Section 2: Part Two: The Effect of Immigration on Microbial Community Dynamics in Full-Scale Anaerobic Digester Systems A six-year investigation...
Published on by Hossein Ataei Far, Deputy Manager of the Research, Technology Development, and Industry Relations Center at NWWEC
🌍Introduction
Anaerobic digestion ♻️ is a widely utilized process for treating organic waste at wastewater treatment plants. Understanding the microbiology involved is crucial for optimizing operational efficiency 🧬. This study investigates microbial communities in 32 full-scale anaerobic digesters across Danish wastewater treatment plants over six years using 16S rRNA gene amplicon sequencing 🔬. The study reveals that many of the most abundant microbial populations are likely inactive 💤 and introduced through the influent, highlighting the risk of misleading analyses if immigration is overlooked 🚫.
🌍Challenges in Taxonomy and Classification
The study found that several abundant operational taxonomic units (OTUs) could not be classified at the genus level using standard taxonomies, complicating the inference of their functions 🧩 and comparison with other studies. As a result, the MiDAS taxonomy was updated 🗂️ to include these abundant phylotypes. Across the digesters, microbial communities were remarkably similar, with only 300 OTUs accounting for 80% of total reads across all plants 🌱. Of these, 15% were identified as non-growing, possibly inactive immigrants 🚶. This identification of abundant and growing taxa sets the stage for targeted characterization 🎯 of key organisms in anaerobic digestion.
🌍Significance of Anaerobic Digestion
Biogas production ⚡ through anaerobic digestion is increasingly adopted as a renewable energy source 🌞, offering the potential for wastewater treatment plants to become net energy producers. The process relies on the synergistic activities of complex microbial communities through four stages: hydrolysis, fermentation, acetogenesis, and methanogenesis. Despite its robustness, anaerobic digestion faces operational challenges such as foaming 🌊 and efficiency drops 📉. A deeper understanding of the microbiology underlying these processes is essential for optimization 🧠.
🌍Linking Microbial Identity to Function
To understand anaerobic digester ecology and its relationship to system function, it is essential to identify active and abundant microorganisms 🔍 and link their identities to their functional roles. Previous 16S rRNA gene amplicon studies have identified a stable set of abundant microorganisms in similarly operated anaerobic digesters ⏳. However, in many cases, the microbial community may include dead or inactive cells ❌ introduced with the influent biomass, which can persist due to DNA, leading to spurious correlations and false conclusions 🚫.
🌍Methodological Considerations
Molecular techniques have been developed to mitigate the influence of DNA from inactive cells in microbial analysis 🧪. However, the complex nature of anaerobic digester sludge samples can lead to unwanted chemical reactions ⚗️ and limited light penetration in the process. An alternative approach is to monitor the microbial composition of the influent 🛢️ to identify organisms whose abundance is maintained by immigration.
🌍Taxonomic Classification and Challenges
Associating phylogeny with function is critical for understanding system ecology 🌱. However, a significant proportion of sequences from previous surveys of anaerobic digesters were not classified at the genus level using standard taxonomies like SILVA, RDP, and Greengenes 🧬. This, combined with biases associated with DNA extraction, primer coverage, and differing taxonomies, hinders cross-study comparisons 🧩. Therefore, standardized methods and a curated taxonomy are essential for meaningful cross-study comparisons and robust biological conclusions 📊. The MiDAS protocols and curated taxonomy, established for activated sludge in wastewater treatment plants, provide a model for standardization in anaerobic digestion.
🌍Community Structure of Anaerobic Digesters: Archaea
The archaeal communities differed significantly between thermophilic and mesophilic reactors 🌡️. In mesophilic reactors, the acetoclastic methanogenic genus *Methanosaeta* was dominant, while thermophilic reactors were dominated by the hydrogenoclastic genus *Methanothermobacter*, followed by *Methanosarcina* 🔬. *Methanobrevibacter* was another abundant archaeon found in both mesophilic and thermophilic reactors, though absent in mesophilic reactors with thermal hydrolysis pre-treatment 🚫.
🌍Community Structure of Anaerobic Digesters: Bacteria
Bacterial communities also showed distinct clustering 🧩 based on reactor temperature and pre-treatment. The dominant phyla included Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Chloroflexi 🌿. Some genera, like *Tetrasphaera* and *Ca. Microthrix*, were abundant in both mesophilic and thermophilic reactors but were absent in mesophilic reactors with thermal hydrolysis pre-treatment, suggesting they were introduced with the influent and not actively growing in the digesters 🛢️.
🌍Survival of Influent Bacteria in the Digesters
The study found that some organisms were present in both influent streams and digesters, while others were detected primarily in one sample type 🧬. No overlap was found between communities in influent streams and reactors with thermal hydrolysis pre-treatment 🚫. By comparing the ratios of mean read abundance in the digester versus the influent streams, the study determined whether immigrating organisms were likely to die off, survive, or grow in the digesters. A clear bimodal distribution of these ratios was observed, highlighting a distinction between organisms that thrive in anaerobic digesters and those that do not 🧠.
🌍Discussion
This study provides an extensive analysis of microbial communities in full-scale anaerobic digesters, emphasizing the importance of considering immigration when analyzing microbial communities 🔍. The updated MiDAS taxonomy allows for more accurate genus-level classification 🧾, improving cross-study comparisons and our understanding of microbial ecology in these systems. The study also underscores the need for standardized methods and primer-free alternatives to capture the full microbial diversity in anaerobic digesters 🧠. By simplifying the complexity of biological processes, these findings will help inform future research and optimization efforts in anaerobic digestion.
**Reference:**
[1] Rasmus H. Kirkegaard et al., (2017), "The impact of immigration on microbial community composition in full-scale anaerobic digesters."
Figure 1. Heatmap of the 20 most abundant bacterial genera: (A) Taxa sorted by the mean read abundance in the influent (primary and surplus sludge), (B) Taxa sorted by the mean read abundance in the anaerobic digesters (mesophilic, thermophilic, and THP). The numbers represent mean read abundances for groups with more reactors and more samples (30–279).