A new strategy for the enrichment of ammonia-oxidizing archaea in wastewater treatment systems: The positive role of quorum-sensing signaling molecules

Highlights

• The addition of exogenous AHLs facilitates the growth of archaea in the wastewater treatment system.

• The enrichment effect of different types and concentrations of AHLs on AOA was different.

• 400 nmol of C₈-HSL and 800 nmol of C₁₀-HSL had the most significant enrichment effect on AOA.

Abstract

Ammonia-oxidizing archaea (AOA) play an important role in natural nitrogen cycle, but are difficult to be enriched in wastewater treatment systems. In this experiment, under ambient temperature and high dissolved oxygen, different types of acyl-homoserine lactones (C₆-HSL, C₈-HSL, C₁₀-HSL, C₁₄-HSL and 3-oxo-C₁₄-HSL) were added to five wastewater nitrification systems to achieve AOA enrichment. Results showed that AOA couldn't be detected in the blank group without the addition of signaling molecules, while the AOA could be detected in all the reactors with the addition. The enrichment effect of AOA was not obvious with added 100 or 200 nmol/L signaling molecules, while the enrichment effect was both obvious with added C₈-HSL of 400 nmol/L and C₁₀-HSL of 800 nmol/L. And relative abundance of AOA increased from undetected in the control group to 1.10 % and 0.96 %, respectively. The exogenous signaling molecules may provide new view for AOA enrichment in wastewater treatment systems.

Introduction

In 1977, Woese proposed the famous three-domain doctrine, in which the Archaea, Bacteria and Eucaryota domains were listed as the three major domains of organisms. The archaea domain mainly includes Crenarchaeota, Euryarcheaota and Thaumarcheota, etc. In 2004, metagenomics studies revealed that the genomes of marine archaea contained structural genes - amoA, amoB and amoC, which are key enzymes in ammonia oxidation (Leininger et al., 2006). Subsequently, the first strain of ammonia-oxidizing archaea (AOA) was isolated and cultured from Seattle Aquarium seawater, confirming that archaea also have the ability to oxidize ammonia at the biometabolic level. Numerous subsequent studies have shown that archaea have stronger environmental adaptations than bacteria under extreme conditions (e.g., low temperature, low dissolved oxygen (DO) and low ammonia concentration levels (Gubry-Rangin et al., 2011; Yin et al., 2018)). Due to the significant differences in phylogeny and genetic composition of a class of mesophilic archaea represented by AOA from the phylum Crenarchaeota and Euryarcheaota, such mesophilic archaea have been classified into a completely new phylum, namely the phylum Thaumarcheota.

With the continuous research on nitrogen cycling processes, many studies have found that AOA usually occupy an important position in ammonia‑nitrogen oxidation processes in natural environments such as deep sea (Mincer et al., 2007), lakes (Liu et al., 2014; Yang et al., 2016), soils (Wang and Huang, 2021). And in some natural environments, the population of AOA are significantly higher than ammonia-oxidizing bacteria (AOB), which breaks the view that AOB is the only microorganism in the process of ammonia oxidation and proves that AOA plays an important role in the nitrogen cycle in nature. AOA can produce AMO for the conversion of ammonia nitrogen, but the physiological, biochemical and genetic characteristics of AOA significantly differ from those of AOB. AOB normally uses AMO to convert ammonia nitrogen to hydroxylamine (NH₂OH), which is then further oxidized to nitrite by hydroxylamine oxidase (HAO) (Bock and Wagner, 2006). Since AOA lacks genes encoding a recognizable AOB-like HAO complex and associated cytochrome c proteins, it is possible that hydroxylamine is oxidized to nitrite via other pathways, and that AOA may have a unique biochemical oxidation of hydroxylamine or that dispersed AMO does not actually produce hydroxylamine (Walker et al., 2010). AOA is usually autotrophic, but also possesses organic carbon metabolic pathways that allow for mixed-nutrient growth. In addition, the presence of AOA has been found in artificial environments. AOA has been found in wastewater treatment plants with low dissolved oxygen and long retention times (Park et al., 2006). In aquaculture wastewater treatment, AOA is effective in removing ammonia nitrogen from wastewater (Lu et al., 2021), especially in seasonal low temperature and in anaerobic sediment layer environments where it plays a major role. This shows that AOA also has a great potential for nitrogen removal processes in domestic wastewater treatment systems. The affinity of AOA for ammonia nitrogen and oxygen can be used to control the conditions of low ammonia nitrogen or low DO, so that it can significantly increase the abundance of AOA and achieve AOA enrichment (French et al., 2012). In addition, high concentrations of spiramycin can also increase the abundance of AOA (Zhang et al., 2015), and Men et al. (2016) had obtained Nitrosococcus viennensis by pure culture. However, the method of pure culture enrichment has serious environmental requirements, and the AOA obtained by pure culture is difficult to be used in actual wastewater treatment. The high concentrations of NH₄⁺-N and DO in wastewater treatment systems are not conducive to the growth of AOA because of the eutrophic environment. New ideas are needed to realize the enrichment culture of AOA in wastewater treatment systems.

It has been found that under certain conditions, a quorum sensing (QS) system may exist in AOA (Montgomery et al., 2013). QS is a cellular communication mechanism commonly found in microbial communities, i.e., microorganisms can spontaneously produce signaling molecules in a certain environment and can sense changes in concentration in the environment. And when the concentration of signaling molecules reaches a threshold, they will be recognized by receptors in microorganisms to regulate their gene expression, thus having the function of coordinating the physiological behavior of the colony and regulating the ecological relationships of the colony. This mechanism is generally common among bacteria. Acyl-homoserine lactones (AHLs) are typically signaling molecules produced by Gram-negative bacteria, in wastewater treatment systems, denitrifying bacteria can spontaneously produce AHLs under certain conditions. Many studies (Li and Zhu, 2014; Mellbye et al., 2016) have shown that the analysis of the effect of exogenous signaling molecules on nitrogen removal bacteria and all have preliminarily shown that QS can modulate nitrogen removal by nitrifying bacteria. Montgomery et al. (2013) was the first found that the same QS mechanism exists in archaea, and in extreme environments, there may be multi-level communication among archaea. Although AOA is archaea, AOA is generally symbiotic with AOB or nitrite oxidizing bacteria (NOB) in nature, and it is likely that the functional genes of related signaling molecules will have gene migration, so AOA may also be affected by AHLs. Therefore, it is potentially feasible to use exogenous signaling molecules to induce the QS mechanism of AOA and thus achieve AOA enrichment.

There are few studies on the enrichment strategy of AOA in wastewater treatment systems, especially the effect of QS on the succession of AOA populations is not clear. In this paper, we propose to induce the QS mechanism of AOA in the wastewater nitrification systems by adding exogenous signaling molecules, analyze the effects of different signaling molecule types and concentrations on the abundance of AOA population, and screen the conditions favorable to AOA enrichment.