Abstract

Amplicon sequencing data and operating data from anaerobic wastewater treatment plants from three Canadian pulp and paper mills were explored using correlation and network modularization approaches to study the microbial community organization and identify relationships between organisms and operating conditions. Each of the digesters contains two or three modules, or functional units, consisting of organisms that cover all trophic stages of anaerobic digestion. The modules function independently from each other, and their relative abundance changes in response to changing operating conditions. The modules show antagonistic responses, with one module associated with stable operation and another module linked to periods of environmental stress. Operating parameters correlated to module abundance include sulfide concentration in the digester influent and biogas sulfide flow rate as well as anaerobic treatment performance metrics such as COD removal efficiency and volatile fatty acid-to-alkalinity ratio. Elevated sulfide levels notably impact the microbial community composition and the anaerobic treatment performance and seem to be the primary driver of process inhibition. The time delay between a change in digester operation and a change in the abundance of microorganisms was investigated using time-lagged operating parameters. This time delay ranged between 2 and 4 days and is likely influenced by the growth rates of the anaerobic microorganisms and the digester hydraulic retention time. The application of lagged parameters appeared to be necessary for identifying numerous correlations that would otherwise have remained undetected. This is because correlations with operating parameters without a time lag tend to be smaller and are often not significant. Digester upsets due to plant shutdown periods and organic overload caused a drastic increase in acetoclastic methanogenesis and the population of acidogenic fermenters and syntrophic acid degraders. In response to impaired process conditions, the same Methanothrix amplicon sequence variants (ASVs) dominated methanogenesis in the digesters of all three mills, with its maximum relative abundance within the archaeal population reaching 68 at mill A, 58 at mill B, and 27% at mill C. The common characteristics of the organisms represented by this ASV should be further investigated for their role in alleviating the impact of digester upset conditions. Across all three mills, biogas production predominantly relied on acetoclastic methanogenesis. Methanothrix were the most abundant methanogenic ASVs, accounting for on average 63% of all archaea in mill A, 52% in mill B, and 73% in mill C over the investigation period. Also, each reactor contained at least three ASVs of high abundance from the archaeal class Bathyarchaeia. The presence of Bathyarchaeia, ranging from 10 to 20% of the total archaeal community in all digesters, may be associated with the higher lignin content present in the mill wastewater.