Hongwei Sun *,Huanan Zhao Baoxia Bai,Yuying Chen Qing Yang ,Yongzhen Peng

1 School of Environmental and Municipal Engineering,Lanzhou Jiaotong University,Lanzhou 730070,China

2 Key Laboratory of Beijing Water Quality Science and Water Environment Recovery Engineering,Beijing University of Technology,Beijing 100124,China

3 Disease Prevention and Control Of fice of Urumqi Railway Bureau,Urumqi 830010,China

Keywords:Land fill leachate Nitrogen removal Denitritation Monod model Fluorescence in situ hybridization

ABSTRACT A novel system coupling an up- flow anaerobic sludge blanket(UASB)and sequencing batch reactor(SBR)was introduced to achieve advanced removal of organic and nitrogen from ammonium-rich land fill leachate.UASB could remove 88.1%of the in fluent COD at a volumetric loading rate of 6.8 kg COD·m-3·d-1.Nitritation–denitritation was responsible for removing 99.8%ofNH4+-Nand 25%oftotalnitrogen in the SBRunderalternating aerobic/anoxic modes.Simultaneous denitritation and methanogenesis in the UASB enhanced COD and TN removal,and replenished alkalinity consumed in nitritation.For the activated sludge of SBR,ammonia oxidizing bacteria were preponderant in nitrifying population,indicated by fluorescence in situ hybridization(FISH)analysis.The Monod equation is appropriate to describe the kinetic behavior of heterotrophic denitrifying bacteria,with its kinetic parameters determined from batch experiments.

1.Introduction

Wastewater usually contains high concentrations of organics and ammonia,so nitrogen land fill leachate without treatment will pollute the environment seriously[1].Cost-effective and highly efficient treatments for leachate are of great interest.

In terms of cost-effectiveness and reusability,biological methods predominate compared to other treatment methods,such as ammonium tripping,ozone oxidization and reverse osmosis[2–7].In biological processes,organic and nitrogen in the leachate can be transformed into carbon dioxide and nitrogen gas,respectively,which means realremoval of organic and nitrogen without secondary pollution.The biological process with an anaerobic–aerobic system is a feasible and sustainable technology for removing organic and nitrogen from land fill leachate[8–11].In the majority ofrecently published papers,organic and ammonium removalhigher than 90%could be achieved,but the totalnitrogen(TN)removalefficiency is not high due to the shortage of carbon source available for denitrification[12,13].

In order to improve the nitrogen removal efficiency,nitritation–denitritation theory has been proposed in recentyears,involving oxidation of ammonium to nitrite and then reduction to nitrogen gas.Compared with conventional biological processes,the nitritation–denitritation process can reduce the amount of aeration by 25%and carbon needed by 40%[14–16].

To enhance the denitritation efficiency,simultaneous denitritation and methanogenesis(SDM)was used in anaerobic reactor[17,18].SDM has become an attractive technology for improving TN removal because it leads to better economic benefit.

In this study,a novel system coupling an up- flow anaerobic sludge bed(UASB)and sequencing batch reactor(SBR)is developed for organic and nitrogen removal from leachate.The main function of the UASB is to improve COD and TN removal through SDM.The SBR is operated under aerobic/anoxic mode to achieve ideal performance for nitrogen removal via nitritation–denitritation.Furthermore,batch experiments are conducted to determine the kinetic model for heterotrophic denitritation.

2.Materials and Methods

2.1.Reactor and operation

Fig.1 shows the experimental system with an UASB and a SBR.The raw leachate sorted in the feed tank was used as the in fluent of UASB.An equalization tank was designed to meet the requirementof continuous ef fluent of UASB and intermittent in fluent of SBR,with leachate in the equalization tank utilized as the in fluent of SBR.The working volume of UASB and SBR was 3 L and 12 L,respectively,which is made of polymethyl methacrylate.For the SBR,dissolved oxygen(DO)and pH meters,mechanicalstirrer,and diffusers connected to an aircompressor were set up.Through the temperature control apparatus,the operation temperature was controlled at(30 ± 2)°C for the UASB,while the SBR reactor was operated atambienttemperature of(20.5–31.4)°C.Nitrified supernatantin the SBR wasreturned to the UASB fordenitrification with 300%of recirculation flow ratio.

Fig.1.Schematic diagram of the novel system coupling UASB and SBR.

In addition,hydraulic retention time of the UASB was 24 h.The SBR had a cycle time of 12 h,with 8 h aerobic,0.5 h settling,0.5 h SNS recycling,2 h anoxic,0.5 h settling,and 0.5 h decanting periods.The exchange volumetric rate was 50%.

2.2.Land fill leachate

Raw leachate from the Liulitun Municipal Solid Waste Sanitation Land fill Site(Beijing,China)was used as the wastewater in this experiment.The main characteristics of the leachate are shown in Table 1.

2.3.Inoculums

Granulated anaerobic sludge from a methanogenic reactor of a beer factor(Beijing,China)was inoculated in the UASB.Inoculums for SBR were aerobic activated sludge from a lab-scale oxidation ditch treating municipal wastewater,which performs nitrogen removal via nitrification–denitrification well.During this experiment,the mixed liquor suspended solid(MLSS)concentrations of UASB and SBR were approximately 55000 and 3500 mg·L-1,respectively.

2.4.Analytical methods

Chemical oxygen demand(COD),ammonium(),nitrate(),nitrite(),MLSS and volatile MLSS(MLVSS)were measured according to the standard methods[19].TN was determined with a TN/TOC analyzer(Multi N/C 3000,AnanltikjenaAG,Germany).Temperature,DO and pH were monitored using pH/Oxi 340i analyzer(WTW Company,Germany).

Fluorescence in situ hybridization(FISH)was performed as specified in Amann[20].Oligonucleotide probes used in this study were EUBmix for the detection of all bacteria,Nso1225 for ammonia-oxidizing β-Proteo bacteria,Ntspa 662 for Nitrospira,and Nit3 for Nitrobacter.The images of FISH samples were captured using an OLYMPUS-BX52 fluorescence microscope(Japan).The quantitative analysis of FISH images was performed using Leica QWIN software,where the relative abundance ofeach group was determined in triplicate as mean percentage of all bacteria.

2.5.Batch tests

Batch experiments were carried out to determine the kinetics of heterotrophic denitritation.In each test,500 ml of nitritation sludge taken from the parent SBR was transferred to batch reactor.Initial nitrite concentrations were adjusted to desired values of 5,10,20,40,60,80 and 100 mg·L-1by adding 10 mg·L-1NaNO2solution.Ethanol was added to the sludge,resulting in an initial C/N ratio in the reactor higher than 4.0.Higher C/N ratio was used to ensure that denitritation was notlimited by carbon source.The pHvalue was keptapproximately constant to 7.0 ± 0.05 through manually adding 0.5 mol·L-1HCl solution.Temperature was controlled at(27 ± 0.4)°C using a water jacket.The MLVSS concentration was controlled at(1250 ± 110)mg·L-1.The rate of nitrite reduction was determined from the measured nitrite profile using linear regression.

3.Results and Discussion

3.1.Performance of the UASB-SBR system on land fill leachate treatment

3.1.1.Organic removal

As shown in Fig.2,during the whole operation period lasting for 113 days,the COD in the raw leachate was(6830 ± 541)mg·L-1,corresponding to an average organic loading rate of(6.8 ±2.3)kg COD·m-3·d-1.A significant decrease in UASB in fluent was caused by the dilution of returned nitrified supernatant.The ef fluent CODofUASB decreased to(802±124)mg·L-1and mostofthe organic matters was removed by denitrification and methanogensis.The low in fluent biodegradable COD in the SBR made nitrification rapid and complete.The final ef fluent COD of the system was below(319±82)mg·L-1and the residual COD mainly involved refractory organic matters.The COD removal efficiency of the system was(95.2±1.2)%.The contribution of the UASB and SBR to total COD removal efficiencywas(88.1±2.3)%and(7.2±2.2)%,respectively.Therefore,the UASB plays a major role in organic removal.

Table 1 Characteristics of the leachate used in this study

Fig.2.COD removal performance of the UASB-SBR system.

Fig.3 shows the ammonium removal in this system.in the raw leachate ranged from 1748 to 2040 mg·L-1,with the average value of(2037 ± 157)mg·L-1,while ef fluentof the system was in the range of(0.1–23.8)mg·L-1,with the average value of(4.4 ± 4.2)mg·L-1,givingremoval efficiency of(99.8 ±0.1)%.The ammonium removal is excellent.According to the pathway of ammonium removal,the operation period of SBR is divided to two stages(stage I and stage II).In stage I,days 0 to 37,ammonium was oxidized to nitrate and nitrite during the aerobic periods of SBR cycles.In other words,nitrate and nitrite co-existed in the reactor.As experiment proceeded,nitrate concentration decreased gradually,while nitrite concentration increased obviously,which means that nitrite began to accumulate in this period.As depicted in Fig.3,the level of nitrite accumulation in the SBR,measured as the amount ofproduced per,reached 94.2%on day 37.This suggests that nitrite pathway is achieved in a short period.In stage II,from days 38 to 113,nitrite was the primary product of nitrification during the aerobic period,which accumulated to about(98.6 ± 11.4)mg·L-1,while the nitrate concentration was always below(4.9 ± 2.6)mg·L-1.A higher level of nitrite accumulation of(95.2±2.3)%was maintained until the end of stage II,suggesting that the nitritation performance of the SBR is good.

Fig.3.Ammonium removal performance of the UASB-SBR system.

Fig.4.Variations of nitrogen and organic in the UASB-SBR system.Raw:raw leachate；UASB-i:UASB in fluent；UASB-e:UASB ef fluent；SBR-Ni:SBR nitritation in fluent；SBR-Ne:SBR nitritation ef fluent；SBR-DNi:SBR denitritation in fluent；SBR-DNe:SBR denitritation ef fluent.

3.2.SDM in the UASB and nitritation–denitritation in the SBR

In order to demonstrate the conversions of nitrogen and organic in the system,typical variations of TN,,,and COD during complete nitritation on day 102 are depicted in Fig.4.Diluted by the returned nitrified supernatant,the in fluent concentrations of TN,and COD in the UASB decreased sharply,while in fluentconcentration increased obviously.In the UASB,the organic matters were super fluous as carbon source for denitritation of returned nitrified supernatant.in the returned nitrified supernatant was about 99.5 mg·L-1,while the ef fluentwas less than 0.5 mg·L-1.Therefore,simultaneous denitritation and methanogenesis appeared in the UASB.Because of further dilution by the remaining sludge,the initial TN,and COD concentrations in the SBR were 158.6,129.8 and 865 mg·L-1,respectively.In the aerobic period,more than 99%of ammonium was oxidized to nitrite,while about 34 mg·L-1TN was removed,likely caused by simultaneous nitritation and denitritation.In the following anoxic period,nitrite was reduced to N2,decreasing TNand COD concentrations simultaneously.The ef fluent TN,NH4+-N and COD values of the system were 18.6,0.2 and 451.4 mg·L-1,respectively,with corresponding removal efficiency of 99.2%,99.9%and 93.3%.This demonstrates that advanced removal of organic and nitrogen is achieved in the system.

3.3.Replenishment of alkalinity consumed in nitritation by denitritation in the UASB and SBR

It is well-known that 7.14 g alkalinity was consumed per gram of ammonium oxidized in nitritation reaction,while one equivalent of alkalinity is produced per equivalent of nitrite reduced,which equals 3.57 g of alkalinity production per gram of nitrite reduced in denitritation,so that by denitritation about one-half of the amount destroyed by nitritation can be recovered[21].

Fig.5 indicates the variations of pHand alkalinity in the same period asthatin Fig.4.With the returned nitrified supernatant,in fluentpHand alkalinity of the UASB decreased compared with those in the raw leachate.The denitritation of nitrite increased both pH and alkalinity in the UASB.The ratio between alkalinity produced and nitrite reduced was 3.32 g CaCO3·()-1,which is slightly lower than the theoretical value of3.57 g CaCO3·()-1.During the nitritation,the decrease in pHand alkalinity was caused by alkalinity reduction and acid production.At the end of nitritation,pH decreased to the lowest point of 7.8,which is known as “ammonia valley”,and alkalinity also decreased to 490.8 mg·L-1.After nitritation completion,the pH value and alkalinity continuously increased with denitritation due to alkalinity production.Moreover,the ratio of alkalinity produced in denitritation to alkalinity consumed in nitritation was calculated as 0.44 g CaCO3·(g)-1,which is lower than the theoretical value of 0.5 g CaCO3·(g)-1.Thus,alkalinity destroyed by nitritation in the SBR can be recovered effectively by denitritation in the UASB and the anoxic phase of SBR.

Fig.5.Variations of pH and alkalinity in UASB-SBR system.

3.4.Denitrifying activity of heterotrophic bacteria

ThesludgesamplesfromSBRreactorwereanalyzedbyFISHinorder to examinethechange ofbacteriumcommunity,especially,the population of ammonia oxidizing bacteria(AOB)and Nitrobacter(NOB).FISH analysis(Fig.6)shows that AOB accounts for 4.5%of eubacterium,while NOB accounts for less than 0.1%.Nitrospira is not detected,and the remaining 95.4%is considered as heterotrophic bacteria.This result strongly suggests that AOB is thepreponderantnitrifyingbacteria inthe sludge.

It should be noted that heterotrophic bacteria were dominant in the sludge system according to the FISH analysis.As a result,batch tests were carried out to determine the denitrifying activity of heterotrophic bacteria at different nitrite concentrations.Fig.7 shows the nitrite concentration profile with time.Under pH 7.0 and initial nitrite concentration of 60 mg·L-1,nitrite concentration decreased gradually,implying that denitritation occurred.The rate of this process was determined as 0.29 g N·(g VSS)-1·d-1through linear regression.The denitritation rates in other batch experiments were determined in a similar way.

Fig.7.NitriteconcentrationprofilemeasuredunderpH7.0andinitialnitriteconcentration of 60 mg·L-1.

Fig.8.Effect of nitrite concentration on denitrifying activity of heterotrophic bacteria in the SBR.

Fig.8 shows that nitrite concentration has significant effect on the denitrifying activity of heterotrophic bacteria.The denitrifying activity increases sharply with nitrite concentration in the low concentration range of 0–40 mg·L-1,but the effect of concentration is less at nitrite concentrations greater than 60 mg·L-1.The data suggest that the Monod kinetic model may be applicable[22]:

Fig.6.FISH results for AOB and NOB in the SBR reactor on day 102.(a)EUBmix target for all bacteria；(b)NSO1225 target for AOB；(c)NIT3 target for Nitrobacter,Ntspa662 target for Nitrospira.

where r and rmaxare the denitritation rate and its maximum value,respectively,g N·(g VSS)-1·d-1；S is the nitrite concentration,mg·L-1；and Ksis the half-saturation constant,mg·L-1.The equation is expressed as r=-0.043exp(-S/25.5)+0.459.The value of Ksis 15.8 mg·L-1and rmaxis 0.435 g N·(g VSS)-1·d-1for the heterotrophic bacteria.

4.Conclusions

The main conclusions of this study are as follows.

1.UASB-SBR system is suitable for advanced treatment of ammoniumrich land fill leachate,achieving COD,TN andremoval ef ficiency higher than 95.2%,99.2%and 99.8%,respectively,with the effluent COD,TN andless than 450,0.5 and 20 mg·L-1,respectively.

2.Simultaneous denitritation and methanogenesis could be achieved in the UASB,which enhances COD and TN removal and replenishes alkalinity consumed during nitritation period of SBR.Nitritation–denitritation could be achieved in the SBR,improving nitrogen removal from leachate.

3.The effect of nitrite concentration on the denitrifying activity of heterotrophic bacteria is described by the Monod model.The kinetic parameter values ofthe half-saturation constantand maximum specific denitritation rate are 15.8 mg·L-1and 0.435 g N·(g VSS)-1·d-1,respectively.