Insights into Microbial Communities in Anaerobic Digesters

Anaerobic Digestion is a process used to treat strong wastewater, especially in industries. This method breaks down waste without oxygen, producing Biogas that can be used as energy. While this process works well, sometimes things do not go as planned, and the treatments can fail. The reasons for these failures can be unclear. Some possible reasons include harmful substances in the wastewater, too much organic matter, missing nutrients, or sudden changes in the type of waste.

Microbes, or tiny living things that cannot be seen by the eye, play a big role in the anaerobic digestion process. For a long time, we had limited information about how these microbes interact and affect the digestion process. With advancements in sequencing methods, we can now learn more about these microbial communities and how they relate to the overall operation of digesters.

Many studies have examined how these microbial communities change during the digestion of organic waste. Some of this research was conducted in smaller lab settings, while others studied larger, full-scale systems. While we have gained valuable insights, there are still many uncertainties and contradictions in previous findings. For example, some researchers found that changing the operation parameters did not significantly alter the microbial community, while others observed constant changes in the community over time.

Few studies have looked into how groups of microbes work together in a digester. These groups, known as modules, can provide a better understanding of how organisms interact and function during the treatment of wastewater. Some studies have already identified modules in other environments. For example, some researchers found groups of microbes in soil that were involved in specific processes like respiration and fermentation. Other studies have identified groups of microbes in digesters that are related to the breakdown of antibiotics.

In our research, we have identified unique modules within digesters that operate somewhat independently and that are linked to various operational parameters. By understanding these correlations, we can better grasp the relationships in anaerobic digesters. However, finding meaningful relationships in microbial communities is challenging. Issues such as uneven sequencing data and the presence of rare organisms can lead to incorrect conclusions.

Computational methods are increasingly being used to analyze these relationships. Recent studies compared different techniques for generating correlation networks in microbial communities. One method, known as Local Similarity Analysis (LSA), showed the best results for identifying correlations over time. This method can reveal both local and time-delayed relationships. Understanding these relationships can improve our knowledge of the interactions between different microbes and their roles in the digestion process.

#Methodology and Data Collection

#Mill Operating Data

Our research focused on three pulp and paper mills that have different types of anaerobic treatment systems. Mill A combines two types of pulp mills, while Mills B and C focus on a specific type known as BCTMP. Each mill treats wastewater using different Reactors, which have unique characteristics and operational parameters.

At Mill A, there are two reactors that operate with a hydraulic retention time (HRT) ranging from 8 to 12 hours. They receive wastewater from three different streams, which have varying flow rates and compositions. Mill B operates three anaerobic hybrid digesters with a longer HRT of 2 to 3 days, and they are fed with a composite of BCTMP wastewater. Mill C has an anaerobic lagoon with an HRT of 12 to 14 days, also utilizing composite BCTMP wastewater.

We collected daily data from these reactors over a period of about 1.5 years. This data included many different parameters such as chemical oxygen demand (COD) removal efficiency, volatile fatty acid (VFA) to alkalinity ratio, and concentrations of various compounds in the wastewater and biogas.

#Sampling and DNA Extraction

To study the microbial communities, we collected samples of anaerobic biomass from the reactors approximately twice a month. These samples were taken at different depths in each reactor to ensure a representative cross-section of the microbial population. Upon collection, the samples were either frozen or cooled during transport to our lab for analysis.

Once the samples arrived, we extracted the total community DNA using a specialized kit. The quality and quantity of the extracted DNA were assessed before further analysis.

#Microbial Community Analysis

The extracted DNA was then sent for sequencing using advanced technologies. We specifically targeted certain regions of the DNA to identify the different types of microbes present in the samples. The raw sequence data was then processed to generate a comprehensive view of the microbial community.

To minimize bias caused by varying sequencing depths, we standardized the datasets to an equal depth before analysis. We then classified the different microbes found in the samples based on their DNA sequences. This classification allowed us to develop a detailed understanding of the microbial community present in each digester.

#Correlation Analysis and Module Identification

To identify relationships between the microbial communities and the operational parameters, we used a specific software designed for correlation calculations. This software helped to determine which microbes were associated with each other and with the various operational conditions of the digesters.

We focused on identifying large, independent modules within the microbial community. These modules contained organisms that interact and function together, supporting the anaerobic treatment process. The modules we identified showed strong correlations with operational parameters, indicating their significance in the digestion process.

#Results and Discussion

#Microbial Community Composition

Initial analyses of the microbial communities showed different patterns across the three mills. At Mill A, the microbial communities were relatively stable over time, indicating that these digesters had adapted well to the wastewater being treated. In contrast, Mill B exhibited greater variability in its microbial community due to its recent start of operation. Mill C showed variation as well but was not as extreme as Mill B.

The most abundant organisms in all the mills were acetoclastic methanogens, which are essential for producing methane in the anaerobic digestion process. These organisms accounted for over half of the total archaeal population in the digesters. Other notable groups included hydrogenotrophic and methylotrophic methanogens, which also play important roles in producing biogas.

In addition to these methanogens, various bacteria were present across the mills. For example, many of the most abundant bacteria belonged to the phylum Bacteroidetes, which are often involved in breaking down complex organic materials in wastewater. Other bacteria, such as those from the Anaerolineaceae family, were also identified as important contributors to the anaerobic digestion process.

#Identification of Functional Modules

Our analyses revealed two to three distinct biological modules in each mill. Each module consisted of organisms that spanned various stages of anaerobic digestion, including hydrolysis, fermentation, acetogenesis, and methanogenesis. The presence of these interconnected modules suggests that the microbial communities work together closely to efficiently treat the wastewater.

The modules exhibited significant correlations with operational parameters, such as pH and concentrations of harmful compounds like sulfide. For instance, one module was positively correlated with better COD removal efficiency and lower levels of volatile fatty acids. This indicates that certain groups of microbes thrive under stable conditions, while others may be more resilient during times of environmental stress caused by higher levels of inhibitory substances.

#Response to Operational Changes

The study also explored how the microbial communities responded to changes in operational parameters over time. We generated lagged versions of various operating conditions to observe their influence on microbial abundance.

In general, we found that the response time between changes in operating conditions and changes in microbial abundance was typically between two to four days. This delay is important for understanding how the microbial community adapts to modifications in the treatment system and can help optimize performance.

#Upset Events and Microbial Community Response

During the study, we identified periods of digester upset, particularly during annual shutdowns or organic overloads. During these periods, the microbial communities shifted significantly in response to environmental stress.

In all three mills, one specific organism, Methanothrix_319, increased in abundance during times of upset. It dominated the archaeal population in each mill, suggesting a resilience to adverse conditions. The ability of this organism to flourish under stressful situations highlights its potential utility in mitigating process upsets in anaerobic digesters.

Analyzing these upset events provides valuable insight into how different microorganisms respond to challenges in the anaerobic digestion process. Recognizing which organisms thrive during these times can inform strategies for improving the stability and performance of digesters.

#Conclusion

The findings of this research shed light on the complex interactions within microbial communities in anaerobic digesters across three pulp and paper mills. Understanding these relationships and identifying distinct modules of organisms can enhance our knowledge of anaerobic digestion processes.

By studying how these microbial communities respond to operational parameters and environmental stress, we can develop better strategies for optimizing biogas production and wastewater treatment. This knowledge is valuable not only for pulp and paper industries but also for other sectors that rely on anaerobic digestion for waste management.

Overall, the insights gained from this study can lead to improvements in the design and operation of anaerobic treatment systems, potentially benefiting a wide range of industrial and municipal applications. Identifying key organisms and their roles within the microbial community may also open up new avenues for bioaugmentation and other interventions that enhance the efficiency of anaerobic digestion processes.