Though freshwater harmful algal blooms have been described and studied for decades, several important dynamics remain uncertain, including the relationships among nutrient concentrations, phytoplankton growth, and cyanotoxin production. To identify when and where nutrients limit phytoplankton, cyanobacteria, and cyanotoxins, we conducted in situ bioassay studies. We added nitrogen (N), phosphorus (P), or N + P across various seasons in water collected from three locations across Utah Lake, one of the largest freshwater lakes in the western U.S. This shallow, hypereutrophic lake provides a powerful testbed for quantifying nutrient-growth-toxin interactions. We assessed a range of parameters over time, including photopigment concentrations, phytoplankton abundance (cell counts), cyanotoxins, and nutrient concentrations. Despite high background nutrient concentrations in lake water, phytoplankton abundance and composition were strongly affected by nutrient addition. Phosphorus limitation was more common in the spring, with N limitation and N + P limitation becoming more common in the fall. Nutrient additions were positively associated with cyanobacteria (Microcystis, Aphanocapsa, Dolichospermum, Merismopedia, Aphanizomenon spp.), eukaryotes (Aulacoseira, Desmodesmus spp.), and two taxonomical categories of phytoplankton (i.e., unicellular and colonial green algae). When detected, anatoxin-a was positively associated with Aphanizomenon and negatively associated with Microcystis spp. However, overall cyanotoxin concentrations were not associated with cyanobacterial cell density but varied seasonally. These findings highlight the importance of considering seasonal nutrient availability dynamics and provide insights into specific nutrient targets, species, and cyanotoxins that play a significant role in the health and management of similar eutrophic lake environments around the world.

Extremophilic biological process (EBP) pretreatment increases substrate availability in anaerobic digestion, but the effect on downstream microbial community composition in industrial systems is not characterized. Changes in microbial communities were determined at an industrial facility processing dairy manure in a modified split-stream system with three reactor types: (1) EBP tanks at 70–72 °C; (2) mesophilic Continuously Stirred Tank Reactors (CSTRs); (3) mesophilic Induced Bed Reactors (IBRs) receiving combined CSTR and EBP effluent. All reactors had a two-day hydraulic retention time. Samples were collected weekly for 60 days. pH, volatile fatty acid and bicarbonate concentrations, COD, and methane yield were measured to assess tank environmental conditions. Microbial community compositions were obtained via 16S rRNA gene sequencing. EBP pretreatment increased acetate availability but led to a decline in the relative abundance of acetoclastic Methanosarcina species in downstream IBRs. Rather, syntrophic methanogens, e.g., members of Methanobacteriaceae, increased in relative abundance and became central to microbial co-occurrence networks, particularly in association with hydrogen-producing bacteria. Network analysis also demonstrated that these syntrophic relationships were tightly coordinated in pretreated digestate but absent in the untreated CSTRs. By promoting syntrophic methanogenesis while increasing acetate concentrations, EBP pretreatment requires system configurations that enable acetoclast retention to prevent acetate underutilization and maximize methane yields.

Infographic summarizing a study on Caldicellulosiruptor bescii pretreatment of manure. It shows background on resistant substrates, an experimental setup with EBP followed by anaerobic digestion (AD) and sampling over 200 days, and results indicating increased hydrogenotrophic methanogens and greater bacterial persistence in AD after pretreatment. Extremophilic biological pretreatment (EBP) of dairy manure using Caldicellulosiruptor bescii at 75°C dramatically alters the microbial community structure and activity in anaerobic digesters (ADs). This study evaluated microbial abundance and activity in manure, EBP, and ADs at industrial scales using 16S sequencing of rDNA and rRNA. Results revealed that bacterial communities remained largely active and resilient throughout the system, but acetoclastic methanogens were sensitive to EBP treatment and failed to reestablish in ADs. In contrast, hydrogenotrophic methanogens survived EBP and persisted in ADs, although these species are poorly suited to metabolize the acetate-rich products generated by C. bescii in pretreatment. We conclude that the acetate utilization of ADs preceded by EBP is likely to improve if supplemented with acetoclastic methanogens, such as those present in raw manure.

Hyperthermophilic pretreatment with Caldicellulosiruptor species (EBP) increases substrate availability in anaerobic digestion, but the effect on downstream microbial community composition in industrial systems is not characterized. Changes in microbial communities were determined at an industrial facility processing dairy manure in a modified split-stream system with three reactor types: 1) EBP tanks at 70–72°C, 2) mesophilic Continuously Stirred Tank Reactors (CSTRs), and 3) mesophilic Induced Bed Reactors (IBRs) receiving combined CSTR and EBP effluent. All reactors had a two-day hydraulic retention time. Samples were collected weekly for 60 days. pH, volatile fatty acid and bicarbonate concentrations, COD, and methane yield were measured to assess tank environmental conditions. Microbial community compositions were obtained via 16S rRNA gene sequencing. EBP pretreatment increased acetate availability but led to a decline in the relative abundance of acetoclastic Methanosarcina species in downstream IBRs. Rather, syntrophic methanogens, e.g., Methanobrevibacter species, increased in relative abundance and became central to microbial co-occurrence networks, particularly in association with hydrogen-producing bacteria. Network analysis also demonstrated that these syntrophic relationships were tightly coordinated in pretreated digestate but absent in the untreated CSTRs. By promoting syntrophic methanogenesis while increasing acetate concentrations, EBP pretreatment requires system configurations that enable acetoclast retention to prevent acetate underutilization and maximize methane yields. Importance: Hyperthermophilic Caldicellulosiruptor pretreatment in anaerobic digestion increased acetate availability while suppressing acetoclastic methanogens and promoting robust syntrophic methanogenic networks. The results offer engineers microbial guidance essential for designing effective multi-stage industrial anaerobic digestion facilities employing EBP methods.

Nutrient enrichment in shallow lakes and ponds can shift systems from clear water, macrophyte dominance to turbid, phytoplankton dominance. Because the identity and threshold of the limiting nutrients vary among sites, system‐specific evidence is needed to guide water quality management. We conducted 3‐day in situ nutrient‐enrichment bioassays in two wetland impoundments adjacent to Great Salt Lake (Utah, USA) to identify the limiting nutrient for phytoplankton growth, quantify growth responses to nitrogen (N) and phosphorus (P), estimate an operational nutrient threshold using Monod kinetics and evaluate bacterioplankton community and functional responses across the same nutrient gradient. Phytoplankton biomass (chlorophyll a ) was used to calculate growth rates, and bacterioplankton communities were profiled by 16S rRNA gene sequencing with inferred functional annotation. Across both wetlands, phytoplankton growth increased with N additions but not with P additions, indicating N limitation under high ambient P. Monod models yielded a half‐saturation constant of approximately 0.7 mg N L ⁻¹ (as NO ₃ –N), which we interpret as an N threshold for phytoplankton growth in these impoundments. Bacterioplankton composition differed by site, with Cyanobiaceae dominating at both locations, and community evenness declining above the N threshold. Inferred metagenomes indicated a higher relative abundance of taxa carrying the microcystin synthetase gene mcyE above the N threshold, suggesting increased cyanotoxin potential. Together, these results support management actions that control N in addition to P and demonstrate the value of bioassay‐derived thresholds for informing numeric criteria in managed wetlands.