title: Microbial communities and off-grid wastewater treatment
Creators: name: Shi, Linghui
Publisher: name: University of Glasgow
Year: 2025

description: Water and sanitation have improved significantly in the past century due to advanced wastewater treatment. Centralised wastewater treatment systems deal with large amount of wastewater in small footprint are always preferred in urban areas. In contrast, decentralised wastewater treatments are more common in rural areas. Among these, septic tanks are one of the most common off-grid solutions, however, their performance various and hardly meets any wastewater discharge standards. This thesis presents a study on the performance and microbial community of small-scale constructed wetland systems treating wastewater from septic tank. Additionally, a lab-based batch experiment to study the change of microbial community and antimicrobial resistance (AMR) during acute amoxicillin exposure.

Three small-scale constructed wetland systems were built and operated in Mexico. Two identical household systems (WS1 and WS2) collected wastewater from single households. They consisted of an equalization tank and a biodigester (AR), followed by a horizontal constructed wetland (CW1), a vertical constructed wetland (CW2) and a holding tank (HT) for the reuse of treated wastewater in gardening. WS3, while similar to WS1 and WS2, received toilet wastewater from a school building and included an additional aeration tank between AR and CW1. All three systems were monitored from the initial three months of the operation, with WS1 and WS2 undergoing an extended monitoring of ten months. Two household systems (WS1 and WS2) were receiving wastewater with high chemical oxygen demand (COD) concentration (average 1131.4mg/L in WS1 and 1894.7mg/L in WS2), NO2 - concentration (average 51.1mg/L in WS1 and 76.7mg/L in WS2) and NO3 - (average 106.4mg/L in WS1 and 214.8mg/L in WS2). Despite higher influent concentrations than full-scale constructed wetland, our constructed wetland system removed at least 73.4% COD, 79.9% NO2 - and 78.8% NO3 - in average. School system (WS3) received wastewater with much lower COD (153.0-587.0mg/L), NO2 - (3.0-54.4mg/L) and NO3 - (13.7-62.9mg/L) concentrations than the household systems, whilst achieved similar removal rates to household systems (58.8-89.9% of COD, 39.3-58.8% of NO2 - and 29.7- 80.6% of NO3 - ).Removal of COD, NO2 - , NO3 - in all three systems were comparable to long-term operational full-scale constructed wetland systems in Mexico from the first month. It took 10 months to achieve a stable removal of COD, NO2 - , NO3 - in both household systems. Phosphate accumulated in the systems in long-term and NH4 + was not removed. Microbial communities were analysed through 16S rRNA sequencing, where AR and HT (influent and effluent of constructed wetlands) were analysed over the monitoring time with additional analysis of within CW1 and CW2 of each system in the last seven months. Microbial community composition in WS3 shared less similarity with household systems, where despite influent was different in two household systems the microbial community compositions in WS1 and WS2 were similar to each other. Bacteroidota was the most dominant phylum in the first three months, while Proteobacteria became the most dominant phylum in the long-term study. Only eight ASVs from the first three months were shared with all 46 most abundant ASVs in long-term study, which suggests the change of microbial community happened overtime. Microbial communities took longer (14 months) to stabilise than pollutant removal, and despite different microbial communities were found in these two systems they achieved comparable pollutant removal rates.

Laboratory preliminary experiments aimed to develop a rapid approach to detect AMR in mixed microbial community from wastewater were undertaken. The growth of anaerobic microbial communities under aerobic condition was monitored by optical density (OD) to mimic transition of wastewater from a septic tank to constructed wetland. A gradient of amoxicillin concentrations (0- 32.0mg/L) was tested, revealing that the growth rate of the microbial community varied across three distinct ranges with increased amoxicillin concentrations in which beneficial range (0-1.5mg/L), detrimental range (1.5- 4.5mg/L) and no further change range (greater than 4.5mg/L). Meanwhile the length of lag phase increased solely with rising amoxicillin concentrations. A further experiment with selected amoxicillin concentrations examined the change of microbial communities through 16S rRNA sequencing and the expression of antimicrobial resistance genes (ARGs) via high throughput quantitative polymerase chain reaction (HT-qPCR), respectively. A shift of the microbial community composition was observed after amoxicillin exposure, where relative abundance of Paenibacillus azoreducens increased while Bacillus cereus group decreased in all amoxicillin exposed microbial communities in both DNA and RNA. However, data were insufficient to conclude that amoxicillin exposure was the only factor for this shift. ARG expression increased with amoxicillin concentration, however ARG abundance was not available due to poor data quality. The results suggest that the length of lag phase may serve as a potential measure for assessing AMR in mixed microbial community, although further work is needed to validate this method.

This study highlights that small-scale constructed wetland systems are capable of removing pollutants from wastewater after septic tank, which could be a potential solution for off-grid sanitation. The potential risk of spreading pathogens and AMR associated with small-scale constructed wetland remain unknown. Further research is needed to understand and assess those risks in the future.