Elsevier

Chemosphere

Volume 217, February 2019, Pages 609-617
Chemosphere

Changes of nitrogen-removal performance and that of the bacterial community in a mixed culture comprising freshwater and marine anammox bacteria under averaged environmental condition

https://doi.org/10.1016/j.chemosphere.2018.11.047Get rights and content

Highlights

  • Marine- and freshwater-anammox bacteria (MAB & FAB) can be cultured together.

  • Mixed culture with MAB and FAB shows satisfactory nitrogen removal performance.

  • The 15 g/L NaCl and 27.5 °C condition is more favorable to MAB than FAB.

  • Anammox bacterial community transition responding to an environmental change.

Abstract

Nitrogen-removal processes using anammox bacteria are expected to achieve high-rate removal while remaining economical, and their practical applications have been investigated. However, anammox bacteria still have unfavorable characteristics for practical use, including susceptibility to a change in environmental conditions. In this study, with an aim of exploring the adaptability of mixed anammox bacteria to environmental conditions, the shift of nitrogen-removal performance and bacterial community in a mixed culture comprising freshwater anammox bacteria (FAB) and marine anammox bacteria (MAB) were investigated by a continuously stirred tank reactor (CSTR). The CSTR inoculated with the mixed anammox bacteria was operated for 180 days under an averaged condition between freshwater and marine conditions with a temperature of 27.5 °C and a synthetic medium with 15 g/L NaCl was used. Nitrogen-removal performance became stable after 114 days and more than 90% of nitrogen that was loaded into the reactor was removed in the range of nitrogen loading rate 0.07–0.42 kg N/m3/d. After operating at 0.42 kg N/m3/d for one month, a biomass sample was taken and its bacterial community was analyzed by clone-library analysis using a partial sequence of 16S rRNA. Among the clones of anammox bacteria that were made by an anammox-bacteria-specific primer, 97% of them were MAB and only 3% were FAB. These results indicate that the bacterial community including anammox bacteria was evidently changed due to environmental conditions and that the averaged condition in this study was suitable for marine bacteria rather than freshwater bacteria.

Introduction

Anammox is a nitrogen-metabolic pathway that was first reported in 1995 (Mulder et al., 1995). In the anammox reaction, ammonium (NH4+) is oxidized by nitrite (NO2), and a small amount of nitrate (NO3) is produced under anoxic conditions. Strous et al. proposed the following Eq. (1) as a typical stoichiometric formula for the anammox reaction obtained in sequencing batch reactors (Strous et al., 1998):NH4++1.32NO2+0.066HCO3++0.13H+1.02N2+0.26NO3+0.066CH2O0.5N0.15+2.03H2O

The anammox reaction is catalyzed by anammox bacteria, which were discovered in 1997 (Strous et al., 1997). Anammox bacteria are anaerobic and autotrophic bacteria that derive energy through the redox reaction with NH4+ and NO2 without an organic-carbon source (Strous et al., 1998). So far, anammox bacteria have been broadly divided into five candidate genera: “Candidatus Brocadia,” “Candidatus Kuenenia,” “Candidatus Jettenid,” “Candidatus Scalindua,” and “Candidatus Anammoxoglobus” (van Niftrik and Jetten, 2012). The species in the genus of “Candidatus Scalindua” have been discovered in marine environments and exhibit a high tolerance for salt (Awata et al., 2012). In addition, the optimal temperatures for the growth of the species within “Candidatus Scalindua” were reported to be typically lower (around 25 °C) than those for the species in other anammox genera (over 30 °C) (Awata et al., 2012; Kawagoshi et al., 2012).

In recent years, numerous studies have been conducted on the application of anammox for nitrogen removal from nitrogen-rich wastewater (van der Star et al., 2007) because of great advantages such as economical and fast nitrogen removal (Jetten et al., 2005). However, anammox processes still have unfavorable characteristics that must be solved prior to practical application. For example, anammox bacteria exhibit quite low growth rates (doubling time is over 10 days) (Strous et al., 1998) and widespread inhibitory factors for their activities have been revealed (Fernandez et al., 2012; Dapena-Mora et al., 2010; Dosta et al., 2008; Guven et al., 2005). Additionally, anammox bacteria are susceptible to damage by rapid changes of environmental conditions, and their recovery from such damage is sometimes difficult for the bacteria (van Niftrik and Jetten, 2012). Such a weakness of anammox bacteria may be improved via adaptation to environmental change (Fernandez et al., 2008; Collins et al., 2005; Toh and Ashbolt, 2002). Although the mechanism of adaptation is not well understood, it is possible that a shift of bacterial flora can contribute to the adaptation process. Kartal et al. successfully acclimatized freshwater-anammox cultures to a high-salinity condition (salt concentration: 30 g/L) and reported that the abundance ratio of halotolerant anammox bacteria and another anammox bacteria varied according to salinity change (Kartal et al., 2006). These results indicate a possibility that different anammox bacteria could live in a reactor and their population may vary according to changes in environmental conditions. On the other hand, Liu et al. reported that freshwater anammox bacteria can live in a high-salinity medium (30 g/L salt) by adapting to the high-salinity condition and can reasonably perform nitrogen removal; however, its nitrogen removal performance abruptly declined at a salt concentration of >30 g/L (Liu et al., 2009).

In consideration of these previous studies and findings, it is expected that a mixed anammox culture comprising different kinds of anammox bacteria could achieve stable nitrogen-removal performance by shifting the dominant bacterial species according to the change in the environmental condition. Therefore, we conceived the construction of a mixed anammox culture by combining marine-anammox bacteria (MAB) and freshwater-anammox bacteria (FAB) to establish the robust anammox culture that can appropriately respond to the changes in salinity and temperature to maintain stable nitrogen-removal performance. Furthermore, changes in the bacterial community of the mixed anammox culture with the environmental condition is very noteworthy information. In this study, we constructed the mixed anammox culture in a continuously stirred tank reactor (CSTR), which can rapidly change the bacterial population in the reactor and revealed its nitrogen-removal performance and the population shift in the bacterial community under the averaged environmental conditions between the culture conditions required for MAB and FAB.

Section snippets

Inoculation of biomass

In our laboratory, biomasses of freshwater anammox bacteria (FAB) and marine anammox bacteria (MAB) were maintained in packed-bed-type reactors with a nonwoven fabric as the biomass carrier (Kawagoshi et al., 2009). The reactors were both operated at a nitrogen-loading rate (NLR) of 0.3 kg N/m3/d for more than six months and showed a stable nitrogen-removal performance: more than 85% of the nitrogen in the influent (medium) had been converted to nitrogen gas. Next, 2.0 g (wet weight) of anammox

First period (0–22 d)

Fig. 2(a–d) show the time course of the setting values of operation condition and measurement results throughout the experimental period: pH values of the medium and effluent are shown in Fig. 2(a); the molar ratios of consumed NO2 to consumed NH4+ (NO2/NH4+) and produced NO3 to consumed NH4+ (NO3/NH4+) are shown in Fig. 2(b); HRT, NLR, NRR, and NRE are shown in Fig. 2(c); the total nitrogen (TN) concentration in the medium and the nitrogen concentrations of NH4+, NO2, and NO3 in the

Nitrogen-removal performance in the mixed culture with suspended marine- and freshwater-anammox biomass under averaged condition

In this study, startup of mixed anammox-bacterial culture inoculated with both the freshwater and marine-anammox biomasses was conducted under an averaged cultivation condition, and the change of bacterial composition was investigated during the culture period. In our previous studies on the startup of anammox cultures (Kawagoshi et al., 2009, 2010), we used a packed-bed reactor with a nonwoven fabric as the biomass carrier and promoted the biomass to make biofilm to reduce the amount of

Acknowledgments

This study was financially supported by Grants-in-Aid for Scientific Research (No. 23560647) from the Japan Society for the Promotion of Science (JSPS), Tokyo.

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