Enrichment culture of marine anaerobic ammonium oxidation （anammox） bacteriaEnrichment culture of marine anaerobic ammonium

GUAN+Yong-jie

【Abstract】The present study investigates the enrichment of anaerobic ammonium oxidation （anammox） bacteria in the marine environment using sediment samples obtained from the East China Sea and discusses the nitrogen removal efficiency of marine anammox bioreactor. Enrichment of anammox bacteria with simultaneous removal of nitrite and ammonium ions was observed in the Anaerobic Sequencing Batch Reactor under a total nitrogen loading rate of 0.37kg-N m-3day-1. In this study， The nitrogen removal efficiency was up to 80% and the molar-reaction ratio of ammonium， nitrite and nitrate was 1.0：1.22：0.22 which was a little different from a previously reported ratio of 1.0：1.32：0.26 in a freshwater system.

【Key words】Anaerobic ammonium oxidation （anammox）； Marine anammox bacteria； Enrichment culture

0 Introduction

Anammox bacteria oxidize ammonium under anoxic conditions with nitrite as the electron acceptor，and conserve energy for CO2 fixation. Compared to conventional nitrification -denitrification-dependent nitrogen removal systems， the anammox process requires less oxygen and no organic material supply. Hence， the costs and CO2 emissions of nitrogen removal are reduced by up to 60% and 90%， respectively[1].Almost a decade ago， anammox reaction in marine sediments was first detected[2]. Later observations from the anoxic water column of the Black Sea also demonstrated the evidence of anammox reaction in oceanic environment[3]. Since their discoveries， marine anammox bacteria have caught much attention. It brought extremely important novelty and expansion to the anammox process， not only， for its high nitrogen removal potential under high salinity conditions， but also for contribution to direct elucidation of the marine nitrogen cycle in nature. However， the wide spread occurrence but low diversity of marine anammox bacteria was another great obstacle along with slow growth rates for the implementation of marine anammox process.Although the presence of anammox bacteria has been confirmed in anoxic seawater environments using molecular biochemical tools[3-5]，So far， very limited work has been reported in the peer-reviewed literature related to the start-up and process control of marine anammox process.The study aims to establish an enrichment culture of marine anammox bacteria obtained from the oceanic sediments and construct an anammox bioreactor for the simultaneous removal of ammonium and nitrite.

1 Materials and methods

1.1 Anammox reactor

We focused on sediment samples obtained from the East China Sea as a promising inoculum for the enrichment of marine anammox bacteria.Marine anammox bacteria were enriched by a Anaerobic Sequencing Batch Reactor （ASBR） with a working volume of 8 L that included a nonwoven fabric as a biomass carrier.The ASBR was provided with a thermostatic jacket to keep the temperature at 30℃.Complete mixture was achieved inside the reactor by means of a two-blade mechanical stirrer with a rotating speed of 100 rpm. The pH value was not controlled and ranged from 7.5 to 8.0. To obtain anaerobic condition， the reactor was deoxidized by an N gas purge for 10 min.The hydraulic retention time （HRT） was fixed at 10h and 24h.

1.2 Feeding composition

The mineral medium fed in this study presents the following composition per litre of nature sea-water：0.191-0.30g NH4Cl， 0.325-0.524 g NaNO2， 1.25 g KHCO3，0.025 g KH2PO4， 0.3 g CaCl2·2H2O， 0.2 g MgSO4·7H2O， 0.00625 g FeSO4， 0.00625 g EDTA.This medium contains ammonia in excess（molar ratio NH4+/NO2-of 1.30） to prevent the presence of nitrite in the reactor because of its strong inhibitory effect on the Anaammox activity.

1.3 Analytical methods

Ammonium was analysed by the phenol-hypochlorite method. nitrite and nitrate analysed by spectrophotometry， pH valuewas measured with a selective electrode Ingold

2 Results and Discussion

The nitrogen loading rate（NLR） fed into the reactor was based on nitrogen removal efficiency（Fig.1）. During the initial period （83 days），NLR was gradually increased from 0.185 to 0.37 kg-N m-3 day-1 with a decrease of HRT from 24 hours to 10 hours .In periods of 114 days to 133 days of duration ，the NLR was initially decreased to 0.232 kg-N m-3 day-1. In the stage （days 133-159） the NRL was maintained constant at 0.116 kg-N m-3 day-1with a increase of HRT from 10 hours to 24 hours .The NLR was then increased gradually to a finally load of 0.37 kg-N m-3 day-1 and the HRT was decreased from 24 hours to less than 10 hours.

Fig. 2 shows the performance of the reactor on the nitrogen compounds （nitrite， nitrate and ammonium） through the experimental period.More than 60 mg-N L-1 nitrite and 50 mg-N L-1 ammonium was removed in the reactor for the first 83 days and subsequently both nitrogen concentrations dropped to less than 40 mg-N L-1.The NHN removal efficiencies were obtained over 70% and 60%， respectively（Fig.3）.Since the expression of anammox activity was assumed to have occurred at this time， the hydraulic retention time（HRT） was fixed at 10h. When the nitrogen loading rate of the influent was increased to 0.37 kg-N m-3day-1， both ammonium and nitrite concentrations in the effluent abruptly increased. This result suggested that the growth rate of the marine anammox bacteria， which was assumed to be similar to that reported for freshwater anammox bacteria[6]could not match the increase in the nitrogen load.

FIG.1. Stepwise changes in total nitrogen loading rate

（concentration ratio of ammonium- and nitrite-nitrogens were 1.0：1.30）

We therefore reduced both the nitrite- and ammonium-concentrations to 50 mg L-1 and 66 mg L-1 to avoid an adverse effect by residual nitrite on anammox bacteria.There was no remarkable increase in nitrogen concentration in the effluent except for nitrate production of approximately10-20 mg-N L-1. In order to supply efficientnutrients and increase Anammox growth capacity， the NLR was increased from 0.116 to 0.232 kg-N m-3day-1 from day 133 to 159. At the same time， the HRT was remained at 24 hours.During this period， the nitrite removal efficiency remained close to100%， while ammonium removal efficiency increased to more than 90% on day 159 .As the HRT dropped from 24 h to 10 h， the NLR increased from to 0.37 kg-N m-3day-1 from day 164. Both nitrogen concentrations dropped to less than 10 mg-N L-1.The NH-N and N-N removal efficiencies were obtained over 90% and the color of the biomass attached to the nonwoven fabric also changed to red suggesting the steady enrichment of anammox bacteria.

FIG.2. Changes in concentrations of ammonium-nitrogen and nitrite-nitrogen in influent and effluent with time

Literature showed that Anammox bacteria were partially inhibited at 50 mg L-1 NO-N land totally inactivated at 100 mg L-1[7]. In this study， nitrogen load was increased unless effluent nitrite exceeded the toxic level. The activity of Anammox biomass remark increased on day 163 when the influent concentrations of ammonium and nitrite increased to 80 and 106 mg L-1（Fig. 2）. The effluent concentration was less than 5 mg L-1.Based on the satisfactory results， there was no big influence in the nitrogen removal efficiency（NRE） with the change of infuuent concentrations. Moreover， the NRE varied from 91.4% to 96.9% aside from the NLR of 0.185 kg-N m-3day to 0.37 kg-N m-3day， indicating that the marine anammox reactor potentially had good tolerance to the change of substrate concentration.

FIG.3. Changes in removal efficiency of ammonium-nitrogen

and nitrite-nitrogen with time

Anammox biomass enriched by calculation of stoichiometric molar ratios of different nitrogen involved in the reaction（Fig. 4）. The experimental values of the average stoichiometric molar ratios of nitrite to ammonium conversion and nitrate production to ammonium conversion were 1.19±0.05 and 0.20±0.02， respectively，.which was a little different from a previously reported ratio of 1.0：1.32：0.26 （10）.

The lower nitrite and nitrate levels in our study indicate a concurrent occurrence of denitrification with anammox in the reactor and thus agree with the presumption that an anammox reaction is coupled to denitrification even in the marine environment[8]. During the whole experimental period， the stoichiometric molar ratio of ammonium conversion， nitrite conversion and nitrate production was calculated to be1.0：1.22：0.22.

FIG.4. Stoichiometric molar ratios of different nitrogen involved

in the reaction

At present， there is little information about marine anammox bacterial culture， however， if such a bacterial culture is further enriched the knowledge will contribute towards elucidation of the nitrogen cycle in the marine environment. Furthermore， since these results indicate the high nitrogen removing potential of the marine anammox bacteria under condition of high salinity， an increased expansion of application of these bacteria is expected in nitrogen removal technology.

3 Conclusions

（1）Anammox bacteria can be enrichment from marine environment and a marine anammox bio reactor that can remove total nitrogen at a rate of over 0.37 kg-N m-3 day-1.

（2）The molar-reaction ratio of ammonium， nitrite and nitrate was1.0：1.22：0.22， which was a little different from a previously reported ratio of 1.0：1.32：0.26.

【References】

[1]Van Dongen， U.， Jetten， M.S.M.， Van Loosdrecht， M.C.M.， 2001.The SHARON-Anammox process for treatment of ammonium rich wastewater. Water Sci. Technol. 44： 153-160[Z].

[2]Kuypers， M. M.， Sliekers， A. O.， Lavik， G.， Schmid， M.， Jorgensen， B. B.， Kuenen， J.G.， Sinninghe Damste， J. S.， Strous， M.， and Jetten， M. S.： Anaerobic ammonium oxidation by anammox bacteria in the Black Sea， Nature， 422， 608-611 （2003）[Z].

[3]Schmid， M. C.， Maas， B.， Dapena， A.， van de Pas-Schoonen， K.， van deVossenberg， J.， Kartal， B.， van Niftrik， L.， Schmidt， I.， Cirpus， I.， Kuenen， J. G.，Wagner， M.， Sinninghe Damste， J. S.， Kuypers， M.， Revsbech， N. P.， Mendez， R.，Jetten， M. S.， and Strous， M.： Biomarkers for in situ detection of anaerobic ammonium-oxidizing（anammox） bacteria. Appl. Environ. Microbiol.， 71： 1677-1684， （2005）[Z].

[4]Tal， Y.， Watts， J. E.， and Schreier， H. J.： Anaerobic ammonium-oxidizing （anammox） bacteria and associated activity in fixed-film biofilters of a marine recirculating aquaculture system， Appl. Environ. Microbiol.， 72： 2896-2904，（2006）[Z].

[5]K. Windey， I. De Bo， W. Verstraete， Oxygen-limited autotrophic nitrification-denitrification（OLAND） in a rotating biological contactor treating high-salinity wastewater， Water Res. 39（2005）： 4512-4520[Z].

[6]J.L. Campos， A. Mosquera-Corral， M. Sanchez， M. Méndez， J.M. Lema， Nitrification in saline wastewater with high ammonia concentration in an activated sludge unit， Water Res. 36（2002）： 2555-2560[Z].

[7]Strous， M.， Van Gerven， E.， Kuenen， J. G.， and Jetten， M.： Effects of aerobic and microaerobic conditions on anaerobic ammonium-oxidizing （anammox） sludge，Appl. Environ. Microbiol.， 63， 2446-2448（1997）[Z].

[8]Dalsgaard， T.， Canfield， D. E.， Petersen， J.， Thamdrup， B.， and Acuna-Gonzalez， J.：N2 production by the anammox reaction in the anoxic water column of Golfo Dulce，Costa Rica， Nature， 422： 606-608（2003）[Z].

[責任編輯：楊玉潔]