Introduction

The first evidence of the anaerobic ammonium oxidizing (anammox) process by bacteria from a denitrifying fluidized bed reactor was confirmed in a laboratory bioreactor of the Netherlands (Mulder et al. 1995). By harnessing the sequencing batch reactor with efficient biomass retaining capability, the physiological characteristics of the anammox process and the possible biochemical reaction mechanism of anammox bacteria are delineated and proposed, in which ammonium is oxidized to hydrazine first by the electron acceptor hydroxylamine; hydroxylamine is originated from nitrite reduction, and hydrazine was finally oxidized to dinitrogen gas, N2 (Jetten et al. 1998). Anammox biochemical process in marine ecosystems was estimated to be responsible for 30~50% marine nitrogen loss (Arrigo 2005; Devol 2003). In addition, comparing to canonical denitrification processes, the application of anammox process contributes to less production of N2O and NO, the gaseous nitrogen intermediates which causes severe greenhouse effects (Jetten et al. 1997). Partial denitrification coupled with anammox is a common technical approach in industrial systems to effectively treat wastewater containing high ammonium and low biochemical oxygen demand (BOD) to decrease the operational cost and produce less greenhouse gases (Den Camp et al. 2006; Jetten et al. 2002). The ecological and industrial significance make anammox process and the micro-organisms to become one of the hot topics in environmental engineering and microbial ecology recently (Jetten et al. 2001, 2005; Kartal et al. 2011b).

Anammox bacteria, deeply branched within Planctomycetales, are responsible for anammox biochemical reactions for the nitrogen transformation (Strous et al. 1999). They occur in anoxic niches, including saline conditions, marine and ocean sediments, marine water column; freshwater wetland, paddy soils, wastewater treatment plants (WWTPs), reactors, groundwater, freshwater, and freshwater sediments and mixed-saline conditions, estuary, and mangrove sediments (Hu et al. 2011; Humbert et al. 2010; Li and Gu 2016a; Oshiki et al. 2016; Sonthiphand et al. 2014). There are additional reports on anammox bacterial populations in extreme and special environments, such as hydrothermal vents, hypersaline sulfidic basins, petroleum reservoirs, marine sponge, fish and polychaete guts (Borin et al. 2013; Byrne et al. 2009; Chan and Gu 2016; Li et al. 2010a; Li and Gu 2016b; Mohamed et al. 2010). Studies on anammox bacteria genome, biology and biochemistry, diversity, and application rely on better practical applications of detecting them from natural and laboratory environments. Currently, no agreement is available on a common gene marker or a set of PCR primer for detecting them in various samples for a comparison of the results available.

The isotope-pairing technique could help the quantitative measurement on anammox activity rates (Risgaard-Petersen et al. 2003; Ward et al. 2009). Molecular probes of fluorescent in situ hybridization (FISH) and catalyzed reporter deposition fluorescent in situ hybridization (CARD-FISH) specific for selective genera have been developed since the beginning of discovery of new anammox bacteria species (Li and Gu 2011; Schmid et al. 2000). Anammox bacteria specific core ladderane lipids and intact ladderane glycerophospholipids of living cells can serve as the bioindicators for general and viable abundance of anammox bacteria (Damsté et al. 2002; Jaeschke et al. 2009; Sinninghe Damsté et al. 2005), but the complicated procedures and sophisticated instruments prevent many from using them practically. PCR primer pairs for qualitative and quantitative detection of anammox bacteria have been developed and applied widely in majority of the studies for distribution and diversity patterns, community composition, and quantification of the metabolic activity by reverse transcription PCR (RT-PCR) (Li and Gu 2011). PCR primers of 16S rRNA gene are the most used in various researches and can offer phylogeny details, while primers targeting functional genes have high specificity and can reflect functional activity (Li and Gu 2011). Up to now, there are four categories of functional gene-based PCR primer pairs developed, targeting central anammox activity enzymes, including hydrazine dehydrogenase (Hzo), nitrite reductase (NirS), hydrazine synthase (Hzs), and cytochrome c biogenesis proteins (Ccs) (Harhangi et al. 2012; Hirsch et al. 2011; Li et al. 2010b, 2011b; Schmid et al. 2008; Wang et al. 2012c; Zhou et al. 2017).

Detection of anammox bacteria in various samples has not been successful as expected, even though the PCR technique has been available for a long time. Since PCR primers were designed in silico and, as a result, their efficiencies and specificities depend on the biomass, sample characteristics and interfering factors for PCR reaction to take place effectively. Many practical applications of the reported PCR primers on sensitive detection of anammox bacteria do not offer acceptable performance unfortunately (Han et al. 2017; Li and Gu 2011; Zhou et al. 2017). Large and comprehensive database of rRNA gene, such as RDP, Greengenes, and SILVA, could serve as a suitable platform to evaluate primer performance in silico (Cole et al. 2005; DeSantis et al. 2006; Quast et al. 2013). Previously published reviews only provide primer details instead of practical information of the primer performance. In this review, an updated summary was made on several aspects: (i) the current diversity and ecology of Scalindua and non-Scalindua genera of anammox bacteria, (ii) evaluation of primer performance both in silico and in practical tests, and (iii) information of PCR performance outcome of general and specific anammox bacteria groups and on different sample types. This mini-review aims to offer PCR information of practical applications and guidance on selection of appropriate PCR primer pairs for different purposes.

Diversity of anammox bacteria

The first identified anammox bacteria were given the provisional name Candidatus Brocadia anammoxidans (Strous et al. 1999). Up to now, there are five genera of anammox bacteria including at least 22 Candidatus species (Fig. 1). The Brocadia includes Ca. Brocadia anammoxidans, Ca. Brocadia fulgida, Ca. Brocadia sinica, Ca. Brocadia caroliniensis, Ca. Brocadia sapporoensis, and Ca. Brocadia brasiliensis (Araujo et al. 2011; Kartal et al. 2008; Narita et al. 2017; Oshiki et al. 2011; Rothrock et al. 2011; Strous et al. 1999). All Brocadia species were enriched in anammox bioreactors (Sonthiphand et al. 2014). Ca. Kuenenia stuttgartiensis is the only identified species in Kuenenia so far (Schmid et al. 2000). Ca. Anammoxoglobus propionicus is the only identified species in Anammoxoglobus, a propionate oxidizing species out-competing other anammox bacteria and denitrifiers in an anammox bioreactor in the presence of propionate (Kartal et al. 2007). Ca. Jettenia asiatica, Ca. Jettenia caeni and Ca. Jettenia moscovienalis are three identified species in the genus Jettenia, enriched in a granular sludge anaerobic anammox reactor from river sediment as inoculum, an active sludge incubated membrane bioreactor (MBR), and an active sludge treating municipal wastewater, respectively (Ali et al. 2015; Nikolaev et al. 2015; Quan et al. 2008).

Fig. 1
figure 1

Phylogenetic tree of 16S rRNA gene of currently defined anammox species (a) and phylogenetic tree of ribosomal protein (12 ribosomal protein alignment) of currently available anammox bacteria genomes. Jukes-Cantor method, gamma distribution of site variation, pairwisely aligning for each sequence pair, and 1000 time bootstrap testing were applied to make the neighbor-joining tree (a). RAxML-HPC v.8 (implemented in CIPRES) with “-m PROTGAMMALG -n 1000” was applied to make the RAxML tree (b)

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Up to now, there are 11 established species in Scalindua, namely Ca. S. sorokinii, Ca. S. richardsii, Ca. S. brodae, Ca. S. wagneri, Ca. S. arabica, Ca. S. pacifica, Ca. S. profunda, Ca. S. sinooilfield, Ca. S. naihaiensis, Ca. S. rubra, and Ca. S. zhenghei (Dang et al. 2013; Fuchsman et al. 2012; Hong et al. 2011; Kuypers et al. 2003; Li et al. 2010a; Schmid et al. 2003; Speth et al. 2017; van de Vossenberg et al. 2008; Woebken et al. 2008; Zhou et al. 2017). However, these tentatively named species could not cover the complete diversity of uncultured Scalindua and regional specific distribution pattern of Scalindua sp. clades, suggesting that more undiscovered endemic groups are still available (Oshiki et al. 2016). Scalindua spp. have a considerably large micro-diversity (Schmid et al. 2007; Woebken et al. 2008). Recent studies suggest that regional specific sub-clusters are evident, while at the same time, for large clades, such as Scalindua arabica clade and Scalindua sorokinii/brodae clade, some species have a globally wide occurrence (Hong et al. 2011; Oshiki et al. 2016; Woebken et al. 2008). Until now, there is still no pure anammox bacterial culture achieved, even though anammox bacterial species could be enriched to nearly pure culture (~ 90% biomass) allowing investigation on their physiological properties possible (Ali et al. 2015; Kartal et al. 2007; Oshiki et al. 2016; Park et al. 2010).

Ecology and environmental influence

Scalindua in marine ecosystem

As for Scalindua spp., many studies showed that they are mainly distributed in marine sediments or water column with a large micro-diversity within the genus (Dang et al. 2013; Schmid et al. 2007; Woebken et al. 2008). However, some non-Scalindua populations are commonly detected in the coastal and estuarine ecosystems and they might be exogenous inputs from adjacent terrestrial river runoff or anthropogenic discharges, thus could serve as bioindicators for anthropogenic/terrestrial input and pollution (Han and Gu 2015). Meanwhile, change of anammox bacteria community composition from sediments of riparian locations, coastal wetlands to marginal sea further showed that such a distribution pattern of diversity was negatively correlated with inorganic nitrogen species from pollution (Dale et al. 2009; Dang et al. 2010; Han and Gu 2015; Li et al. 2011a, c, 2013). Anammox bacterial activities play an important ecological role in nitrogen loss in marine deep sediments (20~80% N loss), continental slope, and estuarine and coastal sea ecosystems (less than 20% N loss); meanwhile, within the anoxic marine water column, anammox process is considered the major contributor to the nitrogen production (Dalsgaard et al. 2005; Jensen et al. 2011; Kuypers et al. 2005; Lam et al. 2009; Thamdrup et al. 2006).

Environmental influence on marine anammox bacteria

Dissimilatory nitrate reduction to ammonium (DNRA) could couple with anammox process by fueling anammox activity with reduced NH4+, while, denitrification in oligotrophic and electron donors limited conditions reduces NO3− to NO2−, and the latter nitrogen compound is also the direct substrate of anammox bacteria (Hu et al. 2011; Jensen et al. 2011). Aerobic ammonia oxidizers in a marine oxygen-limited zone could oxidize NH4+ to NO2− to further deplete oxygen available to create a suitable condition for anammox activity (Lam et al. 2007; Woebken et al. 2007). Both denitrification and availability of ammonium in the marine water column depend on availability of organic matters, thus local availability of organic matters and their mineralization rates are the critical influencing factors on anammox processes (Hu et al. 2011; Lam et al. 2009). In marine sediments and water column, factors favoring anammox existence and activity include low availability of oxygen, low temperature, and stable environmental conditions (Dalsgaard et al. 2005, 2014; Jensen et al. 2008; Oshiki et al. 2016; Russ et al. 2013).

Non-Scalindua in terrestrial ecosystem

In terrestrial ecosystem, anammox process is also an important pathway for nitrogen loss, such as fertilized paddy soil (4–37% N loss), lake riparian zones (< 10% N loss), water column of freshwater lake (~ 13% N loss), contaminated groundwater (18–36% N loss), and constructed wetland (24% N loss) (Erler et al. 2008; Moore et al. 2011; Schubert et al. 2006; Zhu et al. 2011, 2013). Most of the previously investigated sites of freshwater ecosystems were dominated by Brocadia and Kuenenia species (Moore et al. 2011; Zhu et al. 2013). While, in a paddy field, surface soil layers contained Kuenenia, Anammoxoglobus, and Jettenia but deep soil was dominated by Brocadia (Zhu et al. 2011). However, there are still several specific reports of Scalindua appearing or dominating in pristine environments, including freshwater Lake Tanganyika water column (Schubert et al. 2006), wetland (Lee et al. 2016), subsurface oil reservoirs (Li et al. 2010a), and rice paddy in northeast China (Wang and Gu 2013). These indicate a more diverse distribution pattern of anammox bacteria in freshwater column and sediments, resulting from the heterogeneous micro-niches in terrestrial ecosystems favoring the growth of different anammox bacterial groups. The versatile metabolism of Brocadia and Kuenenia on utilizing various electron acceptors and donors also facilitated their adaptation to specific environmental conditions even with limited substrates available for anammox metabolism to take place (Strous et al. 2006).

Salinity is a major factor correlated with the global anammox bacteria distribution pattern and is also the key driving force determining the niche specificity of non-Scalindua and Scalindua (Sonthiphand et al. 2013). Community composition differences between natural and engineered ecosystems could be due to the selection pressure imposed by long-term perturbation to select superior competitors to become dominant (Sonthiphand et al. 2013). Freshwater sediments have the highest diverse anammox bacterial population while marine water column the lowest. In terms of the co-occurrence, Scalindua taxa tend to occur alone in pristine condition, but Brocadia taxa could be found to coexist with all other anammox bacterial genera (Sonthiphand et al. 2013).

PCR primers of 16S rRNA and functional genes

PCR primers of 16S rRNA gene are most frequently used in previous and current studies (Table 1). According to the information summary (Table S1), specific primer pairs to detect Scalindua or non-Scalindua are compatible between in silico and practical tests, though there are still some exceptions. Amx368F-Amx820R is recommended for retrieving non-Scalindua, and Amx368F-BS20R for Scalindua, both with good performance in silico test (Fig. 2). Amx368F-Amx820R has poor in silico coverage (18%) for Scalindua; however, many researches of both marine and terrestrial environments suggest that it can also amplify Scalindua sequences from marine origin samples free of mismatches in the 5’ end and obtain similar community as Amx368F-BS20R from both marine and terrestrial environments (Amano et al. 2007; Humbert et al. 2010; Yoshinaga et al. 2011).

Table 1 PCR primers of 16S rRNA gene for clone library and qPCR of anammox bacteria from various samples
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Fig. 2
figure 2

Properties of 16S rRNA gene primer and primer pairs on different genera of anammox bacteria deduced by in silico primer evaluation. SILVA SSU132 RefNR as database, 2 maximum number mismatches were used in TestProbe 3.0; and SILVA SSU132 RefNR as database, 2 maximum number mismatches and 1 site of 0-mismatch zone at 3’ end were used in TestPrime 1.0. (https://www.arb-silva.de/search). A Perl script was used to calculate primer coverage and specificity for anammox bacterial groups. Primer pairs are the most frequently reported ones in references. “Other Brocadiaceae” stands for Brocadiaceae group which is not clustered in any of the defined genera of Anammoxoglobus, Brocadia, Jettenia, and Kuenenia

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Functional proteins, including cluster 1 of hydrazine oxidoreductase (Hzo) of anammox bacteria, converting N2H4 to N2, nitrite reductase (NirS), catalyzing NO2− to NO, hydrazine synthase (Hzs), converting NH4+ and NO to N2H4, and cytochrome c biogenesis proteins (Ccs), facilitating heme transport into anammoxosome to maintain its reducing state, reflect phylogeny and biochemistry function well (Harhangi et al. 2012; Li et al. 2011b; Schmid et al. 2008; Zhou et al. 2017). Some of them acquired good genera coverage and specificity on general anammox bacteria and selective genera (Table 2 and Table S2). Because of the divergence of protein biochemistry and coding sequence between the anammox bacteria and the others, use of functional gene primers could facilitate detecting specific anammox bacteria and measuring corresponding functional activity by reverse transcription (RT)-PCR (Li and Gu 2011; Zhou et al. 2017).

Table 2 PCR primer pairs based on functional genes of anammox bacteria for clone library and qPCR
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As mentioned previously, primers with mismatches in silico could still offer acceptable PCR results (high specificity); however, it will potentially cause (i) bias of results, reflecting unreal community or (ii) offering non-specific results or (iii) reduced primer coverage. Primer sets of 16S rRNA gene with good performance both in silico and in practical tests are highly recommended for amplifying non-Scalindua, Scalindua, and whole anammox bacteria and the relevant information is summarized in Table 3. Both coverage and specificity are taken into consideration. Nested PCRs are encouraged to increase specificity of complex environmental samples, e.g., soil and sediments with high organic matter, and those with very small anammox bacteria proportion (< 1%) in the total microbial biomass, though it sacrifices coverage (Kartal et al. 2011a).

Table 3 Summary of PCR primer pairs of 16S rRNA and functional genes of anammox bacteria for practically detecting anammox bacteria with good performance and proper use
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PCR primers for different genera

Scalindua species generally appear in marine ecosystem, intertidal or closed niches without human disturbance, e.g., estuary sediment, groundwater, and wetland ecosystems, though some exceptions are possible in terrestrial ecosystems, while non-Scalindua species are completely the reverse. Primer pairs targeting all anammox bacteria and specifically Scalindua, non-Scalindua, Kuenenia, and Jettenia are all summarized, and some of them suitable for both qPCR and PCR for clone library (Table 3). Due to the high divergence of 16S rRNA gene among different genera of anammox bacteria (< 87.1% identity), higher coverage leads to higher primer degeneracy (Junier et al. 2010; Li and Gu 2011). However, high primer degeneracy will probably lead to non-specific amplification from complex sample matrix. A pre-assessment of microbial community will offer a better choice of proper specific primers and improve the PCR outcome. The practical performance of specific primers on different genera also serves as a checklist for researchers to pick PCR primers based on similar situations (Tables S1 and S2).

PCR primers for different samples

For saline environmental category, e.g., marine sediments, marine water column, PCR primers for all anammox bacteria and for specific Scalindua usually have good performance (Tables S1 and S2). These environments tend to be of less diverse community and less PCR inhibitory chemicals, such as humid acids (Schrader et al. 2012). Many researches adopted 16S rRNA and functional genes to quantify Scalindua with good performance on study of biogeochemistry processes and activities (Bale et al. 2014; Lam et al. 2007; Pitcher et al. 2011).

For non-saline environment category, including a wide range of soils, WWTPs, reactors, groundwater, freshwater, and freshwater sediments, and intertidal or brackish environmental category, estuary and mangrove sediments, selection of primers shall consider (i) interests on function or phylogeny, (ii) quantification or qualification, and (iii) pre-assessment of bacterial community as complex or simple, low, or high anammox biomass and marine or terrestrial origin, to achieve better outcomes (Tables S1 and S2). For example, WWTPs and reactors are long-time operated to enrich anammox biomass, thus, less diverse community and simple environmental background will lead to less non-specific PCR amplification, and most of the suggested primer pairs could offer good outcome (Han et al. 2017; Han and Gu 2015). For samples with potential complex background, such as soil, estuary and mangrove sediments, primers with high degeneracy rate could potentially cause over-estimation of anammox bacterial community and yield more non-specific PCR products. Nested PCR procedures are suggested to retrieve anammox bacteria from these environments to improve specificity. Additionally, functional gene primers, specifically targeting functional proteins, could also enhance specificity and avoid over-estimation (Table 3).

Summary and future perspectives

Anammox bacteria are attracting increasingly attention in both research and applications on nitrogen cycle and transformation. The first discovery and applications of anammox bacteria not only fill the missing gap of microbial participants on nitrogen cycle but also improve industrial implementation of nitrogen removal in WWTPs comparing to conventional technologies (Meng et al. 2017). DNA-based molecular detection and quantification methods for anammox bacteria specifically have been applied in many studies in environmental engineering, microbial ecology, and biogeochemistry, and PCR is a basic tool and accessible widely for studying community diversity, abundance, and activity. The evaluation and a summary of performance of 16S rRNA and functional gene PCR primers in practical and in in silico tests in this article serve as a practical guide for researchers to select PCR primers according to specific samples type and/or the group of anammox bacteria of interest for detection and quantification. The effective implementation of PCR primers and PCR techniques will facilitate successful execution on detection of anammox bacteria and therefore contribution to further information on their ecology, diversity, and functions in different ecosystems.

As the rapid development of high throughput sequencing in the recent years, sequencing millions of PCR amplicons is practically feasible and even more suitable for comparing community structures among different samples (Caporaso et al. 2012). To include more phylogenetic information into community structure results by sequence analysis, it will be better to use 16S rRNA gene-based primers to study anammox bacteria, comparing to functional genes. Meanwhile, it will also be easier for users to adapt to currently most frequently used pipelines and softwares, such as QIIME and mothur, to get sequence analyzed (Caporaso et al. 2010; Schloss et al. 2009). The 16S-tag is generated from merging two pair-end reads (PE reads) according to the most currently adopted Illumina platform (Wang et al. 2012b). Since the read length of mainstream version of PE reads is usually 150 bp, the merged 16S-tag will be less than 300 bp. The 16S rRNA gene primer, A438f-A684r, with the targeted PCR product length of 250 bp, is suitable for this purpose with good coverage and specificity.

A general guideline on the selection of most appropriate PCR primers for different situations could be achieved from currently ten major environmental types and such information can serve as basic background information. Since more and more research reports on the practical applications of PCR primers in different types of environmental samples are becoming available, a more detailed, up-to-date, and comprehensive summary of the information on PCR primer performance and PCR outcome is necessary to the research community, especially the new ones into this research field because different choices can be made easily and the results may be biased without in-depth knowledge of the information available comprehensively.