Application of meta-omics technologies reveals microbial ecology in anaerobic digestion

Lin, Tzu-Yu

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https://hdl.handle.net/2142/120524 Copy

Description

Title

Application of meta-omics technologies reveals microbial ecology in anaerobic digestion

Author(s)

Lin, Tzu-Yu

Issue Date

2023-04-19

Director of Research (if dissertation) or Advisor (if thesis)

Liu, Wen-Tso

Doctoral Committee Chair(s)

Liu, Wen-Tso

Committee Member(s)

• Marinas, Benito J
• Wei, Na
• Men, Yujie

Department of Study

Civil & Environmental Eng

Discipline

Environ Engr in Civil Engr

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Anaerobic digestion
• Wastewater treatment
• Microbial

Abstract

Anaerobic digester (AD) is an essential biological process in wastewater treatment to reduce excessive sludge. In AD, wasted sludge is converted to biogas and more biologically stable solids by a series of reactions carried out by microbes. As microbial activity is critical in AD, understanding the factors that affect microbial assembly is beneficial to improve AD performance. Conventionally, microbial studies rely on culture-based approaches or 16S rRNA gene sequencing, restricting the investigation on the microbial functions of the vast majority of uncultivated microbes. Meta-omics technologies enable characterization of microbes in silico, bypassing the necessity of cultivation and PCR amplification. The main objective of this dissertation is to utilize meta-omics to reveal the microbial ecology in AD under the influence of different impact factors. Previously, 16S rRNA gene was coupled with ecogenomics-based mass balance (EGMB) model to evaluate the effect of microbial immigration in AD. The 16S rRNA gene-based EGMB model identified the microbial immigrants by the estimation of microbial growth rate. However, substantial proportion of uncultivated microbes were also revealed. Characterization of these uncultivated microbes is unavailable using 16S rRNA gene sequencing but is attainable through metagenomic analysis. In this dissertation, the effectiveness of both 16S rRNA gene- and metagenome-based EGMB model is firstly verified in a lab-scale bioreactor. The feasibility of metagenome-based EGMB model in full-scale ADs for the characterization of microbial activity and role is further evaluated. Also, temperature and feed type have significant impact on AD microbiome. Unlike mesophilic AD, microbes and their contribution in thermophilic AD is yet to be characterized. Finally, knowledge of the impact of viruses in AD is limited. The viral ecology, the connection between AD viruses and upstream AS viruses, the potential hosts, and the functional genes in viral genomes are yet to be determined. The following sections outline the structure of the dissertation. Chapter 1 introduces the process of wastewater treatment and the key microbial guilds in AD, laying the groundwork for the following chapters. The history of analytical approaches used for characterization of AD microorganisms is discussed, including conventional cultivation methods, culture-independent DNA-based molecular biology techniques, high-throughput 16S rRNA sequencing, and meta-omics. Current knowledge of the impact of environmental conditions (e.g., pH and temperature), operating parameters (e.g., solid retention time and organic loading rate), feed composition (e.g., chemical and biological), and viruses on AD microbiome are reviewed. Objectives of this dissertation are stated in this section. Chapter 2 investigates the applicability and effectiveness of 16S rRNA gene- and MAG-based EGMB model by comparing to qPCR measurements. Mock communities consisted of three known aerobic and facultative anaerobic microbes (Aeromonas sp., Escherichia sp., and Pseudomonas sp.) were added to a lab-scale bioreactor to mimic the microbial immigration events in WWTP. qPCR, 16S rRNA gene, 16S rRNA gene copy number normalization (GCN), and metagenome were used to profile the microbial community and to couple with the EGMB model. The mock communities in the feed profiled by the approaches were all highly comparable. All EGMB models suggested negative growth rates of Aeromonas and Pseudomonas. All EGMB models except 16S rRNA gene-based suggested negative growth rates of Escherichia. Metabolic reconstruction of the contributing populations confirmed their ecological functions in the digestion process. The findings suggested that EGMB model can be used for microbial activity analysis and metagenome-based EGMB model can provide additional information on the potential role of microbes in AD. Chapter 3 estimates the microbial activities in full-scale ADs by evaluating the effect of microbial immigration using EGMB models. Since 16S rRNA gene-based EGMB model cannot imply ecological function of the identified active and inactive populations, the feasibility of metagenome-based EGMB model is evaluated. Our findings suggested that metagenome-assembled genome (MAG)-based EGMB model is comparable to 16S rRNA gene-based EGMB model, both models showed that aerobes and facultative anaerobes were mostly inactive in AD, while obligate anaerobes were active. As high percentage of uncultivated populations presented in AD, gene annotation using metagenomes characterized the respiratory mechanism and ecological function of these uncultivated populations. Chapter 4 investigates the effect of operating temperature and feed type on microbiome in full-scale ADs using metagenomics and metatranscriptomics. Metagenomic sequences from mesophilic and thermophilic AD that were fed with domestic wasted sludge or manure were collected. Core populations in each group were assigned, among thermophilic core populations, 18 out of 37 genera were affiliated with Firmicutes. Metatranscriptomic analysis revealed that uncultivated genera of Firmicutes were actively participating in each step of the thermophilic AD treating domestic wasted sludge. Our findings suggested that the contributing populations in thermophilic ADs were consisted of a limited taxa that were different from the ones in mesophilic ADs, namely, uncultivated Firmicutes and hydrogenotrophic methanogens. Chapter 5 addresses the impact of virus on AD microbiome using metagenomic analysis. With the aid of metagenomics, we uncovered the high diversity of virome in activated sludge (AS), mesophilic AD, and thermophilic AD that were previously undetectable by culture-dependent approaches. High proportion of novel viruses were recovered from the metagenomes, indicating the insufficiency of viral study nowadays. Diversity analysis suggested that the local habitats have more impact on the AD virome than the immigration effect. Host prediction revealed that viruses were mostly linked to site-specific active populations that are involved in syntrophic fatty acid oxidation, methanogenesis, hydrolysis, and fermentation in AD. Viral gene profiling showed that viral genes projected the metabolism of native microbes. We suspect viruses were involved in the digestion process by interacting with site-specific active hosts and by trafficking genes that are horizontally transferred from the active hosts, especially genes related to energy conservation and cofactor metabolism. Chapter 6 summarizes the key findings of this dissertation and discusses the implications to environmental research and engineering applications. Limitations and possible improvements are identified and discussed in detail. Overall, findings in this dissertation contribute to the understanding of the complicate microbial ecology in AD, providing fundamental knowledge for the improvement of AD performance in the future. The identification of active populations in AD may serve as an indicator of AD performance and provide in-depth diagnosis of the cause of poor performance in the perspective of microbiology. The prediction of the metabolism of uncultivated populations obtained from meta-omics analysis provides additional information that could be taken into consideration for isolation. By characterizing the contributing populations and their lifestyle, strategies can be designed to promote the growth of these populations. As viral infection is observed to be very specific to the active populations in AD, future studies can be performed to evaluate the possibility of using phages to manipulate microbial activity in AD.

Graduation Semester

2023-05

Type of Resource

Thesis

Copyright and License Information

Copyright 2023 Tzu-Yu Lin

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