Enhanced pollutant removal from rural non-point source wastewater using a two-stage multi-soil-layering system with blended carbon sources: Insights into functional genes, microbial community structure and metabolic function

Highlights

• Blended carbon sources (BCS) contribute to higher denitrification gene abundances.

• Epsilonbacteraeotra enriched in the surface of BCS to achieve nitrogen removal.

• BCS might release some signaling molecules for enhancing the energy metabolism.

• BCS excite the activities of histidine kinase, glycogen phosphorylase and ATPase.

Abstract

A two-stage multi-soil-layering system with blended carbon sources (MSL–BCS) was constructed at pilot scale for treatment of rural non-point source wastewater. Results showed the MSL–BCS system had effective removal efficiencies with 64% of TN and 60% of TP, respectively. The addition of BCS could result in higher (1.6–3.1 fold) denitrification gene abundances (nirS and nosZ) for enhancing denitrification. High-throughput sequencing approach revealed that the higher abundance (>50%) of Epsilonbacteraeotra (Genus: Sulfuricurvum, Family: Thiovulaceae, Class: Campylobacteria, Phylum: Epsilonbacteraeota) enriched in the surface of BCS, which suggested that Epsilonbacteraeotra are the keystone species in achieving nitrogen removal through enhancing denitrification at oligotrophic level. KEGG analysis indicated that BCS might release some signaling molecules for enhancing the energy metabolism process, as well as stimulate the enzyme activities of histidine kinase, glycogen phosphorylase and ATPase, and thereby the denitrification processes were strengthened in MSL-BCS system. Consequently, this study could provide some valuable information on the removal performance and mechanism of engineering MSL systems packed with BCS to govern the rural wastewater treatment.

Introduction

Rural non-point source (NPS) pollution, such as agricultural, livestock breeding, rural rainwater, rural domestic sewage, and atmospheric deposition (Chen et al., 2018; Shen et al., 2020), is becoming increasingly prominent (Ongley et al., 2010; Sun et al., 2012; Wang et al., 2020). Especially, in rural China, although the strength of NPS is relatively lower (CDO about 50–100 mg/L, TN about 5–10 mg/L, TP about 1–3 mg/L) than that in other wastewater, it is still higher than the surface-water criteria of Environmental Quality Standards for Surface Water (2.0 mg/L of TN for grade V, GB3838-2002, 2002) (Zhu et al., 2018), which has become a serious environmental challenge threatening water ecosystem (De Oliveira et al., 2020; Tang et al., 2020). Therefore, relatively low NPS still needs to be further purificated. Generally, ecological treatment technologies are widely applied to decontaminate rural NPS, which include vegetated drainage ditches, constructed wetlands, vegetation filter strip, vegetation filter belts, artificial floating islands, riparian buffer vegetation, and oxidation ponds (Gu et al., 2020; Yi et al., 2020; Zhou et al., 2019). However, these treatment systems are often time-consuming, and it is difficult to satisfy the purpose of rapid decontamination in some sensitive receiving water (Yi et al., 2020).

As a kind of combination technology, multi-soil-layering (MSL) system, developed by Wakatsuki et al. (1993) in the 1990s, has been widely used in the treatment of rural wastewater. MSL is a soil-based technology, it has been conducted to improve the treatment efficiency of wastewaters via enhancing inherent ability of soil (Guo et al., 2019, 2020). It can overcome the problems of traditional soil-based treatment technologies, such as large area, low removal efficiency and easy clogging (Guo et al., 2018; Tang et al., 2020). The core idea of MSL system is to modularize the soil with alternating the denser soil-mixture-block (SMB) layers and porous permeable layers (PL) to form multiple micro “aerobic-anaerobic” environments for removing pollutants. For instance, Luo et al. (2014) invented a two-stage vertical hybrid system, which was consisted of a trickling filter and an MSL reactor. The MSL reactor had a high removal performance of pollutants with that the removal efficiencies of chemical oxygen demand (COD), NH₄⁺-N and total phosphorus (TP) were 93.1%, 86.3% and 93.2%, respectively. Our previous work also built a hybrid two-step MSL system for the treatment of low C/N ratio polluted river water. Results showed that the COD, NH₄⁺-N and TP were >70%, 99% and 82%, respectively (Wei and Wu, 2018).

Although MSL systems have certain advantages over other soil-based treatment systems, how to maintain a good “aerobic anaerobic” environment is the key to achieve the nitrification-denitrifaication processes in these systems. Recently, some scholars develop the two-step MSL systems, consisted of aerobic and anoxic units, which functioned as nitrification and denitrification, respectively (Luo et al., 2014; Wei and Wu, 2018; Wu et al., 2016). However, due to the lower COD concentration after the treatment of aerobic units, there is an obvious problem with lack of carbon resource in anoxic units, and thereby could significantly weaken the denitrification process of these systems.

The solid-phase denitrification (SPD) system, using solid organic substances as carbon source and biofilm carriers, has been proved to be a promising approach to remove nitrate with high denitrification efficiency and good carbon-release performance in wastewater with low C/N ratios (Sun et al., 2018; Wang et al., 2016; Yang et al., 2020b). Recently, blended carbon sources (eg. PHBV (polyhydroxylbutyrate valerate)-rice hulls and PHBV-sawdust) have been invented to replace the traditional solid carbon sources (i.e. corncob, rice straw, rice hulls, wood chips, PHBV, polybutylene succinate) with cost saving. Our lab-scale results demonstrated that PHBV-sawdust system achieved higher total nitrogen removal efficiency than PHBV system (Yang et al., 2020a). Besides, previous studies focus on the in laboratory-scale solid-phase denitrification systems feeding by synthetic wastewater, limited information is available on constructing pilot-scale MSL system real wastewater treatment. Due to the amplification effect (complicated wastewater characteristics, engineering and climate factors), there might be large gaps between the laboratory-scale and pilot-scale on pollutants removal performance and mechanisms. Thus, building pilot-scale MSL systems is very necessary to test the performance of MSL systems for treatment of real wastewater. And the operation performance of these MSL systems with addition of novel solid carbon source have so far rarely been reported for rural NPS pollution.

Moreover, microbial activities in biochemical processes are recognized as a major contributor to pollutant removal of wastewater treatment (Chen et al., 2020; Wei and Wu, 2018; Zhu et al., 2018). However, few studies focused on the structures of microbial community and distribution characteristics of functional genes in biofilms on MSL system. Meanwhile, microbial metabolism and enzyme activities play a unique role in pollutant degradations (Metcalf et al., 2016). In recent years, phylogenetic investigation of communities by Tax4Fun, just based on 16S rRNA sequence data, has been demonstrated that it could be more effective and accurate in predicting the functional composition of microbial communities when combined with high-throughput sequencing (HTS) (Asshauer et al., 2015).

In this study, combining the advantages of natural biomass carbon source and synthetic biodegradable polymers, a novel blended carbon sources (PHBV-sawdust) was invented, and then apply it to MSL system for treatment of actual rural NPS pollution. Therefore, the objectives of this study were: 1) to investigate the performance of MSL–BCS system on COD, nitrogen and TP removal; 2) to get in-depth understanding into on the distribution characteristics of functional genes in biofilms attached to different carriers in MSL–BCS system, 3) to explore the structures of microbial community, microbial metabolic function and enzymatic activity in biofilms attached to different carriers in MSL–BCS system.