Effect of Hydrocyclones on Floc Size, Morphology, and Microbial Community

Objectives Hydrocyclones can intensify activated sludge processes by retaining larger, faster settling flocs and eliminating smaller, slower settling flocs. This can result in lower sludge volume indices (SVIs), higher mixed liquor solids concentrations, higher solids retention time (SRT), and modified mixed-liquor microbial community structures (Roche et al., 2021; Regmi et al., 2022; Li et al., 2020). While it is clear that hydrocyclones improve SVIs, what factors impact separation, and how separation impacts microbial communities, is less obvious. For example, does the relative abundance of heterotrophs, AOB/NOB, and PAOs determine whether a floc is retained in the hydrocyclone underflow? What is more important for floc retention: floc density, size, or shape? Do hydrocyclones affect protozoa? Such information could help program hydrocyclones to promote a desired treatment objective. In order to answer the above questions, we are using dynamic floc imaging, molecular tools, activity test, and floc density analyses to explore the effect of hydrocyclones at a full-scale wastewater treatment plant. We have completed four sampling events and expect to have four more by the time of the conference. This abstract includes our ongoing results. We expect to have full results in time for WEFTEC. Methodology Samples were collected from a full-scale municipal wastewater treatment plant in the Midwest region of the US. The plant was configured for EBPR and implemented a densified activated sludge (DAS) process using hydrocyclones in November of 2022. Starting in May 2023, sampling campaigns for floc analysis were carried out approximately every two months. Four samples have been collected and analyzed at the time of this abstract submission. Sampling included hydrocyclone influent, overflow, and underflow, and MLSS. Floc characterizations were carried out with an integrated laser diffraction analyzer with synchronous dynamic image analysis, i.e., diffraction and imaging were carried out on a single sample at the same time (Microtrac, Germany). By analyzing thousands of individual flocs in each sample, this provides classical particle size histograms, but also a large range of morphological particle parameters, such as circularity, sphericity, roughness, aspect ratio, roundness, and fractal dimension. Microbial community characterization was carried out by 16S rRNA gene sequencing of VSS from the MLSS, hydrocyclone influent, overflow, and underflow. qPCR and FISH were used to quantify AOB, NOB and PAOs. Findings A plot of SVI30, SVI5, and densification index over time is shown in Figure 1. SVIs decreased, and the densification index increased, over time after implementing the hydrocyclones. Floc size characterizations showed that the 50th percentile area-based diameter (D50) increased progressively from 108 µm (May 2023) to 163 µm (December 2023) (data not shown). Based on floc size histograms (Figure 2):

*Hydrocyclones appeared have little separation effect on the smaller flocs (0 — 50 µm). These were present in similar amounts in both overflow and underflow.

*The impact of hydrocyclones on larger flocs increased with time, i.e., there needed to be more large-sized flocs in the MLSS in order for the hydrocyclones to separate them into the underflow. Based on an analysis of floc circularity (Figure 3).

*Smaller flocs tended to be more circular than larger flocs, both in underflow and overflow

*Larger flocs in the underflow tended to be more circular than similarly sized flocs in the overflow, suggesting that floc morphology can play an important role in retention by hydrocyclones.

*Differences in circularity between underflow and overflow increased progressively over time (data not shown) Sampling sequencing data is available for one sampling date (July 2023). Based on these results, the hydrocyclone underflow had higher abundances of AOB, NOB, and PAOs than the overflow (data not shown). Interestingly, NOB (Nitrospira sp.) were more abundant than AOB. Preliminary qPCR and FISH (Figure 4) results are consistent with the sequencing. FISH is currently being used to differentiate the microbial communities for different floc sizes and shapes. Percoll density separation is also being used to quantify and sequence flocs by density. These results will be included in the final paper and presentation. Significance The results suggest that hydrocyclones separate not only based on floc density, but also size and shape. The role of microbial community is currently being analyzed and will be presented at the conference.