The starvation tolerance of anammox bacteria culture at 35°C

doi:10.1016/j.jbiosc.2015.02.016

Journal of Bioscience and Bioengineering

Volume 120, Issue 4, October 2015, Pages 450-455

https://doi.org/10.1016/j.jbiosc.2015.02.016 Get rights and content

Anammox is an environmental-friendly and cost-effective technology for nitrogen removal. This study provides the nitrogen removal profiles, physiological traits of anammox bacteria culture under the substrate deficiency conditions at the optimal cultivation temperature 35°C. The determined period of starvation tolerance was 4 weeks in the absence of nitrite, 5 weeks in the absence of ammonium, as well as 7 weeks for the absence of these two substrates at 36°C, pH 7–8 and anaerobic conditions. The physiological traits of bacteria consortium were identified through flow cytometry (FCM) analysis, and the ordinal change of increased RNA synthesizing amounts, phosphatidylserine exposure and bacteria death occurred under starvation stress. In addition, the starvation induced the increased protein content in extracellular polymeric substances and the poorer bacteria settling capacity. This study helps to develop a better understanding of anammox process in engineering environment.

Section snippets

Batch test

Four 200 ml vials were applied and each vial was inoculated with 0.16 g VSS anammox bacteria culture. The anammox bacteria Candidatus Brocadia fulgida percentage was nearly 90% by fluorescent in situ hybridization (FISH) analysis (19). All of the vials were fed with synthetic wastewater prepared with tap water including: NaHCO₃ (1.25 g l⁻¹), KH₂PO₄ (0.025 g l⁻¹), CaCl₂ (0.01 g l⁻¹), MgSO₄·7H₂O (0.2 g l⁻¹), FeSO₄·7H₂O (0.01 g l⁻¹), EDTA·2Na (0.006 g l⁻¹). Temperature and pH were controlled at

The profiles of anammox bacteria culture under deficiency conditions of different substrates

The differences in starvation stress under substrate deficiency conditions of different substrates caused different color changes of the culture. The culture color in vial A was dark yellow at the beginning. Afterwards, it changed to pale yellow and then to gray at the end of the starvation stage. Besides, there was also a clear color change of the culture in vials B and C from dark yellow to pale yellow when the tested period was over. This phenomenon was consistent with the previous report

Discussion

The results here illustrated that the decay of anammox bacteria culture was different under different starvation stress. The deficiency of different substrates directly resulted in different starvation tolerance of anammox bacteria culture. The most serious inhibition occurred at presence of sole substrate ammonium, followed by that with nitrite and then no substrate. In fact, both of the substrates ammonium and nitrite at high concentrations have inhibition effects on anammox culture 16, 26.

Acknowledgments

The authors are grateful to the National Natural Science Foundation of China (No. 21261140336 and No. 51308007) for financial support.

References (30)

L.W. Jaroszynski et al.
Impact of free ammonia on anammox rates (anoxic ammonium oxidation) in a moving bed biofilm reactor
Chemophere
(2012)
R.C. Jin et al.
The inhibition of the Anammox process: a review
Chem. Eng. J.
(2012)
W.R.L. van der Star et al.
Startup of reactors for anoxic ammonium oxidation: experiences
Water Res.
(2007)
J. Desloover et al.
Floc-based sequential partial nitritation and anammox at full scale with contrasting N₂O emissions
Water Res.
(2011)
S. Lackner et al.
Full-scale partial nitritation/anammox experiences – an application survey
Water Res.
(2014)
X. Zhang et al.
Biodegradability of biofilm extracellular polymeric substances
Chemosphere
(2003)
C. Tang et al.
Influence of substrates on nitrogen removal performance and microbiology of anaerobic ammonium oxidation by operating two UASB reactors fed with different substrate levels
J. Hazard. Mat.
(2010)
J.A. Torà et al.
Long term starvation and subsequent reactivation of a high rate partial nitrification activated sludge pilot plant
Bioresour. Technol.
(2011)
B. Frolund et al.
Extraction of extracellular polymers from activated sludge using a cation exchange resin
Water Res.
(1996)
B.J. Ni et al.
Evaluation on the microbial interactions of anaerobic ammonium oxidizers and heterotrophs in anammox biofilm
Water Res.
(2012)

N.C.G. Tan et al.

Physiological and phylogenetic study of an ammonium-oxidizing culture at high nitrite concentrations

Syst. Appl. Microbiol.

(2008)

J.M. Carvajal-Arroyo et al.

Inhibition of anaerobic ammonium oxidizing (anammox) enrichment by substrates, metabolites and common wastewater constituents

Chemosphere

(2013)

A. Franco et al.

Granulation in high-load denitrifying upflow sludge bed pulsed reactors

Water Res.

(2006)

J.G. Kuenen

Anammox bacteria: from discovery to application

Nat. Rev. Microbiol.

(2008)

W.R. Abma et al.

Upgrading of sewage treatment plant by sustainable and cost-effective separate treatment of industrial wastewater

Water Sci. Technol.

(2010)

Cited by (19)

Molecular analysis of microbial nitrogen transformation and removal potential in mangrove wetlands under anthropogenic nitrogen input
2021, Science of the Total Environment
Mangrove ecosystems are natural nitrogen removal systems that are primarily mediated by nitrogen cycle microorganisms, but their relative contributions to nitrogen transformation and removal in mangrove sediments under anthropogenic nitrogen input needs further resolution and characterization. Here, we investigated the responses and the relative contributions of ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), anaerobic ammonium oxidizing (anammox) bacteria and denitrifying bacteria after spiking urea into mangrove sediments incubated in a laboratory microcosm experiment for four weeks. During incubation, the diversity, abundances and transcription levels of the hzo genes for anammox bacteria, amoA genes for AOA and AOB, and nirS genes for denitrifying bacteria were monitored using targeted gene clone library analyses and quantitative PCR assays at the DNA and RNA levels. The results showed that mangrove sediments harbour habitat-specific anammox bacteria which related to Candidatus Scalindua and Candidatus Kuenenia clades. Mangrove specific AOA related to deep branched clades within Candidatus Nitrososphaera and Candidatus Nitrosotalea, and AOB related to Nitrosomonas and Nitrosospira were also detected in the collected sediment samples. Growth and activity of AOA were detected at all levels of amendment of nitrogen input, whereas AOB growth was detectable only at the high-level nitrogen input (1.5 mg urea per gram of dry sediment) with no amoA transcripts and lower abundance than AOA. The abundance and transcription levels of the nirS gene were higher (~1000 times) than those of the hzo gene in all groups. Pearson correlation analysis demonstrated that the abundance of both AOA and AOB amoA genes had a significant positive correlation with the nirS gene (p < 0.01). These results indicated that nitrification (primarily mediated by the AOA)-denitrification process played the most important role in nitrogen removal from the amendment of nitrogen short-term input in the mangrove sediments.
Response of FANIR system to starvation stress: “Dormancy”
2020, Water Research
Citation Excerpt :
However, the substrate starvation could give rise to the disintegration of AnGS and the washout of AnAOB, leading to the further decline of NRR (Fig. 2). This was consistent with the results reported in previous literatures (Wu et al., 2015; Li et al., 2019). AnAOB are the functional source of FANIR system.
Anaerobic ammonium oxidation (Anammox) process has been successfully applied in the nitrogen removal from high-strength wastewaters. However, little information is available for its treatment of low-strength wastewaters. In this study, a Famine Anammox NItrogen Removal (FANIR) system was developed to investigate the effect of long-term substrate starvation at the low nitrogen concentration (the influent total nitrogen was set at ∼1 mg/L). The results showed that the response of FANIR system to the starvation stress took on two phases: the functional decline phase (0–54 day) and the functional stabilization phase (62–116 day). Over the two phases, the Nitrogen Removal Rate (NRR) of anammox reactor firstly dropped sharply; and then came to a constant level. The activity and settleability of Anammox Granular Sludge (AnGS) firstly deteriorated seriously, and then stayed in a stable range. The relative abundance of Anaerobic Ammonium Oxidation Bacteria (AnAOB) firstly decreased markedly, and then approached a steady state with the change of dominant genus from Candidatus Brocadia to Candidatus Kuenenia. The abundance of 16S rRNA gene and hzs gene of AnAOB and their transcription level firstly declined largely as well, and then became stable with the 16S rRNA gene, hzs gene, 16S rRNA and hzs-mRNA of AnAOB at 23.9%, 9.1%, 1.2% and 1.0% of the initial value, respectively. To our delight, the behavior of FANIR system in the functional stabilization phase was proved indeed consistent with the feature for AnAOB to enter the dormancy state. These findings are helpful to understand the physiology of AnAOB over the starvation stress and to promote the extension of anammox process to the treatment of low-strength wastewaters.
Efficient nitrogen removal in separate coupled-system of anammox and sulfur autotrophic denitrification with a nitrification side-branch under substrate fluctuation
2019, Science of the Total Environment
In order to achieve efficient nitrogen removal, a separate coupled-system of anaerobic ammonia oxidation (anammox) and sulfur autotrophic denitrification (S⁰-SADN) was established. In this study, the operational feasibility and stability of the coupled-system under substrate fluctuations were investigated. Results showed that the coupled-system improved the total nitrogen removal efficiency (TNRE) to 99.15 ± 0.68%. The tryptophan-like substances in anammox effluent positively impacted the growth of the S⁰-SADN biofilm. This positive cooperativity boosted the S⁰-SADN to achieve rapid 12-day startup and stable operation thereafter. The TNRE was determined at 95.27 ± 1.51% and 93.44 ± 0.96% under excessive nitrite and ammonium, respectively. The coupled-system recovered quickly after 21 days of starvation deterioration. To further treat the excessive ammonium, the nitrification side-branch of the coupled-system improved the TNRE to 99.08 ± 0.68%. Extracellular polymeric substances analysis revealed that the anammox and S⁰-SADN bacteria secreted protein-like substances to resist substrate fluctuation. Microbial community analysis indicated that the stability of bacterial community supported the stability of the coupled-system. These results collectively suggested that the separate coupled-system exhibited excellent performance and provided a platform for practical wastewater treatment in future.
Flow cytometry applications in water treatment, distribution, and reuse: A review
2019, Water Research
Citation Excerpt :
Wu et al. (2015) further stained with pyronin Y to quantify the presence of synthesizing RNA as an indicator of metabolic activity. In the second category (specific compounds), Liu et al. (2013d) used the same staining approach (annexin V + allophycocyanin) as Wu et al. (2015) with FCM to demonstrate that adding Ca2+ had a significant positive effect on restoring a damaged anammox consortium. Foladori et al. (2014) compared FCM to other approaches for investigating the physiological status of bacteria after toxicant addition.
Ensuring safe and effective water treatment, distribution, and reuse requires robust methods for characterizing and monitoring waterborne microbes. Methods widely used today can be limited by low sensitivity, high labor and time requirements, susceptibility to interference from inhibitory compounds, and difficulties in distinguishing between viable and non-viable cells. Flow cytometry (FCM) has recently gained attention as an alternative approach that can overcome many of these challenges. This article critically and systematically reviews for the first time recent literature on applications of FCM in water treatment, distribution, and reuse. In the review, we identify and examine nearly 300 studies published from 2000 to 2018 that illustrate the benefits and challenges of using FCM for assessing source-water quality and impacts of treatment-plant discharge on receiving waters, wastewater treatment, drinking water treatment, and drinking water distribution. We then discuss options for combining FCM with other indicators of water quality and address several topics that cut across nearly all applications reviewed. Finally, we identify priority areas in which more work is needed to realize the full potential of this approach. These include optimizing protocols for FCM-based analysis of waterborne viruses, optimizing protocols for specifically detecting target pathogens, automating sample handling and preparation to enable real-time FCM, developing computational tools to assist data analysis, and improving standards for instrumentation, methods, and reporting requirements. We conclude that while more work is needed to realize the full potential of FCM in water treatment, distribution, and reuse, substantial progress has been made over the past two decades. There is now a sufficiently large body of research documenting successful applications of FCM that the approach could reasonably and realistically see widespread adoption as a routine method for water quality assessment.
The potential of using biological nitrogen removal technique for stormwater treatment
2017, Ecological Engineering
Citation Excerpt :
Accumulated evidence shows that BNR communities are rather resilient to periodic starvation. Studies by Reeve et al. (2016) and Wu et al. (2015) show that the AMX communities were able to quickly resume to normal activity within one day after about two-month starvation. AOB activity decreased slowly during starvation up to 10 days.
Biological nitrogen removal (BNR) may provide permanent elimination of nitrogen pollutants by conversion to nitrogen gas. However, few studies have explored the potential of BNR for the removal of nitrogen from stormwater even though this technique has been broadly applied for wastewater purification. Urban runoff is characterized by its low strength and high dissolved oxygen content, which poses multiple challenges to effective BNR. In this study, mathematical modeling indicated that for most runoff input concentrations, complete nitrogen removal within a 0.5-day hydraulic retention time could be achieved if a critical organic carbon concentration is provided in the influent of steady-state bioretention systems. An appropriate amount of organic carbon is required to simultaneously create spatial aerobic and anoxic conditions for successful stormwater BNR in the layered structures of biofilms that are formed on bioretention media and inhabited with syntrophic BNR communities. Because organic carbon is normally limiting in stormwater denitrification processes, anammox becomes an especially important pathway for stormwater BNR. An analysis of influent runoff concentrations from the National Stormwater Quality Database suggested that 71.1% runoff could have nitrogen pollutant removed via partial nitritation followed by anammox. The adequacy of dissolved oxygen, ammonia and alkalinity in stormwater for successful BNR was also evaluated. It was concluded that adjusting influent organic content to a critical value determined in this study should suffice the stormwater BNR requirement of steady-state bioretention systems. Bioaugmentation is required to expedite the bioretention system startup.
Temperature dependence of nitrogen removal activity by anammox bacteria enriched at low temperatures
2017, Journal of Bioscience and Bioengineering
Citation Excerpt :
These results suggest that the anammox bacteria once enriched at low temperature offer the practical benefit of sustaining their activity robustly and of regaining their activity rapidly, even after idling operations of the reactors. The bacterial community in anammox reactors might be more tolerant to low NLRs at lower temperatures (33). The sludge samples of Low-R1 and Low-R2 demonstrated higher SAAs than that in Mod-R at 10–30°C.
The anaerobic ammonium oxidation (anammox) process, which is applicable at ambient temperature, is necessary to develop more versatile nitrogen removal technologies. In this study, two anammox reactors, Low-R1 and Low-R2 inoculated with activated sludge respectively in Kumamoto and Hokkaido, Japan, achieved nitrogen removal rates (NRRs) of 1.5 kg-N/m³/day at 20°C. The specific anammox activity (SAA) of the Low-R1 and Low-R2 sludge samples had peaks, respectively, of 2.8 ± 0.3 mg-N/g-VSS/h at 25°C and 4.2 ± 0.3 mg-N/g-VSS/h at 30°C and dropped over the optimum temperature. Moreover, the SAA values of the Low-R1 and Low-R2 were higher at 10–25°C and 10–35°C, respectively, than that of an anammox reactor inoculated with activated sludge in Kumamoto operated at 35°C (Mod-R). The apparent activation energy for anammox of Low-R1, Low-R2, and Mod-R were 108 kJ/mol (10–25°C), 73 kJ/mol (10–30°C), and 89 kJ/mol (10–35°C), respectively. Candidatus Kuenenia stuttgartiensis dominated in the Mod-R sludge. The Low-R1 sludge was comprised of Ca. K. stuttgartiensis, Ca. Brocadia caroliniensis and Ca. B. fulgida and uncultured anammox-like or planctomycete-like bacteria. The Low-R2 sludge was comprised of various uncultured anammox-like or planctomycete-like bacteria. As Low-R2 was constructed, enrichment of freshwater anammox bacteria at low temperature with seed sludge collected from cold regions is expected to be an effective strategy for anammox applications under a wide temperature range.

View all citing articles on Scopus

View full text

The starvation tolerance of anammox bacteria culture at 35°C

Section snippets

Batch test

The profiles of anammox bacteria culture under deficiency conditions of different substrates

Discussion

Acknowledgments

Chemophere

Chem. Eng. J.

Water Res.

Water Res.

Water Res.

Chemosphere

J. Hazard. Mat.

Bioresour. Technol.

Water Res.

Water Res.

Syst. Appl. Microbiol.

Chemosphere

Water Res.

Anammox bacteria: from discovery to application

Nat. Rev. Microbiol.

Upgrading of sewage treatment plant by sustainable and cost-effective separate treatment of industrial wastewater

Water Sci. Technol.