterability

tests and GC/MS analyses. Following the promising resultsobtained, another set of experiments with SBR were performedand through them the effect of radiation treatment with 40 kGydose was investigated (R4). Influent strength was increased up to5000 mg dm�3 (R5 and R6). Buffer capacity was also increased(COD:N:P=100:5:30). The set of experimental conditions studiedare summarized in Table 2.

2.8. Monitoring the degradation of complex wastewater structure

Degradation of the complex structure of the wastewater wasfollowed by two different methods, namely filterability and GC/MS analysis. The details of these analyses are given in thefollowing subsections.

2.8.1. Filterability tests

The intention here was to observe any decrease in theresistance of wastewater to filtration which could be likely tooccur with smaller molecules. To this purpose, time for filtrationof wastewater was followed and the filterability of raw waste-water was compared with wastewater pre-irradiated at two dosesnamely 40 and 140 kGy. The filterability test was performedfiltering 50 cm3 of wastewater sample through 0.45 mm pore sizemembrane filters at an applied vacuum pressure of 71 kN m�2.The volume of filtrate was recorded as a function of time. Reportedresults are the averages of at least two measurements.

2.8.2. Gas chromatography mass spectrometry (GC/MS) analysis

GC/MS analyses were performed using a Varian CP-3800 gaschromatograph (Varian Inc, Palo Alto, CA) equipped with a VF-5 ms (30 m x 0.25 mm id x 0.25 mm film thickness) capillarycolumn and a Varian 1200 L Quadrupole MS/MS. Helium was usedas carrier gas. The mass spectrometer was set to scan mass-to-charge ratios (m/z) in the range 50–450 using the electron impactionization mode. 20 cm3 sample is taken, and its pH is adjusted to9 with 1 mol dm�3 NaOH. Then, aqueous solution is extractedthree times with ethyl acetate. Organic phase is dried overNa2SO4. 95% of the solvent is evaporated on a rotary evaporator.Residue is taken to a vial and solvent is evaporated to drynesswith N2 gas. Sample residue in vial is dissolved in methanol,filtered and analyzed.

2.9. Analytical methods

The analytical determinations made in this study wereperformed in accordance with Standard Methods (1998). CODanalyses were performed according to U.S. EPA approved ReactorDigestion Method (HACH, 1997). High range (0–1500 mg dm�3)COD reagent vials were used. Vials were digested for 2 h at 150 1Cin Hach COD reactor model 45600-02 and the results were read

Table 2Operational conditions of the SBR experiments.

Reactor (dm3) Feed wastewater Influent CODa (mg dm�3) SRT (days) Working volume Temperature (oC)

R1 Raw 500 20 2

R2 Raw 2000 20 2 Ambientb

R3 Raw 2000 6 1.6 25

R4 40 kGy 2000 6 1.6 25

R5 Raw 5000 6 1.6 25

R6 40 kGy 5000 6 1.6 25

a Prepared by diluting the wastewater with ordinary tap water.b Summer time room temperature (20–23 oC).

C.B. Bural et al. / Radiation Physics and Chemistry 79 (2010) 519–526 521


with Hach DR/2000 direct reading spectrophotometer at 620 nmwavelength (Hach Company, Loveland, CO, USA). pH measure-ments were performed with a portable pH meter (pH 310, OaktonInstruments, Vernon Hills, IL, USA). Temperature and DO con-centration were measured by a multiparameter meter (HACHSension. 378, USA). Color measurement was performed spectro-photometrically using Hach DR/2000 equipment according toplatinum–cobalt standard method as described in Hach WaterAnalysis Handbook (HACH, 1997). During the microscopic analysisof sludge, samples collected from completely mixed reactors wereexamined with Ernst Leitz Wetzlar model microscope (Germany)using 3.5�15 magnification and Nicolet Almega XR confocalmicroscope (Thermo) using 10�15 magnification.

All the measurements were done at least twice and reportedresults are the average values.

3. Results and discussion

3.1. Aerobic biodegradability of raw alkaloid wastewater- Batch

experiments

Preliminary assays were run in batch reactors in order toevaluate aerobic biological treatability of opium alkaloid contain-ing wastewater. Various pre-designated initial concentrations ofwastewater (COD between 2500 and 26 000 mg dm�3) wereadded into reactors inoculated with unacclimated culture.Samples were taken from reactors at various time intervals andtime course variation of COD concentration was monitored.Results showed that the wastewater was not inhibitory to bacteriaeven at the highest concentrations studied and the organic carbonremoval was variable between 83% and 90% depending on theinitial COD values (data not shown). Overall, the wastewater washighly biodegradable and therefore it was suitable for aerobicbiological treatment. In support of this, a comparison of degrada-tion behaviors for reactors fed with easily biodegradable substratepeptone and alkaloid wastewater (around 4500 mg dm�3 initialCOD) showed a good fit, suggesting again that the wastewater isreadily biodegradable (Fig. 1).

3.2. Effect of irradiation on the biodegradability of raw alkaloid

wastewater

BOD5/COD ratio can be used as a parameter to evaluate thebiodegradability of wastewaters. A wastewater is consideredreadily biodegradable if its BOD5/COD ratio is between 0.4 and0.8 (Al-Momani et al., 2002; Metcalf and Eddy, Inc., 1991). On this

basis, the results of COD, BOD measurements conducted on opiumalkaloid containing wastewater (Table 3) confirm that thewastewater is readily biodegradable. In order to investigate theeffect of irradiation on biodegradability of opium alkaloidcontaining wastewater, BOD and COD measurements wereperformed also on two wastewater samples irradiated at 40 and140 kGy doses, respectively. As can be inferred from Table 3irradiation imparted no significant change in terms of COD, BOD5

and accordingly biodegradability index (BOD5/COD) at the applieddoses. Similarly, Jo et al. (2006) investigated the effect of gammairradiation on biodegradability (BOD5/COD) of pulp wastewatersand stated that the improvement of biodegradability was largelydependent on chemical properties of wastewaters and theabsorbed radiation dose. For cooking and bleaching C/Deffluents (COD values 2261 and 1368 mg dm�3 respectively),they observed that COD values changed only little after gamma-ray treatment at doses from 1 to 20 kGy and the biodegradabilitywas not improved at all. It was stated that this might be caused byformation of recalcitrant organic acids from aromatic compoundsincluded in both effluents. However, they attained abrupt increasein the biodegradability of bleaching El and final effluents at 5 kGy.Barrera-Diaz et al. (2003), who applied gamma irradiation onhighly colored (3750 Pt–Co) industrial wastewater, stated that theradiation effects can be limited due to the colloidal matter thatcontributes to the high color present in the wastewater. On theother hand, Kim et al. (2007) reported that biodegradability(BOD5/COD) of textile effluent increased by an electron beamradiation at 1.0 kGy.

As can be seen from Table 3, although at insignificant level,radiation treatment resulted in slight increase in COD concentra-tion in one case and a small decrease in other. An increase in CODis not unexpected in radiation treatment. Melo et al. (2008)attributed the COD increase after radiation treatment in theirstudy to the radiation-induced degradation that leads to theincrease of the number of low-molecular weight substrates. Thatis radiation treatment increases the amount of chemicallydegradable molecules.

It should be noted here that the opium alkaloid containingwastewater is highly polluted and therefore a large number ofdilutions was needed to reduce the concentrations to measurablelevels. This might have caused some uncertainty in results.

3.3. Treatment of raw alkaloid wastewater in sequencing batch

reactors (SBR)

3.3.1. Gradual adaptation of microorganisms to increasing influent

strength

The biological treatability of opium alkaloid containing waste-water was investigated in instantaneously fed SBRs. The aim wasto feed the reactors with gradually increasing COD concentrationsin order to acclimatize the biomass to opium alkaloid wastewater.For this purpose, the studies were started with a fairly low CODconcentration of 500 mg dm�3 (R1). Macronutrients were sup-plied at a COD:N:P ratio of 100:5:2. Excess sludge (100 cm3) waswasted daily from the mixed liquor to keep the SRT at 20 days.Table 4 shows the results obtained after 32 days of operationwhen the system was seen to reach a steady state, based on MLSS,MLVSS and COD measurements which provided almost constantresults over two consecutive days. As seen in Table 4, over 90%COD removal efficiency was achieved in the system and the sludgewas settling well as indicated by low SVI value of 107 cm3 g�1.

Following that, the influent COD concentration was increasedto 2000 mg dm�3 while keeping the operational conditions thesame as previous (R2). Upon increasing the influent COD, sludgesettlement problem was observed, and therefore steady-state

Fig. 1. Soluble COD degradation profiles for reactors fed with peptone and alkaloid

wastewater.



conditions could not be achieved. After one SRT period (20 days),worsened settling was still a problem, hence remedial actionswere taken. In one of the two identical reactors that run in parallelSRT was decreased to 10 days by doubling the daily sludgewastage. In the other reactor addition of micronutrients besidesthe macronutrients nitrogen and phosphorus was accomplished.A further reduction in SRT to 6 days in a later set of study had nopositive effect on settleability either (data not shown). Despite theefforts the problem could not be overcome. Experiment washalted as it was not possible to operate (fill and draw) the systemanymore due to sludge settlement problem. The problem was theformation of small flocs due to lack of filamentous backbones.Only few filaments existed.

3.3.2. Effect of irradiation on the complex structure of the raw

alkaloid wastewater

It was supposed that the sludge settleability problem observedwith R2 might originate from the composition of wastewateritself. Thus it was decided to alter its complex structure prior tobiological treatment. Once the chemical structure of wastewaterchanges, microbial community—the activated sludge—fed on thiswastewater may accordingly change, affecting the settleabilitycharacteristic. Therefore, gamma irradiation was considered onceagain, this time to help degrade the complex structure of alkaloidwastewater. Degradation was monitored via filterability tests andGC/MS analyses. Filterability test results revealed that the timerequired to filter the original raw wastewater was 943 s whereas itwas decreased to 73 and 28 s when the wastewater waspretreated with irradiation by 40 and 140 kGy, respectively. Thatis, irradiation proved effective in altering the complex structure ofmolecules in opium alkaloid containing wastewater.

Moreover, the breakdown of compounds via radiation treat-ment was qualitatively assessed by comparison of peak heights forsome identified alkaloid compounds (morphine, codein, papaver-ine, thebain, protopine, laudanosine and noscapine) on GC/MSchromatograms of raw and irradiated wastewaters. Visual com-parison of the overlaid chromatograms (Fig. 2) indicated asignificant decrease in all peaks after radiation treatmentproving that the compounds were degraded to a certain extentupon irradiation.

3.3.3. Effect of irradiation on SBR operation

Two SBRs were set up and seeded with mixed sludge. One ofthe reactors, R3, was fed with original wastewater and R4 withwastewater irradiated by 40 kGy. Experiment was started withinfluent strength of 2000 mg dm�3 COD for both reactors.COD:N:P was supplied at a ratio of 100:5:30. By the increasedbuffer capacity, pH was kept at 7.270.1 during the course of theexperiment. SRT was maintained at 6 days. Obtainment of almost

constant results for SVI, MLSS (variedo10%) and COD removal(variedo15%) in 4–5 consecutive measurements conducted in atleast 3 SRT periods indicated the attainment of steady state.

Steady state was achieved in a time period of 3 SRT in R3. SVIstarted to decrease after 6 days of operation and became constantat a level around 5173 cm3 g�1 by the attainment of steady-statecondition (Table 5). Starting from day 12 sludge rising problemwas observed in the settling period. Nitrogen bubbles in settledsludge were easily detectable. In order not to lose biomass,settling time was reduced from 120 to 30 min which was enoughfor the biomass to settle well. Volume exchange ratio (VER) (i.e.volume of the effluent withdrawn to total working volume of thereactor) was 75% during steady-state period. Up to 80% CODremoval efficiency was achieved with a steady-state average of 767 5% for the influent overall average COD concentration about20107255 mg dm�3.

R4 showed better settling characteristics in comparison to R3,indicated by lower SVI attained around 2272 cm3 g�1 (Table 5).Sludge was settling faster too. Over 45 min settling time, sludgerising problem was observed as in the case with R3. Therefore,time allocated for the settling period was kept at 30 min whichwas enough to separate the settled sludge from the effluent.

Table 4Results obtained with R1 (COD=500 mg dm�3; SRT=20 days).

Days Influent COD (mg dmg dm�3) % COD removal MLSS (mg dm�3) MLVSS (mg dm�3) SVI (cm3 g�1)

32 51072 9171 1865720 147574 10771

Fig. 2. Sample overlaid chromatogram showing degradation of Codein upon

irradiation.

Table 3COD, BOD5 values for raw and irradiated alkaloid wastewater.

Date of sample Wastewater COD (mg dm�3) BOD5 (mg dm�3) BOD5/COD

April 2006 Raw 40310 19500 0.48

Irradiated—40 kGy 42013 17750 0.42

Irradiated—140 kGy 40029 18000 0.45

April 2007 Raw 41640 22375 0.54

Irradiated—40 kGy 39560 20500 0.52



Because some biomass was lost at the start period of this reactordue to sludge rising, R4 was allowed to run one SRT period longerthan R3 and then steady-state condition was observed to beattained. VER was again 75%. Similar to the results obtained in R3,up to 80% COD removal was achieved with a steady-state averageof 7674% for the influent overall average COD concentration of19307135 mg dm�3.

Following the collection of data, the feed COD concentrationfor both reactors were increased to around 5000 mg dm�3 (R5and R6) and the reactors were allowed to run until systemperformances re-stabilize to a new steady state. Results obtainedare presented in Table 6.

After the increase of feed concentration to about 5000 mgdm�3 in R5, sludge rising problem continued to prevail. One-houranoxic cycle was implemented from day 6 to day 12 in order todenitrify the wastewater before aerobic oxidation. However, nopositive effect was observed, and especially between day 8 andday 14 settling period had to be kept around 20–30 min not tolose biomass due to rising. This settling time was enough for thesludge to settle well. However, SVI could not be calculatedbetween these days as it requires the volume of sludge settledin 30 min. COD removal efficiency was as high as 77% in thatperiod. Sludge rising was not observed visually at day 15, but thensludge settling relatively worsened with SVI values of 99 cm3 g�1

and sludge was bulking. Here it should be noted that, though SVIvalue (99 cm3 g�1) may indicate acceptable settling condition,this low SVI value was due to high biomass concentration retainedin the reactor in that period. The attainment of high MLSSconcentration (9500 mg dm�3) during that period might be dueto accumulated COD in reactor caused by lower volume of effluentwithdrawal due to worsened settling. Though COD:N:P ratio waskept at 100:5:30 again, it was observed that effluent pH valueswere at levels 8.570.1. This high pH was supposed to be related toworsened settling, but no further amount of buffer was added. Inorder to continue safe reactor operation, time allocated forsettling period was raised to its former duration (120 min) andkept at that value over the remaining experiment period. Thisbulking period lasted for 14 days from day 15 through day 28. VERdecreased to 50% in that period (i.e. around half of reactor volumewas being withdrawn and fed daily). No operator intervention wasmade and reactor was allowed to reach a steady state. From day26 onward SVI showed a decreasing trend with an improvementin sludge settlement characteristic. Finally, reactor was allowed torun around 9 SRT periods and steady-state data were gatheredbetween day 48 and day 52. As seen in Table 6, SVI value leveled at59 cm3 g�1 indicating good settling behavior and the average CODremoval efficiency at steady state was 79%. Microscopicalobservations of the reactor contents revealed that a moderate

number of filamentous bacteria and a high amount of protozoawere present in the reactor. The presence of filamentousorganisms in moderate amount is desirable as they providebackbone for flocs according to Sezgin et al. (1978). Protozoa(ciliates) are known to feed on the bacteria that are dispersed inthe liquid phase and crawl over the surface of the flocs, helping toclarify the effluent and to flocculate the suspended matter andbacteria (Curds et al., 1968; Curds, 1973). Flocs formed wereirregularly shaped (Fig. 3).

Moreover, GC/MS analysis was also performed on bothinfluent and effluent samples taken at steady-state period. Thetwo chromatograms, presented in Fig. 4, revealed that aremarkable alteration in wastewater composition was achievedin SBR. Signals of alkaloid compounds observable in raw influentsample partly (thebain) or totally disappeared after biologicaltreatment. This indicates that aerobic SBR was able to eliminatethe main alkaloid pollutants that are present in wastewater,except thebain. Several unidentified peaks that are observed inchromatograms consist of nonalkaloid organic molecules such asresidue from organic solvent, and some impurities originatingfrom poppy seed.

In R6, sludge rising problem continued 6 more days (1 SRTperiod) after the increase of feed COD concentration to 5000 mgdm�3. Nitrogen bubbles were easily observable. No biomass waslost as 20–30 min settling time was enough for the sludge to settlewell. In the next SRT period settleability of sludge worsened andbulking condition prevailed (days 8–12). During that period, thetime allocated for settling period increased to 120 min; VERdecreased to levels as low as 16%. As a result MLSS concentrationincreased due to COD accumulation from former cycles, as in thecase with R5. No operator intervention was made during thatperiod and the reactor was allowed to reach a steady state.

From day 13 onwards an improvement in sludge settlementwas observed indicating that the reactor was reaching a steady-state condition. In sum, reactor was allowed to run around 6 SRTperiods and the steady-state data were gathered in days 32–35.Average COD removal efficiency at steady state was 73%. Thisvalue is only slightly lower than the original-fed R5. One possiblereason for this might be the difference in bacterial communitiesgrew on original and irradiated wastewaters.

As can be seen in Table 6, SVI value leveled at 25 cm3 g�1,which indicates very good settling behavior. Visual observationrevealed a clear supernatant after SVI test. Microscopicalobservations of the reactor contents revealed a moderate numberof filamentous organisms and a high density of protozoa. Incomparison to R5, the density of protozoa (free swimmingciliates) was lower which could be due to the better flocformation, i.e. lower concentration of bacteria dispersed in the

Table 5Results obtained with R3 and R4 (COD=2000 mg dm�3; SRT=6 d).

Reactor Influent COD(mg dm�3) % COD removal MLSS (mg dm�3) MLVSS (mg dm�3) SVI (cm3 g�1)

R3—Raw 20107255 7675 46157100 3265775 5173

R4—40 kGy 19307135 7674 46557120 30207100 2272

Table 6Results obtained with R5 and R6 (COD=5000 mg dm�3; SRT=6 d).

Reactor Influent COD (mg dm�3) % COD removal MLSS (mg dm�3) MLVSS (mg dm�3) SVI (cm3 g�1)

R5—Raw 48107205 7971 80307115 5640745 5971

R6—40 kGy 47757135 7371 80007245 53007150 2571



liquid phase. More compact, larger and denser flocs were observedin this reactor (Fig. 3).

Furthermore, GC/MS analysis performed on samples takenfrom influent and effluent of the reactor showed that the peakscorresponding to alkaloid compounds were fully disappearedupon SBR treatment (Fig. 4).

As a conclusion, after modifying the operational conditionsincluding addition of phosphate buffer, it was possible toovercome sludge settlement problem, and influent COD concen-tration was raised up to 5000 mg dm�3. Effective COD removal

efficiencies were attained for both reactors and irradiation helpedimproving sludge settlement. The populations of protozoa andfilamentous organisms required for proper flocculation were ableto grow under cured conditions. As was expected, the averageMLSS concentrations increased in both SBRs after the increase ofinfluent COD to 5000 mg dm�3. The SVI values for both reactorswere very low partly because of the high concentrations of solidsmaintained in the SBRs. The SBR fed with irradiated wastewaterconsistently provided lower SVI values than the one fed withoriginal.

Fig. 3. Sample microphotographs of the sludge for (a) R5 (raw) (3.5x), (b) R6 (40 kGy) (3.5x), (c) R5 (raw) (10x) and (d) R6 (40 kGy) (10x).

Fig. 4. GC chromatogram of samples taken from influent and effluent of SBRs fed by (a) raw alkaloid wastewater and (b) radiated wastewater; 1—codaine; 2—morphine;

3—thebaine; 4—laudanosine; 5—papaverine; 6—noscapine (Influent COD=5000 mg dm�3).



4. Conclusion

In SBR, 79% and 73% COD removal efficiencies were achievedfor reactors fed with raw and 40 kGy irradiated wastewater,respectively, having an influent COD of 5000 mg dm�3. Thereactor fed with pre-irradiated wastewater consistently producedbetter settling sludge indicated by lower SVI in comparison to noradiation case. Improvement in sludge sedimentation by irradia-tion is an important achievement in biological wastewatertreatment. It is a fact that the one of the major causes of improperoperations of aerobic biological treatment processes is the sludgesettleability problem. Of course, sedimentation can be improvedusing chemicals (flocculants), but this could be the temporarymeans of control. Furthermore, using such chemicals is known toincrease the volume of sludge to be handled, which is animportant disadvantage of such means of control.

Furthermore, GC/MS analysis performed on samples takenfrom influent and effluent of SBRs fed with raw and 40 kGyirradiated wastewaters (COD=5000 mg dm�3) showed that alka-loid compounds almost fully disappeared after biological treat-ment, only thebain remained in the non-irradiated wastewater.Complete removal of thebain could be achieved only with radiatedwastewater.

As a result, it was shown that opium alkaloid containingwastewater with COD around 5000 mg dm�3, which could beattained by an anaerobic pre-treatment, can be successfullytreated by aerobic SBR in combination with gamma irradiation.Radiation treatment improves the settling characteristics andprovides more compact sludge which is considered advantageousnot only with regard to the proper operation of SBR system butalso with regard to sludge handling. To our knowledge, it is thefirst demonstration of such an effect of irradiation in aerobicbiological wastewater treatment.

Acknowledgements

The authors wish to express their gratitude to both Middle EastTechnical University Research Fund (BAP-LTP), InternationalAtomic Energy Agency (TUR/8/017) and Turkish Atomic EnergyAuthority (A3.H2.P2.03), which financially supported this work.Mr.Yuksel Mert from TAEK is also acknowledged for his assistanceduring the irradiation and GC/MS analysis.

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