Anoxic Granular Activated Sludge Process For Simultaneous Denitrification and Perchlorate Reduction: Process Performance and Anoxic Granulation Strategy

Introduction
Granular sludge (GS) is an emerging biological wastewater treatment technology that is gaining world-wide attention due to its high rates of removal of both nutrients and recalcitrant organic matter from wastewater [1]. Of the various types of GS, anoxic GS (AxGS) is the least studied when compared to aerobic GS (AGS) and anaerobic GS (AnGS), where thus the formation mechanisms of AxGS are even more ambiguous. Anoxic granules comprising microbial communities mediating anaerobic ammonium oxidation (anammox) represent the bulk of previous literature on AxGS. However, AxGS bioreactors exclusively operated for denitrification have received less academic inquiry. Multiple studies have reported GS formation under denitrifying conditions [2]–[5], however, the complete characterization of the anoxic granulation process was not reported in these studies and thus the formation mechanisms of denitrifying AxGS still remain unclear and subject to debate. Therefore, this study seeks to characterize the anoxic granulation process under denitrifying conditions with perchlorate present as a competing electron acceptor to infer possible formation mechanisms and assess the temporal variation in biomass characteristics as the loose flocs compact into dense microbial aggregates. Microbial ecology was also assessed to determine the temporal distribution in phylotypes as anoxic granulation ensued.
Objectives
The main objectives of this study include:
(1) bioprocess optimization for concurrent denitrification and perchlorate reduction;
(2) characterizing the anoxic granulation process; and
3) to study the microorganisms comprising the AxGS biomass.
Significance
Findings from this study will contribute to the advancement of AxGS process bioreactors by demonstrating at the lab-scale the application this biotechnology.
Status
An AxGS process bioreactor has been operational for over 350-d, where dense anoxic granules have been cultivated. Bioprocess optimization has been executed, resulting in the consistent removal of >99% of the influent perchlorate and nitrate, while concurrently oxidizing >90% of the influent COD.
Methodology
A Plexiglass bubble-column bioreactor (2.625' diameter, 27' height) was fabricated to cultivate anoxic granules. The reactor was seeded with a mixture of hypersaline lake sediments, river sediments, anaerobic digester sludge solids, and aerobic activated sludge solids, previously enriched for denitrifying and perchlorate reducing organisms. Enrichment of perchlorate-reducing and denitrifying organisms was performed by providing perchlorate and nitrate as electron acceptors along with acetate as the electron donor in a basal medium described by Song et al. (2019). The anoxic granulation process was characterized through a combination of sludge settling analyses, solids concentrations, extracellular polymeric substance (EPS) extraction and analyses, microscopy, and microbial methods. Complete cycle analyses were conducted so that specific rates could be expressed. Microbial ecology of the AxGS biomass was characterized by high-throughput 16S rDNA amplicon sequencing as described by Stein et al. (2021). All anions were quantified using a Compact 930 IC Flex ion chromatograph (IC; Metrohm, USA). COD was quantified according to standard methods [8] and dissolved organic carbon (DOC) was quantified using a Shimadzu, TOC-V Total Organic Carbon (TOC) Analyzer (Shimadzu, USA). Findings Reactor Performance The overall performance of the AxGS bioreactor in terms of perchlorate, nitrate, and COD removal efficiencies, as well as chloride accumulations in the effluent, is summarized in Figure 1. The AxGS biomass efficiently reduced both the influent perchlorate and nitrate, while simultaneously oxidizing the influent COD. Average perchlorate, nitrate, and COD removal efficiencies have been 97.3±7.2%, >99.9%, and 90.1±3.07%, respectively. Average chloride accumulation in the effluent has been 153±23.7 mg/L, which is representative of an 80.3% (mol/mol) molecular conversion of the influent perchlorate to chloride. The initial mixed liquor suspended solids (MLSS) and mixed liquor volatile suspended solids (MLVSS) concentrations in the bioreactor were 1.09 g/L and 0.93 g/L, respectively. The biomass concentrations in the reactor steadily increased until reaching an average steady-state MLSS and MLVSS concentration of 3.11±1.15 g/L and 2.76±1.01 g/L, respectively, while maintaining a solids retention time (SRT) of 20-d. The 5-min sludge volume index (SVI5) of the AxGS biomass rapidly decreased in the first 150-d of reactor operation as the loose flocs gradually transformed into dense microbial aggregates. The SVI5 stabilized at a steady-state value of 24.9±11.7 mL/g. Both the specific protein and carbohydrate content of the EPS comprising the AxGS biomass steadily increased as the floccular biomass transformed into compact aggregates. Additionally, the protein fraction of the EPS became >90% as the anoxic granules matured and reached steady-state. Figure 1 also displays the results of a complete cycle analysis conducted over a standard reactor operational cycle under steady-state conditions. Nitrate was reduced to below detection 6-h into the cycle, where specific reduction rates of 6.12±1.68 mg NO3-N/g VSS•h were recorded. DOC became limiting 6-h into the cycle, where specific oxidation rates of 8.60±2.66 mg DOC/g VSS•h were recorded. Perchlorate was reduced to below detection before the effluent was withdrawn from the reactor, where specific reduction rates of 8.62±2.36 mg ClO4-/g VSS•h were recorded. Specific chloride production rates of 2.18±0.60 mg Cl-/g VSS•h were also recorded. Microscopy Figure 2 displays micrographs of the AxGS biomass captured throughout reactor operation. Initially, the biomass was comprised of loose flocs consisting of a dispersed web of EPS. The loose flocs then gradually underwent compaction until irregularly-shaped, distinguishable anoxic granules were observed on reactor operational day 97, where the EPS matrix became compact and smooth. The anoxic granules then became much larger and more compact, while also taking on a more symmetrical, spherical shape starting on reactor operation day 146. The EPS matrix of the granules became slightly rougher starting on reactor operational day 146 and remained that way. Microbial Ecology Figure 3 displays the taxonomic classification at the phylum (panel 'a') and genus (panel 'b') level for the microbial community comprising the AxGS biomass throughout reactor operation. At the Phylum level, Proteobacteria (94.1-98.2%) were dominant at all sampling events. Minor phyla identified were Acidobacteriota (0.2–1.54%) and Bacteroidota (0.43–4.74%). At the genus level, Dechloromonas (49.5–90.6%) and Azospira (3.16-40.8%) were the clear dominant genera throughout reactor operation, both of whom have members known to be perchlorate reducing and denitrifying organisms [9]–[11]. Minor genera identified were Holophagaceae (0.01-1.33%), Lentimicrobium (0.25-4.16%), Dechlorosoma (0.36-9.17%), Denitratisoma (0-5.46%), and Thauera (0.35-6.44%).