Darwin’s theory of evolution says it is the one most responsive to change that survives, while the neutral theory born a hundred years later proposed that most evolutionary changes are caused by random genetic drift. Thus, ecologists have since long tried to answer whether deterministic or stochastic processes contribute more to biodiversity patterns.

Microorganisms are the most diverse forms of life on earth. Understanding the influencing factors of microbial diversity and community patterns in the environment, especially in engineered systems with service functions, can help us predict the microbial changes, and control system functioning.

Dr. Ju’s team took the South-to-North Water Diversion Middle Route Project as a part of their research. By using molecular ecology techniques such as high-throughput sequencing, quantitative amplification and model fitting, the group analyzed the dynamic changes of microorganisms in the world’s longest water diversion canal, and evaluated the growth and extinction of microorganisms in the canal based on the newly proposed local growth factor analysis. The results were published in the journal “Water Research” on October 1^st, with Dr. Lu Zhang, a postdoc fellow in Feng’s Lab as the first author, and Dr. Feng Ju, a principal investigator at School of Engineering, Westlake University as the corresponding author.

As of December 2021, the South-to-North Water Diversion Middle Route Project has transferred over 44 billion cubic meters of water from the Danjiangkou Reservoir to the north. It is the world’s largest inter-basin water transfer project with a main canal of 1432 kilometers in length. It an artificial system created to alleviate water shortages in Henan, Hebei, Beijing and Tianjin.

Microorganisms are an important part of the water canal ecosystem, and they are among key factors that determine whether the water quality of the main canal can meet health standards. However, there have been no in-depth research reports on the microbial processes in the South-to-North Water Diversion Project and other long-distance water transfer projects. Additionally, water transfer canals resemble rivers, but they are very different from natural water bodies. So, how is the microbial diversity in these canals different from natural water bodies?

This study was aimed to answered three major questions:

1. How do canal microorganisms spatio-temporally change with water flow and seasons?

2. How do stochastic and deterministic processes affect the dynamics of microorganisms in the canal?

3. Which immigrating microorganisms from the Danjiangkou Reservoir can adapt to the new environment and thrive in the artificial canal?

With the support of the government, together with collaborators from Changjiang Water Resources Protection Institute, Dr. Ju’s research team carried out four samplings over a year at 19 different sites along the main canal and at the Danjiangkou Reservoir. The extracted DNA from the waters were then analyzed using absolute quantitative metagenomic approaches. Other quantitative metagenomic techniques were previously documented and applied by Dr. Ju’s team elsewhere (Bio-101: e2003693 / The ISME Journal. 2019 Feb; 13(2): 346–360).

The microbial composition in the main canal water varied significantly across the seasons and with water flow.

The research team found that bacteria and micro-eukaryotes (including fungi, protozoa, and eukaryotic algae) in the main canal water both showed considerable seasonal variation in their biomass, richness, and diversity, which was more obvious than changes with water flow.

Figure 1. Microbial biomass and diversity changed across the seasons.

Driven by the varying climatic conditions and hydraulic constructions, microbial communities along the canal were constantly shifting.

Figure 2. The composition of micro-eukaryotic communities and their changes with water flow at different sampling sites.

Stochastic factors exerted greater impacts on micro-eukaryotes than bacteria

The research team further discovered through multivariate statistical analyses that water temperature, pH, dissolved oxygen, total nitrogen, and fluoride concentrations were all important deterministic factors that affected the structure of the microbial communities. However, these factors cannot explain all the variance in microbial diversity among samples (Figure 3 right), which is mainly attributed to the influence of stochastic processes (such as diffusion, individual birth and death, etc., which occur randomly).

Figure 3. The influence of deterministic factors on the structure of the microbial community in the main canal

The null model-based normalized stochasticity ratio (NST) estimation revealed that, the micro-eukaryotic community was dominated by stochastic processes, while bacteria were more affected by the deterministic factors. In addition, the research team found based on neutral model analysis that the influence of stochasticity on microbiota assembly was the smallest in May (Figure 4. right, R² represents the neutral model fit, i.e., the degree to which the neutral model can predict microbial diversity patterns). May is also when the lowest water level in Danjiangkou occurs related to seasonal flood control, so the shortage in water storage may enhance selection pressure on microorganisms in the source water and the investigated main canal.

Figure 4. The relative influence of stochastic factors varies with object, space, and time

First use of local growth factor analysis to reveal microbial growth and extinction

When microorganisms immigrate from a natural habitat to an engineered environment, which ones will adapt, and which others will die out? This is a question of both theoretical significance and water quality concern. To address this question, the research team proposed a local growth factor method based on absolute quantitative sequencing analysis and evaluated the growth and extinction of microorganisms along the main canal.

According to the local growth factor analysis, several cyanobacterial lineages (such as Cyanobium PCC-6307; cyanobacteria are typically responsible for harmful algal blooms) and potentially pathogenic bacteria (such as Acinetobacter spp.) significantly reduced in the main canal, indicating the auto-purification potential of the main canal water. On the contrary, Luteolibacter sp., Limnohabitans sp. and Cryptophyceae were gradually enriched along the canal, which were likely involved in carbon and nitrogen cycling in the canal water.

The South-to-North Water Diversion Middle Route Project went into service on December 12^th, 2014. The canal water ecosystem is still at its early forming stage. This study for the first time revealed the dynamic changes and influencing factors of microorganisms in engineered canal systems, which can play indispensable roles in water ecological monitoring. The results also provide an important foundation for effective water quality control and sustainable system management for large water diversion projects.

In this 1432-km engineered canal, was it all the fittest survive? The answer is: not exactly. Although the influence of stochasticity adds to challenge in microbial and system prediction, knowledge about when and where stochasticity plays more important roles as well as how it works improves our understanding of the microbial processes in engineered systems, and facilitate sustainable system management and decision making.