Ecological niche differentiation among anammox bacteria

Journal Pre-proof Ecological niche differentiation among anammox bacteria Lei Zhang, Satoshi Okabe PII:

S0043-1354(20)30004-X

DOI:

https://doi.org/10.1016/j.watres.2020.115468

Reference:

WR 115468

To appear in:

Water Research

Received Date: 22 September 2019 Revised Date:

3 December 2019

Accepted Date: 2 January 2020

Please cite this article as: Zhang, L., Okabe, S., Ecological niche differentiation among anammox bacteria, Water Research (2020), doi: https://doi.org/10.1016/j.watres.2020.115468. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Enrichment and possible pure culturing of anammox bacteria Initial enrichment

>99.8% pure culture

ca. 90% pure culture

pure culture ??

Percoll density gradient centrifugation

Up-flow column biofilm reactor

MBR culture

Limiting dilution with Antibiotics AHL

further enrichment of anammox bacteria

What shapes community compositions of anammox bacteria ? Direct microbial competition study

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MINIREVIEW for submission to Water Research

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Revised manuscript WR52109R1

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Ecological niche differentiation among anammox bacteria

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By

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Lei Zhanga, 1 and Satoshi Okabea,*

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Category: Review paper

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a

Division of Environmental Engineering, Faculty of Engineering, Hokkaido University,

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North 13, West 8, Sapporo, Hokkaido 060-8628, Japan.

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1

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Jiangsu Province, China.

Current address: School of Environmental and Civil Engineering, Jiangnan University, Wuxi,

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*

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E-mail: [email protected].

Corresponding author: Satoshi Okabe

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Abstract Anaerobic ammonium oxidizing (anammox) bacteria can directly convert

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ammonium and nitrite to nitrogen gas anaerobically and were responsible for a substantial

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part of the fixed nitrogen loss and re-oxidation of nitrite to nitrate in freshwater and marine

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ecosystems. Although a wide variety of studies have been undertaken to investigate the

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abundance and biodiversity of anammox bacteria so far, ecological niche differentiation of

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anammox bacteria is still not fully understood. To assess their growth behavior and

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consequent population dynamics at a given environment, the Monod model is often used.

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Here, we summarize the Monod kinetic parameters such as the maximum specific growth rate

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(µmax) and the half-saturation constant for nitrite (KNO2-) and ammonium (KNH4+) of five

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known candidatus genera of anammox bacteria. We also discuss potential pivotal

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environmental factors and metabolic flexibility that influence the community compositions of

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anammox bacteria. Particularly biodiversity of the genus “Scalindua” might have been

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largely underestimated. Several anammox bacteria have been successfully enriched from

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various source of biomass. We reevaluate their enrichment methods and culture medium

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compositions to gain a clue of niche differentiation of anammox bacteria. Furthermore, we

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formulate the current issues that must be addressed. Overall this review re-emphasizes the

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importance of enrichment cultures (preferably pure cultures), physiological characterization

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and direct microbial competition studies using enrichment cultures in laboratories.

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Keywords

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Anammox bacteria; niche differentiation; growth kinetics; enrichment cultures; microbial

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competition.

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Introduction Anammox bacteria can catalyze the oxidation of ammonium with nitrite as the

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electron acceptor under anoxic conditions (van de Graaf et al., 1996). So far 19 species under

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6 candidatus genera (Brocadia, Kuenenia, Jettenia, Scalindua, Anammoxoglobus and

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Anammoximicrobium) have been reported from various natural and man-made ecosystems

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(Oshiki et al., 2016; Khramenkov et al., 2013).

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Anammox process has received considerable attention as a cost-effective and

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environment-friendly nitrogen removal process from wastewater (Ali and Okabe, 2015;

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Kartal et al., 2010). More importantly, anammox bacteria have been detected ubiquitously in

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anoxic environments and found to be responsible for 24 - 67% of N loss in marine sediments

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(Thamdrup and Dalsgaard, 2002) and 20 - 40% in the suboxic water columns of the Black

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Sea and Gulfo Dulce (Dalsgaard et al., 2003; Kuypers et al., 2003). Recent studies

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demonstrated even greater percentages (up to 100%) of marine N loss. These experimental

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evidences reveal that anammox bacteria are one of key players in the global nitrogen cycle

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(Hamersley et al., 2007; Kuypers et al., 2005; Schmid et al., 2007; Trimmer et al., 2013).

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An important ecological question remains to be answered is what shapes

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community compositions of anammox bacteria? Due to intra- and inter-specific competition

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occurred in natural environment, anammox bacteria have been considered to occupy different

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niches in the natural environment in correlation with the physiological properties of

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individual species or group of species (Kartal et al., 2007b). Hence, enrichment or pure

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culture of anammox bacteria followed by physiological characterization is essential to

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understand the ecological niche differentiation of anammox bacteria (Koops and

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Pommerening-ro, 2001; Zhang et al., 2017a). Given that among 19 anammox species being

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identified, 10 species have been enriched, and their physiological characteristics have been

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characterized to date (Oshiki et al., 2016). Thus, direct microbial competition studies can be

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performed using those individual enrichment cultures. Individual populations can be

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quantified using 16S rRNA gene-targeted quantitative polymerase chain reaction (qPCR)

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assays (Ali et al., 2015; Awata et al., 2013; Koops and Pommerening-ro, 2001; Narita et al.,

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2017; Oshiki et al., 2011). In fact, population shifts among different anammox species have

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been frequently observed in laboratory reactors: for instance, from “Ca. B.

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fulgida”-dominated population to “Ca. Brocadia. sp.40” (Park et al., 2010b), from “Ca. B.

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fulgida” to “Ca. K. stuttgartiensis” (Park et al., 2015), from “Ca. Brocadia sp.” to “Ca. K.

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stuttgartiensis” (van der Star et al., 2008), and from “Ca. B. anammoxidians” to “Ca.

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Anammoxoglobus propionicus” (Kartal et al., 2007b). In addition, mage-data analysis on

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anammox 16S rRNA gene sequences detected so far revealed diverse geographical

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distributions and suggested possible driving factors (Sonthiphand et al., 2014), which must be

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experimentally verified by direct microbial competition studies under controlled laboratory

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conditions (Ali et al., 2018; Zhang et al., 2017a).

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Currently, most of the wastewater treatment plants are seeded with mature

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anammox granules to reduce the start-up time (van der Star et al., 2007). Selection of suitable

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seeding sludge could be a key to rapid and efficient start-up of annamox processes (Kartal et

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al., 2006). If we know the niche differentiation, the most suitable anammox species can be

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selected for targeting wastewater. Therefore, here we provide an overview of physiological

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characteristics of anammox bacteria and discuss potential factors determining ecological

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niche differentiation among different anammox species. For details on the natural 4

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distributions of anammox bacteria reported so far, see a recent review by (Oshiki et al.,

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2016).

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1. Microbial growth kinetics Information on intrinsic microbial growth kinetics, i.e., the maximum specific

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growth rate (µmax) and half-saturation constant (Ks), is indispensable to predict the growth

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behavior under given conditions based on the Monod model (Bollmann et al., 2002; French et

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al., 2012; Füchslin et al., 2012; Kindaichi et al., 2006; Kovarova-Kovar and Egli, 1998;

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Martens-Habbena et al., 2009; Ngugi et al., 2016; Nogueira and Melo, 2006; Nowka et al.,

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2015; Zhang et al., 2017a). Based on the Monod equation, the growth properties of anammox

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species could be predicted using the reported values of Ks and µmax (Fig. 1). For instance, “Ca.

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Brocadia sp.40” most likely outcompete other freshwater anammox bacteria under almost all

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conditions (when assuming NO2- is a limiting substrate and there is no specific inhibitor(s)

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for “Ca. Brocadia sp.40”). Under high NO2- conditions like wastewater treatment, “Ca.

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Brocadia sinica” becomes a good competitor for “Ca. Brocadia sp.40”, suggesting that both

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Brocadia species are suitable for wastewater treatment. On the other hand, it seems that

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diverse anammox species could coexist under low substrate conditions like natural

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environments. This speculation can be supported by the geographical distribution patterns of

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anammox bacteria revealed by anammox 16S rRNA gene sequences data (Sonthiphand et al.,

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2014). However, choice of 16S rRNA gene-targeting primer sets can significantly bias the

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community composition of anammox bacteria as recently described by Orschler et al. (2019).

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Therefore, abundance and diversity of anammox bacteria reported so far must be carefully

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evaluated and compared.

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So far, direct evidence of a correlation between substrate availability and species

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abundance of anammox bacteria in natural environment is not available. Such information

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could be very helpful to understand the niche differentiation.

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1-1. Maximum specific growth rate (µmax) µmax of anammox bacteria was mainly determined by either controlling the sludge

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retention time (SRT) in membrane bioreactor (MBR) or measuring the biomass yield and

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maximum specific substrate consumption rate due to the lack of pure culture (Lotti et al.,

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2015; Oshiki et al., 2011). µmax of “Ca. B. sinica”, “Ca. J. caeni” and “Ca. S. japonica” were

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recently reevaluated by directly measuring the time course increase of 16S rRNA gene copy

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numbers using newly developed qPCR assays, yielding the fastest µmax ever reported for these

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species so far (Zhang et al., 2017b) (Fig. 2). “Ca. B. sinica” and “Ca. Brocadia sp.40”

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exhibited the highest µmax (0.33 d-1) among anammox bacteria, which could be advantageous

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of being dominant population. Thus, µmax of other anammox species needs to be reevaluated

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using appropriate cell cultures (i.e., planktonic free-living cells with high purity) and

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methodology (e.g., more accurate cell quantification methods).

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1-2. Half-saturation constant (Ks)

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Availability of nitrite is more relevant to the niche differentiation among anammox

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bacteria, since ammonium is usually more abundant than nitrite in both natural or man-made

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ecosystems (Pitcher et al., 2011; van der Star et al., 2007). Thus, Ks is the key parameter for

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microbial growth and consequent niche differentiation. A wide range of Ks values for

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ammonium (KNH4+: 3.0 - 640 µM) and nitrite (KNO2-: 0.2 - 370 µM) have been reported for

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different anammox species (Fig. 3 and Fig. 4) (Ali et al., 2015; Awata et al., 2013;

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Carvajal-Arroyo et al., 2013; Kartal et al., 2007b, 2008; Lotti et al., 2014; Narita et al., 2017;

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Oshiki et al., 2013; Oshiki et al., 2017; Strous et al., 1999b; van der Star et al., 2008).

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However, it seems that most of the Ks values were determined using granular and/or

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aggregated biomass, in which substrate transport limitations occurred (Lotti et al., 2014).

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Therefore, the reported affinity constants (KS) were more likely apparent but not the intrinsic

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half-saturation constants. Among the reported Ks values, some of them were determined from

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planktonic enrichment cell cultures (“Ca. K. stuttgartiensis”, “Ca. B. sinica”, “Ca. Brocadia

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sp. 40”, “Ca. S. japonica” and “Ca. B. sapporoensis”) (Awata et al., 2013; Lotti et al., 2014;

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Narita et al., 2017; Oshiki et al., 2013; van der Star et al., 2008), thus which are of highly

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importance.

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1-3. Maintenance coefficient (m)

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Maintenance (m) has been defined as the energy consumed for functions other than

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the production of new cell material (Pirt, 1965). This value is considered to be growth rate

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independent and deeply involved in the microbial competition (Füchslin et al., 2012; van

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Bodegom, 2007). The relative proportion of energy assigned for maintenance becomes more

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significant when cells are cultured at low growth rates than at high growth rate. For example,

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the maintenance energy was a critical factor to determine the successful growth under low

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substrate concentration in co-culture of Escherichia coli and Chelatobacter heintzii (Füchslin

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et al., 2012). The population dynamics could be estimated using an extended form of the

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Monod model incorporating a finite substrate concentration at zero growth rate (Smin), 7

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alternative expression of maintenance energy.

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In a competition study between “Ca. B. sinica” and “Ca. J. caeni”, “Ca. B. sinica”

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was outcompeted by “Ca. J. caeni” under only low nitrogen loading rates even though the

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Monod model predicted the dominance of “Ca. B. sinica” (Zhang et al., 2017a). This

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discrepancy might suggest the importance of Smin. “Ca. B. sinica” requires high maintenance

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energy (high Smin) than “Ca. J. caeni”, thereby “Ca. B. sinica” could not sustain their

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population at low nitrogen loading rates. Since information of maintenance energy

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requirement was mostly missing for anammox bacteria, they should be determined in the

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future.

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2. Oxygen tolerance Anammox bacteria anoxically oxidize ammonium with nitrite as an electron

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acceptor, which was supplied by aerobic ammonia-oxidizing bacteria (AOB) and/or archaea

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(AOA). Thus, their affinities for ammonia and oxygen and oxygen tolerance of anammox

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bacteria play a crucial role in their association with AOB and/or AOA. A limited information

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on oxygen tolerance of anammox bacteria is available. Anammox bacteria had higher KNH4+

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values than AOA but these values were comparable with those of AOB (Fig. 3). Anammox

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bacteria were inhibited by oxygen at very low levels (inhibition coefficient that gives the half

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maximum inhibition (Ki, O2) is 0.092 µM) (Straka et al., 2019). This suggests that the

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cooperation of anammox bacteria and AOA is only possible when ammonia and oxygen

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diffuse in the counter direction, such as stratified lakes and oceans (Straka et al., 2019).

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Association of AOB and anammox bacteria could be expected at relatively high NH4+ and O2 8

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concentrations like wastewater treatment. Anammox bacteria were considered to be microaerotolerant instead of being strict

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anaerobes (Jensen et al., 2008; Lam et al., 2009). The oxygen tolerance determined from

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enrichment cultures varies depending on species (0 - 200 µM O2 for “Ca. K. stuttgartiensis”;

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< 63 µM O2 for “Ca. B. sinica”; < 1 µM O2 for “Ca. B. anammoxidans”; 120 µM O2 for “Ca.

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B. caroliniensis”; 70 µM O2 for “Ca. B. fulgida”) (Oshiki et al., 2015). Despite their

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significant contribution to the oceanic nitrogen cycle, the oxygen tolerance of marine

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anammox species, “Ca. Scalindua”, is currently missing. In marine environments (i.e.,

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oxygen minimum zones (OMZs)), anammox activity was found up to 10 µM O2 (Jensen et al.,

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2008) and 20 µM O2 (Kalvelage et al., 2011). However, it should be noted that the oxygen

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tolerance is highly dependent upon types of biomass (i.e., planktonic free-living or

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aggregated biomass), and careful assessment of biomass type is essential.

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Oxygen inhibition seems to be reversible. Anammox activity of

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Brocadia-dominated biomass gradually recovered to the original level after micro-aerobic

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conditions (< 0.02 mg O2 L−1) was achieved (Seuntjens et al., 2018). The recovery time was

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dependent on the degree (i.e., exposure O2 concentration and time) of perceived inhibition.

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This result indicates that anammox bacteria residing in partial nitritation/anammox (PN/A)

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granules and marine snow probably experience repeated O2 inhibition and recovery. However,

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our understanding is still very limited. Oxygen inhibition and recovery needs to be further

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investigated for different anammox species.

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3. Salinity 9

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Salinity has been suggested as the most important discrimination factor governing

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the geographical distributions of anammox bacteria. “Ca. Scalindua” dominated in saline

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environments (mostly marine) while other genera were mostly found in freshwater

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environments (Sonthiphand et al., 2014). All enrichment cultures of “Ca. Scalindua” are

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obligately halophilic (requirement of salt for growth). They formed a cluster that

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phylogenetically distant from other anammox genera (Oshiki et al., 2016).

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Biogeographical distributions of anammox bacteria have shown that “Ca.

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Scalindua” was the most abundant genus in river estuary area, while abundances of “Ca.

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Brocadia” and “Ca. Kuenenia” were negatively correlated with salinity concentration

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(Amano et al., 2007; Dale et al., 2009). “Ca. Brocadia” and “Ca. Kuenenia” were mostly

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detected from wastewater treatment plants and soil environment (Humbert et al., 2010;

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Schmid et al., 2005). Their detection in saline environments has been considered as transient

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accumulation (Dale et al., 2009). Interestingly, first two “Ca. Scalindua” species, “Ca.

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Scalindua brodae” and “Ca. Scalindua wagneri”, were retrieved from a wastewater treatment

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plant (WWTP) treating landfill leachate in Pitsea, UK, instead of marine environment. In

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addition, “Ca. Scalindua”-related 16S rRNA genes and the hydrazine synthase (hzsA) genes

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were detected in two anoxic hypersaline (up to 24 % salinity) sulphidic basins in Eastern

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Mediterranean Sea (Borin et al., 2013). These evidences suggest that “Ca. Scalindua” are

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distributing from freshwater environment (e.g. river estuary) to hypersaline environments,

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indicating that “Ca. Scalindua” might constitute the most diverse genus among anammox

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bacteria. It is therefore essential to explore eco-physiological and functional diversity of “Ca.

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Scalindua”.

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To better describe the growth behavior with salinity inhibition, the salinity effect 10

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must be quantitatively determined and incorporated in the Monod equation. The Monod

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model has been integrated with the Gauss equation using the least-square non-linear

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regression for evaluation of salinity-induced kinetics (Wu et al., 2019). The model simulation

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revealed that “Ca. Scalindua” dominated over “Ca. Kuenenia” only at salinity > 3.0% when

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nitrite concentration (SNO2−) was higher than nitrite affinity constant (KNO2−). Conversely,

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high nitrite affinity of “Ca. Scalindua” leads to their predominance in all salinities under

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nitrite-depleted conditions (KNO2− ≥ SNO2−) (Fig. 4) (Wu et al., 2019).

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Generally, to survive under saline conditions, microorganisms must make

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osmolarity balance between the inside and outside cells, either synthesize/uptake organic

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compatible solutes or accumulate inorganic ions (K+), so-called “salt-in” strategy (Ventosa et

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al., 1998). For non-halophiles, this adaptation also involves the manipulation of intracellular

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protein structures to function under elevated intracellular ion concentrations (Dennis and

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Shimmin, 1997). Depending on sort of osmolyte, this process could be extremely energy

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intensive, particularly for autotrophs (Oren, 1999). For example, to create same osmolarity,

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de novo biosynthesis of trehalose (a 12-carbon disaccharide sugar) requires 750 times more

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ATPs than accumulating KCl (Fig. 6), which significantly reduces energy allocated to growth.

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Therefore, to successfully survive under saline environment, an energy-efficient

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osmoregulation strategy is preferred. Although experimental evidence for osmoregulation in

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anammox bacteria is still not available, only some information has been inferred from the

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genomes. Based on protein isoelectric point distributions derived from the genome of “Ca. S.

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brodae” and “Ca. S. profunda” (Speth et al., 2015; van de Vossenberg et al., 2013), it was

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suggested that both species might employ a “salt-in” strategy for salinity adaptation. However,

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the genome of “Ca. S. rubra” suggests the use of a compatible solutes synthesis strategy to 11

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cope with high salinity (Speth et al., 2017). It was observed that addition of yeast extract

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improved the activity of “Ca. S. japonica” (personal commination). This may suggest that

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anammox bacteria could directly up take the osmolytes or precursors including amino acids

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in yeast extract to enhance osmoregulation, which is less energy intensive than de novo

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biosynthesis (Nieto et al., 1998; Oren, 2011; Youssef et al., 2014). Further study is apparently

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required to verify the osmoregulation strategies of anammox bacteria, the salinity effect on

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kinetic properties, and interspecies competition at various salinities to assess its effect on the

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niche differentiation.

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4. Aggregation ability Anammox bacteria exhibit high tendency to form aggregates or biofilms (Ali et al.,

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2018), in which different microenvironments are developed due to substrate diffusion

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limitation and high microbial activity (Fig. 7). Microelectrode measurements showed

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considerable heterogeneity with respect to physicochemical parameters even in small

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microbial flocs, granules, and thin biofilms (Ali et al., 2016; Kindaichi et al., 2007a; Okabe et

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al., 1999). This spatial heterogeneity makes competing species to coexist in the same

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aggregates, but different locations such as surface and inner part of the aggregate (Fig. 7) (Ali

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et al., 2016; Kindaichi et al., 2007b). Apparent half-saturation coefficient in activated sludge

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flocs is highly dependent on the microcolony size, biomass density, and spatial biomass

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distribution (Lotti et al., 2014; Picioreanu et al., 2016). The reversion of AOB and NOB

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apparent oxygen half-saturation coefficients (KO) was caused by high biomass density and

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resulting O2 concentration gradients inside the microcolonies (Picioreanu et al., 2016).

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Therefore, it is very important to describe more detailed microbial spatial distributions and

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directly link them to in situ analyses of microenvironments to accurately interpret the niche

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differentiation of anammox bacteria in aggregated biomass.

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Aggregates and biofilms also provide a protective niche as a physical barrier

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against physical washout (Weissbrodt et al., 2012), predators (Kumar et al., 2017), oxygen

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and inhibitory chemicals (Monier and Lindow, 2003; Nogueira and Melo, 2006). Aggregation

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abilities of three different anammox species (“Ca. B. sinica”, “Ca. J. caeni” and “Ca. B.

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sapporoensis”) were quantitatively evaluated based on cell surface properties and

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extracellular polymeric substances (EPS) production (Ali et al., 2018). It was found that “Ca.

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B. sinica” possesses the most hydrophobic cell surface, leading to the best aggregation

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capability (Ali et al., 2018). In addition, “Ca. B. sinica” has the highest maximum specific

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growth rate (Zhang et al., 2017b). These superior eco-physiological features suggest that “Ca.

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B. sinica” is the most suitable inoculum for wastewater treatments. This speculation is

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supported by the geographical distribution based on the 16S rRNA gene sequence analysis

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showing the most frequent detections of this species in engineering ecosystems (Sonthiphand

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et al., 2014). Aggregate formation, however, makes it extremely difficult to understand the

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mechanisms or factors involved in niche differentiation.

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5. Organic matter Anammox bacteria have been considered to be a chemolithotroph upon the

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discovery (Strous et al., 1999a). However, some anammox bacteria could oxidize short chain

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fatty acids, which was coupled with reduction of nitrate and/or nitrite to ammonium, 13

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disguised as denitrifiers alternatively (Kartal et al., 2007a). For example, “Ca. A.

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propionicus” can oxidize propionate at a rate of 0.64 ± 0.05 µmol per gram protein per

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minute, which was more than 5 times higher than “Ca. B. anammoxidans” and “Ca. K.

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stuttgartiensis” (Kartal et al., 2007b). While “Ca. B. fulgida”, an autofluorescent anammox

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bacteria, can oxidize acetate at a rate of 0.95 ± 0.04 µmol per gram protein per minute, which

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is higher than other anammox species (Kartal et al., 2008). Both species became the dominant

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species in anammox community in the laboratory reactors fed with an anammox medium

293

containing organic acids. According to those studies, the addition of organic acids does not

294

affect the biomass yield and specific growth rate. The question now is whether the organic

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matter can be used as energy or carbon source or both. Laureni et al. (2015) have reported

296

that “Ca. B. fulgida” did not directly incorporate or store the amended acetate and glucose

297

based on microautoradiography-combined with fluorescence in situ hybridization

298

(MAR-FISH) analysis. However, this metabolic capability might be species specific therefore

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test using other anammox species should be conducted. Further study on fatty acids oxidation

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by anammox bacteria is also needed to understand how this metabolism gives certain

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anammox species physiological advantages over others.

302 303 304

6. Culture conditions for enrichment Cultivation conditions are important for enrichment of anammox species because

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they reflect their potential niche differentiation. Here we summarized the inoculum types and

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culture conditions used for enrichment of each anammox species (Table 1).

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Anammox bacteria have been enriched from various source of biomass so far, 14

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including denitrifying sludge (“Ca. J. caeni” and “Ca. B. sinica”) (Fujii et al., 2002; Tsushima

309

et al., 2007), activated sludge (“Ca. B. flugida” and “Ca. A. propionicus”) (Kartal et al., 2008,

310

2007b), nitrifying biomass (“Ca. K. stuttgartiensis”) (Egli et al., 2001), marine sediments

311

(“Ca. S. japonica” and “Ca. Scalindua sp.”) (Kindaichi et al., 2011; van de Vossenberg et al.,

312

2008). Composition and concentrations of the feed substrate might determine the outcome of

313

enrichment (Park et al., 2010a, 2010b). For example, “Ca. B. flugida” and “Ca. A.

314

propionicus” were enriched from the same activated sludge by supplying an inorganic

315

anammox medium with acetate (1 - 30 mM) and propionate (0.8 - 15 mM), respectively

316

(Kartal et al., 2008, 2007c).

317

In addition, various types of reactors were used for enrichment of different types of

318

biomass; sequencing batch reactor (SBR) for granular biomass, membrane bioreactor (MBR)

319

for planktonic biomass, and up-flow anaerobic bioreactor (UAB) for attached biomass (i.e.,

320

biofilms). No correlation between the reactor type and enriched anammox species was

321

observed (Table 1).

322

In our laboratory, we first used up-flow column reactors with non-woven fabrics as

323

biomass carrier for initial enrichment of “Ca. B. sinica” from activated sludge, yielding about

324

90 % pure biofilm biomass after about 6-month anoxic cultivation (Tsushima et al., 2007)

325

(Fig. 8). A secret key to successful enrichment is that contaminated other bacteria should be

326

washed-out by applying high flow rate (i.e., short hydraulic retention time (HRT)) and

327

meanwhile maintaining low NO2 concentration to avoid NO2- inhibition. Oxygen

328

contamination must be avoided especially at the beginning of enrichment. Thereafter,

329

enriched biofilm biomass was homogenized and inoculated into to MBRs to obtain

330

planktonic free-living biomass (with >95% purity) for physiological and biochemical studies 15

331

(Oshiki et al., 2013; Zhang and Okabe, 2017). In this way, we have succeeded in enriching

332

four different anammox species: “Ca. B. sinica” (Oshiki et al., 2011), “Ca. J. caeni” (Ali et al.,

333

2014), “Ca. S. japonica” (Awata et al., 2013; Oshiki et al., 2017), and “Ca. B. sapporensis”

334

(Narita et al., 2017). However, a certain species may not be easily cultured as planktonic cells

335

in MBR (Trigo et al., 2006). On the other hand, it seems that “Ca. S. japonica” forms less

336

aggregates or biofilms. It is therefore interesting to investigate which anammox species can

337

be enriched in what form of biomass?

338

Most freshwater anammox species were enriched at temperature range from 27°C

339

to 37°C while marine anammox species were enriched at a temperature range from 15°C to

340

25°C. Maximum activities were observed at around 37°C for freshwater anammox species

341

(Ali et al., 2015; Oshiki et al., 2011) and below 30°C for marine species (Awata et al., 2013).

342

Therefore, besides salinity, temperature could be another pivotal factor between marine and

343

freshwater anammox species. pH has been found to regulate the abundance, diversity and

344

activity of AOB and AOA in paddy soils (Li et al., 2015). Since all anammox species were

345

enriched at pH of 6.8 - 8.5 so far, it is very interesting if certain anammox species be detected

346

or enriched from acidic or alkalic environments.

347

Finally, an inorganic medium that was originally used by van de Graaf et al. (1996)

348

has been used for these enrichment cultures with some modifications of metal concentrations

349

(Table 1). For example, various iron (5.0 (van de Graaf et al., 1996), 6.3 (Strous et al., 1998),

350

6.8 (Hsu et al., 2014) or 9.0 mg L-1 (Quan et al., 2008), respectively) and copper (0.25 mg L-1

351

or no addition) (Ali et al., 2015; van de Graaf et al., 1996) concentrations were used.

352

Anammox bacteria can carry out nitrate dependent ferrous iron oxidation (Oshiki et al., 2013).

353

In addition, Fe (II) addition significantly enhanced the specific growth rate of “Ca. J. caeni” 16

354

from 0.12 d-1 at a regular Fe (II) level (0.03 mM) to 0.17 d-1 at 0.09 mM (Liu and Ni, 2015).

355

Iron is deeply involved in anammox metabolism, and vast majority of cellular iron is in the

356

form of cofactors within Fe-S proteins and multi-heme cytochromes (Ferousi et al., 2017;

357

Kartal and Keltjens, 2016). High percentages of intracellular Fe contents (41% and 39% for

358

“Ca. J. caeni” and “Ca. B. sinica”, respectively) were reported (Ali et al., 2015). Copper was

359

also found to be the dominant metal component in anammox bacteria (39% and 42% for “Ca.

360

J. caeni” and “Ca. B. sinica”, respectively), which is involved in enzymes of reductive

361

acetyl-CoA pathway for carbon fixation (Ali et al., 2015; Kartal et al., 2011b).

362

It is also interesting that some anammox bacteria were enriched using ground water

363

instead of demineralized water (Ali et al., 2015; Kindaichi et al., 2011; Tsushima et al., 2007).

364

Indeed, certain anammox species have been detected at groundwater environment

365

(Sonthiphand et al., 2014). Composition of groundwater (most likely metal elements,

366

minerals and alkalinity) varies depending on geographical regions and weather conditions

367

(Dragon and Marciniak, 2010). Therefore, it deserves to investigate the composition of trace

368

elements in groundwater and compared with those in demineralized water for enrichment of

369

anammox bacteria. In fact, “Ca. B. sinica”-dominated biomass has been enriched and

370

maintained for many years using groundwater in author’s laboratory (Hokkaido University,

371

Sapporo Japan). However, the dominant anammox population shifted to “Ca. K.

372

stuttgartiensis” after the Ca. B. sinica”-dominated biomass was cultured by exactly same

373

manner and conditions except using the local groundwater at Nagaoka National College of

374

Technology, Niigata Japan. Furthermore, when the “Ca. K. stuttgartiensis”- dominated

375

biomass was sent back to Hokkaido University and cultured with Hokkaido University’s

376

groundwater, the dominant population shifted back to “Ca. B. sinica” again (unpublished our 17

377

data). Since the only difference was the groundwater used for culture medium, it clearly

378

indicates that there are as-yet-unknown factor(s) in groundwater which determines the

379

dominant anammox species.

380 381 382

7. Interspecific competition Interspecific competition, different anammox bacterial species compete for the

383

same resources (e.g. a limiting substrate (nitrite or ammonium) or living space), which

384

consequently results in the exclusion of an inferior species (or decline in the population) in

385

the habitat and niche differentiation. An important question is what shapes the community

386

compositions? Among various properties such as growth kinetics, metabolic flexibility, and

387

environmental factors, growth kinetics play an important role in the competition. Recently,

388

the interspecies competition for a limiting substrate (nitrite) between “Ca. B. sinica” and “Ca.

389

J. caeni” were experimentally investigated using planktonic enrichment cultures in MBRs and

390

gel-immobilized enrichment cultures in column reactors (Zhang et al., 2017a). As predicted

391

by the Monod model (Fig. 1), “Ca. B. sinica” outcompeted “Ca. J. caeni” under high nitrogen

392

loading rates (NLR). However, “Ca. J. caeni” could proliferate and dominate under low

393

NLRs, which contradicts the model prediction. This discrepancy is probably attributed to

394

insufficient accuracy of growth kinetic parameters especially KNO2- values and lack of

395

information on maintenance rate as mentioned above. More study is essential to consolidate

396

the kinetic parameters of anammox bacteria for better understanding of niche differentiation.

397

In gel-immobilized biomass (gel beads), both species coexisted, but spatially separated; “Ca.

398

J. caeni” was mainly present in the inner part (low NO2- environment), whereas “Ca. B.

18

399

sinica” was present throughout the gel beads. This spatial distribution may imply the niche

400

differentiation of two species in natural environments. More studies with different species

401

combinations and different culture conditions are required to understand the niche

402

partitioning among anammox species.

403 404

8. Other factors

405

Other variables might be involved in constituting the niche differentiation of anammox

406

species. The inhibitory concentration levels of nitrite (100 – 768 mg L-1), nontoxic organic

407

matters (counted as COD, 237 mg COD L-1 – 290 mg COD L-1), sulfide (> 5 mM), phosphate

408

(>50 mM), methanol, and others have been reported (see references (Oshiki et al., 2016; Jin

409

et al., 2012) and references therein), but data are yet too limited for a species specific

410

differentiation.

411 412

9. Outlook

413

Isolation of pure anammox bacteria.

414

Although isolation is laborious and time-consuming, pure cultures are obviously

415

needed for accurate measurement of growth kinetics and physiological traits and biochemical

416

studies as stated above (Bollmann et al., 2010). It is still not clear the missing element(s) in

417

the isolation of anammox bacteria. The culturability of slow-growing fastidious bacteria from

418

environmental samples has been improved by a simple modification in the preparation of

419

agar media (i.e., autoclaving the phosphate and agar separately) (Kato et al., 2018). Other 19

420

efforts including simulating environment and coculturing targets have been proven successful

421

in pure-culturing certain microorganisms (see detailed review (Stewart, 2012)).

422

Pure cultures are still not available for anammox bacteria. However, several

423

enrichment planktonic cell cultures have been established using membrane bioreactors

424

(MBRs) so far (van der Star et al., 2008; Oshiki et al., 2013; Kartal et al., 2011; Lotti et al.,

425

2014; Zhang and Okabe, 2017). These enrichment cultivations, however, require an

426

enormous amount of time to achieve free-living planktonic cells in most studies; usually

427

more than 100 - 200 days (Ali et al., 2015; Lotti et al., 2014; Oshiki et al., 2013; van der Star

428

et al., 2008). In addition, detailed mechanism of achieving planktonic free-living cells in

429

MBR is not well understood. Recently, a more rapid and systematic cultivation technique was

430

developed by applying polyvinyl alcohol-sodium alginate (PVA-SA) gel immobilization

431

technology (Zhang and Okabe, 2017). In this new approach, anammox bacterial cells were

432

firstly grown in the PVA-SA gel beads, which provides easily dispersible biomass, and then

433

the dispersed biomass was inoculated to MBR. The combination of PVA-SA gel

434

immobilization technique and MBR cultivation can provide planktonic cell cultures with high

435

purity (>95%) within 35 days (Fig. 5), which will significantly accelerate the cultivation of

436

planktonic anammox cells and consequently studies on physiology and biochemistry of

437

anammox bacteria.

438

To further purify anammox bacterial cells (more than 99.5% purity), a percall

439

density gradient centrifugation technique has been applied for the planktonic MBR cultures

440

(Kartal et al., 2011a; Strous et al., 1999b, 1999a). In this method, percoll solution and

441

anammox culture were mixed and then centrifugated, resulting in an anammox cell

442

suspension with more than 99.8 % purity (Kartal et al., 2011a). This percoll separated “almost 20

443

pure” anammox cells could carry out the anammox reaction and CO2 fixation and thus can be

444

used for further purification or isolation. For isolation, we commonly carry out a limiting

445

dilution of the percall separated cell culture. However, anammox activity is highly dependent

446

on the cell density. Minimum cell densities required to obtain a detectable anammox activity

447

were reported to be at least 1010 - 1011cells mL-1 (Strous et al., 1999a), > 106 cells mL-1

448

(Oshiki et al., unpublished data), and 0.4 g-anammox biomass-VSS L−1 (De Clippeleir et al.,

449

2011). Thus, the cell density of the diluted Percoll separated culture must be higher than 106

450

cells mL-1. However, since the percoll separated “almost pure” anammox culture is not pure

451

yet (ca. 99.8%), it still contains >103 cells/mL of other contaminated cells, which hampers the

452

isolation of anammox bacteria. Therefore, growth of the contaminated microorganisms must

453

be supressed by adding antibiotics such as penicillin G and others which inhibit other

454

contaminated bacteria but not anammox bacteria (Hu et al., 2013), promoting selection of

455

anammox bacteria. Furthermore, the cell density dependent anammox activity seems to be

456

regulated by acyl-homoserine lactones (AHL)-mediated quorum sensing system (Sun et al.,

457

2018; Tang et al., 2015; Tang et al., 2019). The release of three AHL (AHLs; C6-HSL,

458

C8-HSL, C12-HSL) by anammox bacteria was confirmed (Tang et al., 2015). Furthermore,

459

external addition of AHLs to low biomass concentration cultures significantly increased the

460

anammox activity (De Clippeleir et al., 2011; Tang et al., 2019). In addition, nitric oxide (NO)

461

could act as a signaling molecule, which has been suggested from metagenome analysis

462

(Kartal et al., 2011a; Strous et al., 1999b), and promote growth of anammox bacteria (Hu et

463

al., 2019; Kartal et al., 2010). Thus, an alternative approach for further isolation could be a

464

limiting dilution of the percoll separated anammox culture (the cell density could be below

465

106 cells/mL) followed by careful incubation with addition of signaling molecules like AHL

466

and/or NO and specific antibiotics to inhibit the growth of other contaminated bacteria. Since 21

467 468

this isolation method has never been tested yet, it is important to try if it works or not. Alternatively, anammox bacteria may associate with syntrophs. Metabolic networks

469

between anammox bacteria and the member of Chlorobi on degradation of protein and

470

extracellular peptides and nitrate recycling into nitrite were proposed for naturally aggregated

471

granules based on metagenomic analysis (Lawson et al., 2017). However, there was no

472

experimental study to confirm this hypothesis yet. In anammox granules, nitrate could be

473

reduced to nitrite by heterotrophic denitrifiers using either organic acids or hydrogen (those

474

are produced by fermentation of organic matter) as the electron donor, which makes

475

additional nitrite available for anammox bacteria (Speth et al., 2016). Hence, hydrogen

476

forming microorganisms and denitrifiers may establish a syntrophic association with

477

anammox bacteria. It was experimentally confirmed that anammox bacteria produced and

478

excreted a large amount of organic matter, which was directly utilized by coexisting

479

heterotrophs (Kindaichi et al., 2012). Therefore, such syntrophic association with

480

heterotrophs may not be excluded in enrichment or isolation processes, otherwise the growth

481

of anammox bacteria could be inhibited by accumulation of metabolites (Kindaichi et al.,

482

2012). To enrich anammox bacteria, the accumulation of organic matter-derived from

483

anammox bacteria must be minimized by washing out the metabolites from reactors or

484

exchanging culture medium frequently with efficient biomass retention. This is a main reason

485

why we have been using an up-flow column reactor (Fig. 8) and MBR for enrichment of

486

anammox bacteria because MBR can be operated at very short hydraulic retention time (HRT)

487

with 100% biomass retention. That is, HRT and solid (biomass) retention time (SRT) can be

488

controlled separately in MBR.

489 22

490 491

Cooperative and competitive microbial interaction Anammox bacteria must live with both cooperative and competitive microbial

492

interactions created in granular and aggregated biomass of wastewater treatment processes

493

and marine and freshwater sediments. Since nitrite is scare as compared with ammonium in

494

typical wastewater, aerobic AOB and/or AOA partially oxidize ammonium to nitrite using

495

oxygen, creating a suitable anoxic condition and supplying nitrite for anammox bacteria

496

(oxygen and nitrite-centered cooperative interaction). On the other hand, anammox bacteria

497

must compete aerobic AOB and/or AOA for ammonium and NOB and denitrifiers for the

498

produced nitrite (ammonium and nitrite-centered competitive interaction). These cooperative

499

and competitive interactions are alternative driving factors determining dominant anammox

500

bacteria. Obviously, anammox bacteria representing the higher oxygen tolerance and higher

501

affinity to ammonium and nitrite can advantageously compete with other N-cycle

502

microorganisms. Therefore, kinetic parameters especially half-saturation constants for

503

ammonium (KNH4+) and nitrite (KNO2-) are of importance (Fig. 3 and Fig. 4) but are lacking

504

because of a limited number of pure and enrichment cultures (Nowka et al., 2015). The KNH4+

505

values of anammox bacteria are slightly higher than those of AOA but lower than AOB (Fig.

506

3). The KNO2- values of anammox bacteria are comparable with those of NOB (Fig. 4). This

507

implies that NOB more likely scavenge nitrite and convert to nitrate if oxygen is available,

508

which is an unwanted case for wastewater treatment. For the competition for ammonium

509

among anammox bacteria, AOB and AOA, ammonium could be supplied from anaerobic

510

degradation (remineralization) of organic matter occurring inside of marine and freshwater

511

sediments, which creates counter diffusion of nitrite from the surface and ammonium from

512

the inner part. 23

513

These cooperative and competitive interactions may take place at oxic/anoxic

514

interface (usually only a few 10 µm) (Fig. 7). A clear vertical stratification of these N-cycle

515

microorganisms was created in a partial nitritation-anammox granule (Ali et al., 2016) (Fig.

516

7D). AOB were restricted to the outermost of the granule, which were followed by NOB in a

517

thin oxic region, and underneath anammox bacteria dominated. Thus, AOB convert

518

ammonium into nitrite, providing substrate for anammox (Ali et al., 2016). Anammox

519

bacteria can tolerate trace amount of oxygen (e.g. 20 µM) (Kalvelage et al., 2011). However,

520

a part of nitrite was scavenged by the tightly clustering NOB. In this granule, active nitrogen

521

transformation occurred within outer ca. 300 - 500 µm, which was obviously regulated by

522

oxygen availability. The cooperative and competitive interactions with other N-cycle

523

microorganisms could shape up the dominant anammox population. Interestingly, anammox

524

bacteria (“Ca. Bracadia sp.”) were found to be co-aggregated with recently discovered

525

complete ammonia oxidizing (comammox) bacteria, which were affiliated with the genus

526

Nitrospira, in flocs at very low oxygen concentrations (van Kessel et al., 2015). Recent

527

kinetic analysis of a comammox strain (Nitrospira inopinata) revealed its higher affinity with

528

ammonium (0.65 µM) and lower affinity with nitrite (372 µM) as compared with anammox

529

bacteria (Kits et al., 2017) (Fig. 3 and Fig. 4). Both bacteria compete for ammonium, but

530

comammox bacteria create anoxic environment for anammox bacteria. It was also

531

experimentally demonstrated that anammox bacteria could cooperate with AOA by supplying

532

nitrite and a trace amount of oxygen (Yan et al., 2012). Since interaction between anammox

533

bacteria and these N-cycle microorganisms is delicate and complicated, mathematical

534

modeling could be a useful tool for assessment of niche separation of anammox bacteria

535

(Picioreanu et al., 2016; Straka et al., 2019).

24

536 537 538

Utilization of organic nitrogen compounds

Although anammox contributes significantly to fixed N loss in OMZs,

539

ammonium is scarce in the OMZs. Dissimilatory nitrate reduction to ammonium (DNRA),

540

heterotrophic nitrate reduction, and remineralization of organic matter could be potential

541

ammonium sources for anammox. However, still a substantial part (54% to 77%) of

542

ammonium whose sources still cannot be identified (Lam et al., 2009). For nitrite, anammox

543

bacteria retrieve 67% of nitrite from nitrate reduction and < 33% from aerobic ammonium

544

oxidation (Dalsgaard et al., 2012). Thus, it is speculated that organic nitrogen compounds

545

such as urea and cyanate might be alternative ammonium sources for anammox, because

546

these compounds were commonly detected in the OMZs due to microbial degradation of

547

dissolved organic matter (Zehr and Ward, 2002). Genomic analysis revealed that the genes

548

encoding for urease (degrades urea to ammonium) have not been found in anammox

549

bacteria so far, and genomic information on cyanate utilization is also limited. Recently, it

550

was reported that the genes encoding for urease were transcribed together with the genes for

551

core anammox metabolisms in the OMZs where the highest anammox rates were observed

552

(Ganesh et al., 2018). This evidence implies that organic nitrogen compounds were potential

553

alternative ammonium sources for anammox bacteria. The capability of urea utilization may

554

drive the niche differentiation of anammox bacteria in ocean and provide fundamental

555

evidence for urea degradation in wastewater treatment using specific anammox species. AOB

556

possessing urease activity could proliferate successfully at oligotrophic environment with

557

scarce ammonium concentrations (Koops and Pommerening- Röser, 2001) and formed a

558

distinct phylogenetic cluster among Nitrosomonas (Pommerening-Röser et al., 1996). 25

559

However, it is still entirely unclear if anammox bacteria can degrade urea and

560

cyanate to ammonium themselves or rely on other coexisting organisms. Future studies are

561

required to assess how these organic nitrogen compounds are utilized by anammox bacteria

562

under what environmental conditions.

563 564

Application of integrated multi-omics analysis

565

Multi-omic techniques such as metagenomics, metatranscriptomics, and metabolomics

566

analysis are often seen as an indispensable platform for microbiological studies. Integrated

567

multi-omics analysis have begun to unravel the metabolic functions and interactions of

568

anammox and coexisting heterotrophic bacteria in anammox granules (Lawson et al.,

569

2017). Despite the potential of multi-omics analysis, the importance of culture-based

570

studies cannot be neglected. Cultures have provided the basis for our understanding of the

571

microbiology and are necessary for physiological studies. Therefore, we re-emphasize that

572

omics data must be combined with culture-based studies to confirm the newly discovered

573

microbial functions and interactions (e.g., substrate cross-feeding) through -omics

574

analysis.

575 576 577

10. Conclusions Many studies of anammox have been undertaking since anammox bacteria were

578

discovered in the early 1990s (for about 30 years), which focused mainly on engineering

579

applications (nitrogen removal from various wastewaters). As a result, there are now more

26

580

than 120 full-scale anammox plants around the world. However, knowledge about their

581

ecology, physiology, and biochemistry is still largely limited due to mainly lack of pure and

582

enrichment cultures. To improve the efficiency and stability of anammox processes, further

583

research is obviously required. To assess niche differentiation of anammox bacteria in natural

584

and engineered ecosystems, it should be emphasized that kinetic parameters, especially

585

half-saturation constants for nitrite and ammonium, metabolic flexibility, and effects of

586

environmental factors must be evaluated using planktonic free-living enrichment cultures.

587

Other physiological traits such as maintenance coefficient must be experimentally determined

588

further and linked to population dynamics in competition experiments. The application of

589

"omics" in studying niche differentiation in anammox could be included in

590 591

Acknowledgements

592

This research was financially supported by Institute for Fermentation, Osaka (IFO)

593

and JSPS KAKENHI Grant Number 19H0077609, which were granted to Satoshi Okabe. Lei

594

Zhang was supported partly by the Monbukagakusho Honors Scholarship for Privately

595

Financed International Students offered by the Ministry of Education, Culture, Sports,

596

Science and Technology (MEXT), Japan.

597 598

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Zehr, J.P., Ward, B.B., 2002. Nitrogen cycling in the ocean : New perspectives on processes and paradigms. Appl. Environ. Microbiol. 68, 1015–1024. Zhang, L., Narita, Y., Gao, L., Ali, M., Oshiki, M., Ishii, S., Okabe, S., 2017a. Microbial

954

competition among anammox bacteria in nitrite-limited bioreactors. Water Res. 125,

955

249–258.

956

Zhang, L., Narita, Y., Gao, L., Ali, M., Oshiki, M., Okabe, S., 2017b. Maximum specific

44

957 958 959

growth rate of anammox bacteria revisited. Water Res. 116, 296–303. Zhang, L., Okabe, S., 2017. Rapid cultivation of free-living planktonic anammox cells. Water Res. 127, 204–210.

960

45

Specific growth rates [µ, d-1]

"Ca. Kuenenia stuttgartiensis"

"Ca. Brocadia sinica"

"Ca. Brocadia sp. 40"

"Ca. Jettenia caeni"

0.4

"Ca. Brocadia anammoxidans"

"Ca. Brocadia caroliniensis"

"Ca. Scalindua japonica"

"Ca. Brocadia sapporoensis"

0.05

B

A 0.04 0.3 0.03 0.2 0.02 0.1 0.01

0.0

0.00 1

10

100

1000

10000

0

0.2

0.4

0.6

0.8

1

Nitrite concentration (µM)

Figure 1 Simulated specific growth rates (µ) of various anammox bacterial species as a function of nitrite concentration (nitrite was considered as a limiting substrate). (A) Nitrite concentration ranged from 1 to 10,000 µM. (B) Nitrite concentration ranged from 0 to 1 µM. Average values of µmax and Ks were applied.

46

Maximum specific growth rates [µmax, d-1]

0.4

0.3

0.2

0.1

nd

nd

0.0

Figure 2 Maximum specific growth rates (µmax) of anammox bacteria. For anammox bacteria, different genus was indicated in red (“Ca. Kuenenia”), green (“Ca. Brocadia”), blue (“Ca. Scalindua”) and brown (“Ca. Jettenia”). nd: no experimental data available. 47

AOB, AOA and comammox bacteria

Km [µM NH4++NH3]

10000

Anammox bacteria

A

B

1000

100

10

1 nd

nd

nd

nd

nd

0.1

Figure 3 Affinity constants (Ks) to ammonium of ammonium oxidizing archaea (AOA, red), comammox bacteria (orange) and ammonium oxidizing bacteria (AOB, green) (A), which was obtained from Martens-Habbena et al. (2009), and anammox bacteria (B). Information of AOA and comammox bacteria was obtained from Straka et al. (2019) and Kits et al. (2017), respectively. Nitrification in bulk ocean (light blue), grass ecosystem (light green), heterotrophs (Toluene as substrate, light gray), marine phytoplanktons (black) were also plotted. For anammox bacteria, different genus was indicated in red (“Ca. Kuenenia”), green (“Ca. Brocadia”), blue (“Ca. Scalindua”) and brown (“Ca. Jettenia”). nd: no experimental data available. 48

NOB and comammox bacteria

Anammox bacteria

10000

B

A

Km [µM NO2-]

1000

100

10

1 nd

nd

0.1

Figure 4 Affinity constants (Ks) to nitrite among nitrite oxidizing bacteria (NOB, green) and comammox bacteria (orange) (A), which is adapted from Nowka et al. (2015), and anammox bacteria (B). Information of comammox bacteria was adapted from Kits et al. (2017). For anammox bacteria, different genus was indicated in red (“Ca. Kuenenia”), green (“Ca. Brocadia”), blue (“Ca. Scalindua”) and brown (“Ca. Jettenia”). nd: no experimental data available. 49

A

B

20 µm

Figure 5 Enrichment planktonic cell culture of “Ca. B. sinica” in a membrane bioreactor (MBR) (A). Enriched planktonic “Ca. B. sinica” cells in yellow (combination of TRITC-labeled AMX820 probes counterstained with FITC-labeled EUB 338 mix probes for most bacteria); scale bar = 20 µm.

50

ATP required [mmol]

1000

750

500

250

0

Figure 6 Energy requirement for the synthesis of selected organic compatible solutes from CO2 and accumulation of KCl to create same osmolarity for autotrophic microorganisms under 4M salt condition. Adapted from (Oren, 1999).

51

Figure 7 Steady state concentration profiles of NH4+, NO2-, NO3- (A) and DO (B) in nitritation-anammox granular biomass. Dashed line represents a liquid-granule interface. Gary bars are showing spatial distributions of the net volumetric consumption rates of DO. (C) Spatial distributions of net volumetric consumption rates of NH4+ (red bars) and NO2- (blue bars) and net volumetric production rates NO3- (green bars). (D) Confocal laser scanning microscope (CLSM) images showing the in situ spatial organization of anammox bacteria and coexisting bacteria in a nitritation-anammox granule. FISH was performed with Alexa488-labeled Nso190 probe (green) for ammonium oxidizing bacteria (AOB) and Cy3-labeled Ntspa662 (red) for nitrite oxidizing bacteria (NOB) and Alexa647-labeled Amx820 probe (blue) for anammox bacteria. Scale bar = 5 µm. All figures were adapted from Ali et al. (2016). 52

Figure 8 An up-flow column reactor with non-woven fabric sheet as biomass carrier was used for initial enrichment of anammox bacteria. If the effluent NO2- concentration is < 5 mg/L, the nitrogen loading rate (NLR) was gradually increased by increasing the flow rate not NH4+ and NO2- concentrations. HRT could be decreased to 0.5 h, under which contaminated other bacteria are likely washed out. Only attached anammox bacteria grow because inorganic anammox medium is supplied. Oxygen contamination must be carefully avoided especially at the beginning of enrichment. 53

Table 1 Species specific enriching conditions. Species

“Ca. B. anammoxidans”

“Ca. B. fulgida”

“Ca. B. sinica”

“Ca. B. sapporoensis”

“Ca. K. stuttgartiensis”

“Ca. A. propionicus”

“Ca. S. brodae”

“Ca. S. profundal”

“Ca. S. japonica”

“Ca. S. sp.”

“Ca. J. caeni”

“Ca. J. asiatica”

Enrichment level (%)

74

80

70, 90

>90

90, >90

80, 65±5

~90

~90

85±4.5, >90

<67

72.8, >90

50

Reactor type

SBR*

SBR, MBR

UAB, MBR

MBR

Flask, SBR, MBR

SBR, batch mode reactor

SBR

SBR

UAB, MBR

UAB

UAB, MBR

UAB

Form of biomass

Granule

biofilm aggregates

suspended planktonic cells

Floc

Granule

30, 25, 37

33, 27 – 30

23

15 – 18

25

Biofilm, suspended planktonic cells 30, 37

Granule

33

Biofilm, suspended planktonic cells 20

Biofilm

32 – 33

Granule, suspended planktonic cells 30, 35, 38

Granule

Temp. (°C)

Biofilm, suspended planktonic cells 37, 30

30 – 35

pH

7.0 – 8.0

7.0 – 7.3

7.0 – 8.6

6.8 – 7.5,

7.0 – 8.0

7.0 – 8.0

7.0 – 8.0

7.0 – 8.0

7.3 – 7.6

8.0

7.5 – 8.0

8.0 – 8.5

Inoculum

Denitrifying FBR sludge

Activated Sludge

Denitrification sludge, Anammox biofilm

Full-scale anammox reactor sludge, anammox UAB sludge

Activated sludge, landfill leachate

Marine sediment (Gullmar Fjord)

Marine sediment

Marine sediment, Anammox biofilm

Marine biofilm

Laboratory scale denitrification reactor sludge

River sediment

Location of enrichment

Delft (NLD)

Nijmegen (NLD)

Sapporo (JPN)

Delft (NLD), Sapporo (JPN)

Nijmegen (NLD), Taichung (TWN)

Nijmegen (NLD)

Nijmegen (NLD)

Hiroshima (JPN)

Tsu (JPN)

Kumamoto (JPN), Sapporo (JPN)

Shanghai (CHN)

Medium composition **

A1, A2

A1

A1, A6

A5, B1

Nitrifying RBC, anammox SBR sludge, full-scale anammox reactor sludge Dübendorf, (CHE), Santiago de Compostela (ESP), Delft (NLD) A3, A4

A1, B4

C1

C1

C2, C3

C4

B1, B2

B3

54

Type of water

Demineralized water

Not described

Ground water

Demineralized water, Ground water

Demineralized water

Demineralized water

Demineralized water

Demineralized water

Ground water

Deep sea water

Ground water

Not described

Ammonium (mM)

5

3-45

6.2-24.6, 16-30

60, 1-15

6, 26.8, 120

2.5-45, 13.1

1-45

1-45

0.58-6.3, 5-10

3.6-10.9

17.9, 2.5-16.8

15

Nitrite (mM)

5

3-45

3.7-22.8, 16-30

60, 1-15

6, 26.8, 120

2.5-45, 11.9

0.5-45

0.5-45

1.8-6.3, 5-10

3.6-14.1

17.9, 2.5-20

15

Nitrate (mM)

-

6

-

-

-

6, 0

0-1.5

0-1.5

-

-

-

Other

-

1-30 mM Acetate

-

-

-

0.8-15 mM propionate

-

-

Sulfide to adjust pH

- (3 mg-N/L in ground water) -

SRT (d)

-

-

125, 30-60

12-15, 60

-

-

-

-

-

-

-

-

Reference

(Strous et al., 1998; van de Graaf et al., 1996)

(Kartal et al., 2008)

(Mamoru Oshiki et al., 2013; Oshiki et al., 2011; Tsushima et al., 2007)

(Lotti et al., 2014; Narita et al., 2017)

(Dapena-Mora et al., 2004; Egli et al., 2001; van der Star et al., 2008)

(Hsu et al., 2014; Kartal et al., 2007b)

(van de Vossenberg et al., 2008)

(van de Vossenberg et al., 2008)

(Kindaichi et al., 2011; Mamoru Oshiki et al., 2013)

(Nakajima et al., 2008)

(Ali et al., 2015; Fujii et al., 2002)

(Quan et al., 2008)

Influent substrate

-

*Abbreviations of reactor SBR: sequencing batch reactor; MBR: membrane bioreactor; UAB: up-flow anaerobic biofilm; EGSB: expanded granular sludge bed; FBR: fluidized bed reactor; RBC: rotating biological contactor **Medium composition A group: variations of synthetic mineral medium proposed at first A1. KHCO3, 500 mg L-1; KH2PO4, 27.2 mg L-1; MgSO4.7H2O, 300 mg L-1; CaCl2.2H2O, 180 mg L-1; trace element solution I (EDTA, 5 g L-1; FeSO4, 5 g L-1) 1 mL L-1; trace elements solution II (EDTA, 15 g L-1; ZnSO4.7H2O, 0.43 g L-1;CoCl2.6H2O, 0.24 g L-1; MnCl2.4H2O, 0.99 g L-1; CuSO4.5H2O, 0.25 g L-1; NaMoO4.2H2O, 0.22 g L-1; NiCl2.6H2O, 0.19 g L-1; NaSeO4.10H2O,

55

0.21 g L-1; H3BO4, 0.014 g L-1), 1 mL L-1 (van de Graaf et al., 1996) A2. KHCO3, 1.25 g L-1; NaH2PO4, 50 mg L-1; CaCl2.2H2O, 300 mg L-1; MgSO4.7H2O, 200 mg L-1; FeSO4, 6.25 mg L-1; ethylenediamine tetraacetic acid, 6.25 mg L-1; trace elements solution I and II (same as A1), 1.25 mL L-1 (Strous et al., 1998) A3. KHCO3, 250 mg L-1; K2HPO4, 174.2 mg L-1; MgCl2, 42.6 mg L-1; CaCl2, 55.5 mg L-1; trace element solution I (EDTA, 10 g L-1; FeSO4, 5 g L-1) 2 mL L-1; trace elements solution II (EDTA, 15 g L-1; ZnSO4.7H2O, 0.43 g L-1;CoCl2.6H2O, 0.24 g L-1; MnCl2.4H2O, 0.99 g L-1; CuSO4.5H2O, 0.25 g L-1; NaMoO4.2H2O, 0.22 g L-1; NiCl2.6H2O, 0.19 g L-1; NaSeO4.10H2O, 0.21 g L-1; H3BO4, 0.014 g L-1), 1 mL L-1 (Egli et al., 2001) A4. KHCO3, 1.5 g L-1; KH2PO4, 24.5 mg L-1; MgSO4.7H2O, 49.4 mg L-1; CaCl2, 147 mg L-1; EDTA, 14.6 mg L-1; yeast extract, 1.0 mg L-1; trace elements solution II (same as A1), 1 mL L-1 (van der Star et al., 2008) A5. KHCO3, 0-1.5 g L-1; KH2PO4, 24.5-2041 mg L-1; MgSO4.7H2O, 49.4 mg L-1; CaCl2, 73.5-147 mg L-1; EDTA, 14.6 mg L-1; vitamin solution, 0-10 mL L-1; trace elements solution I and II (same as A1), 1 mL L-1 (Lotti et al., 2014) A6. KHCO3, 24.4 mg L-1; KH2PO4, 24.4 mg L-1; MgSO4.7H2O, 60 mg L-1; CaCl2, 51 mg L-1; yeast extract, 1.0 mg L-1; trace elements solution I and II (same as A1), 0.5 mL (Mamoru Oshiki et al., 2013) B group: Other medium for freshwater anammox bacteria B1. NaHCO3, 84 mg L-1; KH2PO4, 54 mg L-1; MgSO4.7H2O, 1 mg L-1; CaCl2.2H2O, 1.4 mg L-1; FeSO4.7H2O, 9 mg L-1; EDTA, 5 mg L-1; NaCl, 1 mg L-1; KCl, 1.4 mg L-1 B2. KHCO3, 125 mg L-1; KH2PO4, 54 mg L-1; MgSO4.7H2O, 9 mg L-1; CaCl2.2H2O, 1.4 mg L-1; FeSO4.7H2O, 9 mg L-1; EDTA, 5 mg L-1; NaCl, 1 mg L-1; KCl, 1.4 mg L-1 B3. KH2PO4, 10 mg L-1; trace elements (not described in detail) (Quan et al., 2008) B4. KHCO3, 1.36 mg L-1; NaH2PO4, 65.2 mg L-1; MgSO4.7H2O, 217 mg L-1; CaCl2.2H2O, 326 mg L-1; FeSO4.7H2O, 6.79 mg L-1; EDTA, 6.79 mg L-1; HCl, 1 mM; trace elements solution II (same as A1), 1 mL (Hsu et al., 2014) C group: media for marine anammox bacteria

56

C1. Red sea salt, 33 g L-1; FeSO4, 0-9 µM; KH2PO4, 0-0.2 mM; NaHCO3 (van de Vossenberg et al., 2008) C2. SEALIFE (artificial sea salt), 35 gL-1; KHCO3, 500 mg L-1; KH2PO4, 27 mg L-1; MgSO4.7H2O, 300 mg L-1; CaCl2.2H2O, 180 mg L-1; trace elements solution I and II (same as A1), 1 mL (Kindaichi et al., 2011) C3. SEALIFE (artificial sea salt), 35 gL-1; KHCO3, 24.4 mg L-1; KH2PO4, 24.4 mg L-1; MgSO4.7H2O, 60 mg L-1; CaCl2, 51 mg L-1; yeast extract, 1.0 mg L-1; trace elements solution I and II (same as A1), 0.5 mL (Mamoru Oshiki et al., 2013) C4. KHCO3, 125 mg L-1; KH2PO4, 54 mg L-1; FeSO4.7H2O, 9 mg L-1; EDTA, 5 mg L-1 (Nakajima et al., 2008)

57

Highlights Ecological niche differentiation of anammox bacteria is still not fully understood. We summarized the Monod kinetic parameters (µmax and Ks) of anammox bacteria. We re-evaluated their enrichment methods and culture medium compositions. We formulated the current issues of anammox studies in this review article.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Satoshi Okabe, Ph.D. (Corresponding author) Division of Environmental Engineering, Faculty of Engineering, Hokkaido University Kita 13 Nishi 8, Kita-ku, Sapporo, Hokkaido 060-8628, JAPAN Phone and fax: 81-(0)11-706-6266 E-mail: [email protected]