On resolving ambiguities in microbial community analysis of partial nitritation anammox reactors

Abstract

PCR-based methods have caused a surge for integration of eco-physiological approaches into research on partial nitritation anammox (PNA). However, a lack of rigorous standards for molecular analyses resulted in widespread data misinterpretation and consequently lack of consensus. Data consistency and accuracy strongly depend on the primer selection and data interpretation. An in-silico evaluation of 16S rRNA gene eubacterial primers used in PNA studies from the last ten years unraveled the difficulty of comparing ecological data from different studies due to a variation in the coverage of these primers. Our 16S amplicon sequencing approach, which includes parallel sequencing of six 16S rRNA hypervariable regions, showed that there is no perfect hypervariable region for PNA microbial communities. Using qPCR analysis, we emphasize the significance of primer choice for quantification and caution with data interpretation. We also provide a framework for PCR based analyses that will improve and assist to objectively interpret and compare such results.

Figure 1

Comparison of the three most widely used EUB primer pairs based on the literature assessment, primer pair 1 (1055f-1392r), primer pair 2 (338f-518r) and primer pair 3 (341f-543r) for the primer coverage and abundance of the microbial groups most relevant for the PNA process (abundance is defined as theoretically calculated value). AnAOB (anaerobic ammonium oxidizing bacteria), (AOB) ammonium oxidizing bacteria, Nitrobacter and Nitrospira (nitrite oxidizing bacteria) and EUB (total eubacteria).

Figure 2

Distribution of the diversity of primer pairs based on the literature survey using certain keywords (i.e., “qPCR”, “anammox”, “wastewater”), for different microbial groups and the percentage of usage. (1) AnAOB: Anaerobic ammonium oxidizing bacteria; (2) AOB: ammonium oxidizing bacteria; (3) NOB: nitrite oxidizing bacteria; and (4) DNB: denitrifying bacteria. (1) 16S: 16S rRNA gene; (2) amoA: ammonia monooxygenase; (3) hzs: hydrazine synthase; (4) nirK: copper-containing nitrite reductase; (5) nirS: cytochrome cd1 type nitrite reductase; (6) nxra: nitrite oxidoreductase, alpha subunit; and (7) nxrb: nitrite oxidoreductase, beta subunit.

Figure 3

Relative abundance profiling of three samples: (1) TUD_1, (2) TUD_2, and (3) TUD_3, based on the 16S rRNA amplicon sequencing, targeting multiple hypervariable regions of 16S rRNA gene.

Figure 4

Percentage dissimilarity based on qPCR between absolute abundance of eubacterial population measured using three different primer pairs: (1) pp1 (1055f-1392r), (2) pp2 (338f-518r), and (3) pp3 (341f-543r). In legend pp1-pp2: percentage dissimilarity between pp1 and pp2, pp2-pp3: percentage dissimilarity between pp2 and pp3, pp3-pp1: percentage dissimilarity between pp3-pp1 (percentage dissimilarity was measured between a range of 0–100%, the higher the percentage greater the difference between measured absolute abundance).

Figure 5

Relative abundance based on qPCR of anaerobic ammonium oxidizing bacteria (AnAOB), ammonium oxidizing bacteria (AOB), Nitrobacter (NOB) and Nitrospira (NOB) which is normalized to the abundance of total eubacteria (EUB), measured using three different primer pairs targeting two different hypervariable regions; error bar represents the standard deviation between qPCR technical triplicate runs.

Figure 6

Decision tree framework for qPCR analysis and 16S amplicon sequencing for PNA systems.

Figure 7

Schematic showing the approach used to extract PNA studies, which were used for the assessment.