Implications of the operation of continuous granular activated carbon filters on the effluent quality.

Granular activated carbon (GAC) filtration is a commonly used method for advanced wastewater treatment. Filters can be operated continuously or discontinuously, with continuous operation not requiring feed flow interruption for backwashing and circulation (B/C). This study investigated the influence of B/C on the effluent quality of continuous filters. Two continuous GAC filters were operated for 1.5 years, with analysis of dissolved substances and particulate matter in the influent and effluent. The results indicated that various B/C modes had no impact on the removal of dissolved organic carbon and organic micropollutants (OMP), achieving an OMP removal of over 70% after 5,600 treated bed volumes (m3 treated wastewater per m3 GAC). However, it was evident that continuous B/C over 2-4 h resulted in increased turbidity, total suspended solids over 30 mg/L and total phosphorus concentrations of 1.3 mg/L in the filter effluent. Additionally, the study demonstrated that longer and more intensive B/C processes resulted in GAC size degradation with AC concentrations of up to 6.9 mg/L in the filter effluent, along with a change in GAC particle size. Furthermore, the importance of pre-filtration in reducing particulate matter in the filter influent and decreasing hydraulic head loss could be demonstrated.

Are K-strategists yield-strategists in disguise? An example from autotrophic nitrogen removal.

The behavior of heterotrophic bacteria growing in systems with low or no external supply of chemical oxygen demand (COD) has become more relevant within the wastewater context. Growth strategies help to clarify how bacteria behave and adapt to different environmental conditions. In the case of substrate limited conditions, research has been mainly focused on the k-strategy, whereas another important strategy: the yield strategy has not been explored intensely. Some authors have, however, demonstrated the implications of bacteria pursuing the yield strategy when living in structured environments and facing low-substrate concentrations. This study uses a one-dimensional biofilm model to study the influence of the affinity constant, the maximum growth rate, and the growth yield on the heterotrophic formation of dinitrogen gas (N2 ) in a completely autotrophic partial nitritation anammox system. The effect of these parameters on the composition and the diversity of the heterotrophic community is also evaluated. In a first scenario, heterotrophic bacteria are allowed to grow only on the COD produced by biomass decay. In a second step, the competition with a second group of heterotrophs using external COD as electron donor is assessed. For both evaluated scenarios, the results suggest that the yield plays a crucial role in the heterotrophic biomass and dinitrogen gas formation. Moreover, in the case of the community diversity the yield seems to be the decisive parameter. Finally, we conceptually compared the K and the yield strategy and give some insight to the possibility of both either being closely related or even being the same strategy.

Targeted metagenomics reveals extensive diversity of the denitrifying community in partial nitritation anammox and activated sludge systems.

The substantial presence of denitrifiers has already been reported in partial nitritation anammox (PNA) systems using the 16S ribosomal RNA (rRNA) gene, but little is known about the phylogenetic diversity based on denitrification pathway functional genes. Therefore, we performed a metagenomic analysis to determine the distribution of denitrification genes and the associated phylogeny in PNA systems and whether a niche separation between PNA and conventional activated sludge (AS) systems exists. The results revealed a distinct abundance pattern of denitrification pathway genes and their association to the microbial species between PNA and AS systems. In contrast, the taxonomic analysis, based on the 16S rRNA gene, did not detect notable variability in denitrifying community composition across samples. In general, narG and nosZa2 genes were dominant in all samples. While the potential for different stages of denitrification was redundant, variation in species composition and lack of the complete denitrification gene pool in each species appears to confer niche separation between PNA and AS systems. This study suggests that targeted metagenomics can help to determine the denitrifying microbial composition at a fine-scale resolution while overcoming current biases in quantitative polymerase chain reaction approaches due to a lack of appropriate primers.

Combination of 15N Tracer and Microbial Analyses Discloses N2O Sink Potential of the Anammox Community.

Although nitrogen removal by partial nitritation and anammox is more cost-effective than conventional nitrification and denitrification, one downside is the production and accumulation of nitrous oxide (N2O). The potential exploitation of N2O-reducing bacteria, which are resident members of anammox microbial communities, for N2O mitigation would require more knowledge of their ecophysiology. This study investigated the phylogeny of resident N2O-reducing bacteria in an anammox microbial community and quantified individually the processes of N2O production and N2O consumption. An up-flow column-bed anammox reactor, fed with NH4+ and NO2- and devoid of oxygen, emitted N2O at an average conversion ratio (produced N2O: influent nitrogen) of 0.284%. Transcriptionally active and highly abundant nosZ genes in the reactor biomass belonged to the Burkholderiaceae (clade I type) and Chloroflexus genera (clade II type). Meanwhile, less abundant but actively transcribing nosZ strains were detected in the genera Rhodoferax, Azospirillum, Lautropia, and Bdellovibrio and likely act as an N2O sink. A novel 15N tracer method was adapted to individually quantify N2O production and N2O consumption rates. The estimated true N2O production rate and true N2O consumption rate were 3.98 ± 0.15 and 3.03 ± 0.18 mgN·gVSS-1·day-1, respectively. The N2O consumption rate could be increased by 51% (4.57 ± 0.51 mgN·gVSS-1·day-1) with elevated N2O concentrations but kept comparable irrespective of the presence or absence of NO2-. Collectively, the approach allowed the quantification of N2O-reducing activity and the identification of transcriptionally active N2O reducers that may constitute as an N2O sink in anammox-based processes.

High-throughput profiling of antibiotic resistance genes in wastewater: comparison between a pond system in Namibia and an activated sludge treatment in Germany.

There are increasing concerns about wastewater treatment plants (WWTPs) acting as hotspots for antibiotic resistance genes (ARG). However, their role largely depends upon the treatment methods and antibiotics in the wastewater. To better understand these influences, we compared the occurrence and fate of ARG between a pond system in a developing country (Namibia) and an advanced WWTP (activated sludge system) in a developed country (Germany). A targeted metagenomic approach was used to investigate the wide-spectrum profiles of ARGs and their co-occurrence patterns at both locations. In total, 93 ARG subtypes were found in the German influent wastewater, 277 in the Namibian influent wastewater. The abundant ARG types found in Namibia and Germany differed, especially for multidrug resistance genes. The differences in occurrence and reduction can help to understand the performance of simple WWTP such as pond systems common in Namibia, where direct contact with wastewater is a potential risk for contamination.

Looking deeper - exploring hidden patterns in reactor data of N-removal systems through clustering analysis.

In this work, clustering analysis of two partial nitritation-anammox (PN-A) moving bed biofilm reactors (MBBR) containing different types of carrier material was explored for the identification of patterns and operational conditions that may benefit process performance. The systems ran for two years under fluctuations of temperature and organic matter. Ex situ batch activity tests were performed every other week during the operation of these reactors. These datasets and the parameters, which were monitored online and in the laboratory, were combined and analyzed applying clustering analysis to identify non-obvious information regarding the performance of the systems. The initial results were consistent with the literature and from an operational perspective, which allowed the parameters to be explored further. The new information revealed that the oxidation reduction potential (ORP) and the anaerobic ammonium oxidizing bacteria (AnAOB) activity correlated well. ORP also dropped when the reactors were exposed to real wastewater (presence of organic matter). Moreover, operating conditions during nitrite accumulation were identified through clustering, and also revealed inhibition of anammox bacteria already at low nitrite concentrations.

NMR investigation of water diffusion in different biofilm structures.

Mass transfer in biofilms is determined by diffusion. Different mostly invasive approaches have been used to measure diffusion coefficients in biofilms, however, data on heterogeneous biomass under realistic conditions is still missing. To non-invasively elucidate fluid-structure interactions in complex multispecies biofilms pulsed field gradient-nuclear magnetic resonance (PFG-NMR) was applied to measure the water diffusion in five different types of biomass aggregates: one type of sludge flocs, two types of biofilm, and two types of granules. Data analysis is an important issue when measuring heterogeneous systems and is shown to significantly influence the interpretation and understanding of water diffusion. With respect to numerical reproducibility and physico-chemical interpretation, different data processing methods were explored: (bi)-exponential data analysis and the Γ distribution model. Furthermore, the diffusion coefficient distribution in relation to relaxation was studied by D-T2 maps obtained by 2D inverse Laplace transform (2D ILT). The results show that the effective diffusion coefficients for all biofilm samples ranged from 0.36 to 0.96 relative to that of water. NMR diffusion was linked to biofilm structure (e.g., biomass density, organic and inorganic matter) as observed by magnetic resonance imaging and to traditional biofilm parameters: diffusion was most restricted in granules with compact structures, and fast diffusion was found in heterotrophic biofilms with fluffy structures. The effective diffusion coefficients in the biomass were found to be broadly distributed because of internal biomass heterogeneities, such as gas bubbles, precipitates, and locally changing biofilm densities. Thus, estimations based on biofilm bulk properties in multispecies systems can be overestimated and mean diffusion coefficients might not be sufficiently informative to describe mass transport in biofilms and the near bulk.

Assessing the influence of biofilm surface roughness on mass transfer by combining optical coherence tomography and two-dimensional modeling.

Imaging and modeling are two major approaches in biofilm research to understand the physical and biochemical processes involved in biofilm development. However, they are often used separately. In this study we combined these two approaches to investigate substrate mass transfer and mass flux. Cross-sectional biofilm images were acquired by means of optical coherence tomography (OCT) for biofilms grown on carriers. A 2D biofilm model was developed incorporating OCT images as well as a simplified biofilm geometry serving as structural templates. The model incorporated fluid flow, substrate transfer and biochemical conversion of substrates and simulated the hydrodynamics surrounding the biofilm structure as well as the substrate distribution. The method allowed detailed analysis of the hydrodynamics and mass transfer characteristics at the micro-scale. Biofilm activity with respect to substrate fluxes was compared among different combinations of flow, substrate availability and biomass density. The combined approach revealed that higher substrate fluxes at heterogeneous biofilm surface under two conditions: pure diffusion and when high flow velocity along the biofilms surface renders the whole liquid-biofilm interface to be highly active. In-between the two conditions the substrate fluxes across the surface of smooth biofilm geometry were higher than that of the heterogeneous biofilms.

Determining the flow regime in a biofilm carrier by means of magnetic resonance imaging.

Biofilms on cylindrical carrier material originating from a lab-scale moving bed biofilm reactor (MBBR) were investigated by means of Magnetic Resonance Imaging (MRI). The aim of this study was to determine the local flow velocities at the inner face of the biofilm carrier. To get an insight into the mass transport processes, flow velocity maps of blank and with biofilm cultivated carriers were measured. A single carrier was placed in a tube in three different orientations and exposed to flow velocities of 0.21, 0.42, and 0.64 mm/s. The interplay of the biofilm morphology and the local flow pattern was then analyzed including the effect of the orientation of the carrier in relation to the upstream flow angle. Within this study, the biofilm carrier can be understood as an interconnected system of four sections in which the incoming fluid volume will be distributed depending on the biomass occupation and morphology. In sections with high biofilm occupation, the flow resistance is increased. Depending on the orientation of the carrier in the flow field, this effect leads to flow evasion through less covered sections showing higher flow velocities and consequently the risk of biofilm detachment. However, there was no clear correlation between biofilm coverage and flow ratio.

Influence of seasonal temperature fluctuations on two different partial nitritation-anammox reactors treating mainstream municipal wastewater.

Partial nitritation-anammox (PN-A) has gained increasing interest for municipal wastewater treatment in recent years due to its high energy-saving potential. Moving the PN-A technology from side- to mainstream exhibited a set of challenges. Conditions are quite different, with much lower ammonium concentrations and temperatures. Biomass retention becomes highly important due to the even lower growth rates. This study compared two laboratory-scale reactors, a sequencing batch reactor (SBR) and a moving bed biofilm reactor (MBBR), employing realistic seasonal temperature variations over a 1-year period. The results revealed that both systems had to face decreasing ammonium conversion rates and nitrite accumulation at temperatures lower than 12°C. The SBR did not recover from the loss in anammox activity even when the temperature increased again. The MBBR only showed a short nitrite peak and recovered its initial ammonium turnover when the temperature rose back to >15°C. The SBR had higher biomass specific rates, indicating that suspended sludge is less diffusion-limited but also more susceptible to biomass wash-out. However, the MBBR showed the more stable performance also at low temperatures and managed to recover. Ex situ batch activity tests supported reactor operation data by providing additional insight with respect to specific biomass activities.

Start-up of a full-scale deammonification SBR-treating effluent from digested sludge dewatering.

This study shows the start-up and operation of a full-scale sequencing batch reactor (SBR) with a volume of 550 m³ for deammonification of reject water from sludge dewatering over the first 650 days of operation. The SBR was operated with discontinuous aeration and achieved an optimum of around 85% of ammonium removal at a load of 0.17 kg m⁻³ d⁻¹. The application of batch tests for the activity measurement of aerobic ammonium and nitrite oxidizing bacteria and anaerobic ammonium oxidizing bacteria were proven to support the identification of setbacks in reactor operation. Furthermore, the calculation of the oxygen uptake rates from online oxygen measurements helped to explain the overall reactor performance. The aeration regime is a key parameter for stable operation of such an SBR for deammonification. At aeration/non-aeration time ranges from 6-9 min, the best results with respect to turnover rates and low nitrate production were achieved. Compared with the nitrification/denitrification SBR operated in parallel with methanol as the carbon source, a significant reduction in costs for energy and chemicals was achieved. The costs for maintenance slightly increased.

Low temperature partial nitritation/anammox in a moving bed biofilm reactor treating low strength wastewater.

Municipal wastewater collected in areas with moderate climate is subjected to a gradual temperature decrease from around 20 °C in summer to about 10 °C in winter. A lab-scale moving bed biofilm reactor (MBBR) with carrier material (K3 from AnoxKaldnes) was used to test the tolerance of the overall partial nitritation/anammox process to this temperature gradient. A synthetic influent, containing only ammonium and no organic carbon was used to minimize denitrification effects. After stable reactor operation at 20 °C, the temperature was slowly reduced by 2 °C per month and afterward held constant at 10 °C. Along the temperature decrease, the ammonium conversion dropped from an average of 40 gN m(-3) d(-1) (0.2 gN kgTSS h(-1)) at 20 °C to about 15 gN m(-3) d(-1) (0.07 gN kg TSS h(-1)) at 10 °C, while the effluent concentration was kept <8 mg NH4-N l(-1) during the whole operation. This also resulted in doubling of the hydraulic retention time over the temperature ramp. The MBBR with its biofilm on 10 mm thick carriers proved to sufficiently sustain enough biomass to allow anammox activity even at 10 °C. Even though there was a minor nitrite-build up when the temperature dropped below 12.5 °C, reactor performance recovered as the temperature decrease continued. Microbial community analysis by 16S rRNA amplicon analysis revealed a relatively stable community composition over the entire experimental period.

Response of different nitrospira species to anoxic periods depends on operational do.

The exploitation of a lag phase in nitrate production after anoxic periods is a promising approach to suppress nitrite oxidizing bacteria, which is crucial for implementation of the combined partial nitritation-anammox process. An in-depth study of the actual lag phase in nitrate production after short anoxic periods was performed with varied temperatures and air flow rates. In monitored batch experiments, biomass from four different full-scale partial nitritation-anammox plants was subjected to anoxic periods of 5-60 min. Ammonium and the nitrite that was produced were present to reproduce reactor conditions and enable ammonium and nitrite oxidation at the same time. The lag phase observed in nitrite oxidation exceeded the lag phase in ammonium oxidation after anoxic periods of more than 15-20 min. Lower temperatures slowed down the conversion rates but did not affect the lag phases. The operational oxygen concentration in the originating full scale plants strongly affected the length of the lag phase, which could be attributed to different species of Nitrospira spp. detected by DGGE and sequencing analysis.

Looking deeper into the effects of scouring and aeration on membrane aerated biofilms: Analysis of nitrogen conversion, oxygen profiles and nitrous oxide emissions.

This study investigated the effects of aeration and scouring strategies on the performance of Membrane Aerated Biofilm Reactors (MABRs) and the distribution of oxygen and nitrous oxide in the biofilm. Four flat sheet MABRs were operated with synthetic feed under different conditions: two with intermittent aeration (iMABR) and two with continuous aeration (cMABR). Scouring was induced by bubbling dinitrogen gas through the reactor bulk at low and high frequencies (LF and HF). In the iMABRs, a partial nitritation biofilm initially developed, but the biofilm adapted to the aeration strategy over time and became nitrifying. The cMABRs directly developed a nitrifying biofilm without a significant phase of partial nitritation. Limiting oxygen availability improved the overall performance with regards to total nitrogen (TN) removal by providing a better environment for anaerobic ammonium oxidation (Anammox) while limiting complete nitrification. Oxygen profiles were measured in the iMABR over time at different biofilms depths, showing that intermittent aeration led to various oxygen concentrations and temporal variations in the oxygen availabilities at different depths of the biofilm. Also, N2O emissions from the MABRs differed greatly between the different systems, but still remained lower compared to other reactor configurations for nitrogen removal, making the MABR technology a worthy alternative. The results showed large differences between the operating strategies of the MABRs and can help to gain more insight into the specific properties of MABRs for nitrogen removal.

Scattered and transmitted light as surrogates for activated carbon residual in advanced wastewater treatment processes: Investigating the influence of particle size.

The use of powdered activated carbon (PAC) is a common process in advanced wastewater treatment to remove micropollutants. Retention and separation of PAC is essential as PAC loaded with micropollutants should not be released into the environment. Determining the activated carbon (AC) residual in the effluent poses a challenge, as there is currently no on-line measurement method. In this study, the correlation between turbidity, measured by scattered light, and absorption at wavelength of 550 nm (Absorption550 nm), measured by transmitted light, was investigated in relation to the AC residue. Linear correlations for turbidity (R2 = 0.95) and Absorption550 nm (R2 = 1.00) to AC concentrations were observed in both laboratory and full-scale experiments in a pilot plant where superfine PAC was added prior to Pile Cloth Media Filtration (PCMF). Decreasing the particle size (d50) while maintaining the same AC concentration leads to increased turbidity: Therefore, a fourfold reduction in d50 results in a 2- to 3-fold increase in turbidity, whereas a 30-fold reduction in d50 leads to a 6-to 8-fold increase. Furthermore, the original wastewater turbidity led to a parallel shift in the linear correlation between turbidity and AC. Coagulant doses of up to 400 mg Me3+/g AC resulted in a 50% reduction in turbidity. However, higher concentrations from 400 to 1,000 mg Me3+/g AC resulted in increased turbidity with only a 30% reduction compared to the initial turbidity. The study also highlights the significance of AC particle size in optical measurements, impacting result accuracy.

Quest for Nitrous Oxide-reducing Bacteria Present in an Anammox Biofilm Fed with Nitrous Oxide.

N2O-reducing bacteria have been examined and harnessed to develop technologies that reduce the emission of N2O, a greenhouse gas produced by biological nitrogen removal. Recent investigations using omics and physiological activity approaches have revealed the ecophysiologies of these bacteria during nitrogen removal. Nevertheless, their involvement in| |anammox processes remain unclear. Therefore, the present study investigated the identity, genetic potential, and activity| |of N2O reducers in an anammox reactor. We hypothesized that N2O is limiting for N2O-reducing bacteria| |and an| |exogeneous N2O supply enriches as-yet-uncultured N2O-reducing bacteria. We conducted a 1200-day incubation of N2O-reducing bacteria in an anammox consortium using gas-permeable membrane biofilm reactors (MBfRs), which efficiently supply N2O in a bubbleless form directly to a biofilm grown on a gas-permeable membrane. A 15N tracer test indicated that the supply of N2O resulted in an enriched biomass with a higher N2O sink potential. Quantitative PCR and 16S rRNA amplicon sequencing revealed Clade II nosZ type-carrying N2O-reducing bacteria as protagonists of N2O sinks. Shotgun metagenomics showed the genetic potentials of the predominant Clade II nosZ-carrying bacteria, Anaerolineae and Ignavibacteria in MBfRs. Gemmatimonadota and non-anammox Planctomycetota increased their abundance in MBfRs despite their overall lower abundance. The implication of N2O as an inhibitory compound scavenging vitamin B12, which is essential for the synthesis of methionine, suggested its limited suppressive effect on the growth of B12-dependent bacteria, including N2O reducers. We identified Dehalococcoidia and Clostridia as predominant N2O sinks in an anammox consortium fed exogenous N2O because of the higher metabolic potential of vitamin B12-dependent biosynthesis.

Microbial activity of suspended biomass from a nitritation-anammox SBR in dependence of operational condition and size fraction.

Single-stage nitritation-anammox combines the growth of aerobic ammonium-oxidizing bacteria (AOB) and anaerobic ammonium oxidizing bacteria (AnAOB) in one reactor. The necessary compromise of their milieu conditions often leads to the growth of nitrite-oxidizing bacteria (NOB). For this study, a sequencing batch reactor (SBR) for nitritation-anammox was operated for 180 days with sewage sludge reject water (removal capacity, 0.4 kg N m(-3) day(-1)). The growth of NOB was favored by enhanced oxygen supply rather than extended aerobic phases. Suspended-type biomass from this SBR was taken regularly and sieved into three size fractions (all of them <1,000 µm). Batch experiments as well as fluorescence in situ hybridization were performed to study the distribution and activity of AnAOB, AOB, and NOB within those size fractions. Both the measured conversion rates and detected abundances decreased with increasing size fraction. The highest anammox conversion rates (15 g NH4 (+)-N per kilogram VSS per hour) and the highest abundances of Brocadia fulgida were found in the medium size fraction (100-315 µm). The batch experiments proved to be accurate tools for the monitoring of multiple processes in the reactor. The results were representative for reactor performance during the 6 months of reactor operation.

Comparing the performance and operation stability of an SBR and MBBR for single-stage nitritation-anammox treating wastewater with high organic load.

Single stage nitritation-anammox reactors have gained increasing attention for their application in municipal and industrial wastewater treatment. The most commonly used system in municipal reject water treatment is at present the sequencing batch reactor (SBR), the moving-bed biofilm reactor (MBBR) is the second most common. However, little is known about their applicability to industrial wastewaters with high C/N ratios. This study presents a comparative approach to evaluate the performance of these two systems by changing the influent from reject water (C:N ratio 1:1) stepwise to an industrial wastewater (C:N ratio 3:1). An intentionally induced temperature drop that led to nitrite accumulation was also tested. The results showed that the MBBR (1.9 kg-N m(-3) d(-1)) was superior to the SBR (0.5 kg-N m(-3) d(-1)) with at maximum up to four times higher volumetric nitrogen removal rates. Both systems accumulated nitrite (> 100 mg-N L(-1)) during the temperature drop from 30 degrees C to as low as 18 degrees C (MBBR) and 20 degrees C (SBR), which subsequently resulted in almost complete loss in the removal capacities. However, the previous removal rates could be re-established in both systems within approximately 40 days. In comparison, the MBBR showed the more stable and higher performance even though higher nitrite concentrations (up to 500 mg-N L(-1)) were encountered. Overall, MBBR operation and handling was also easier and the system was more robust to disturbances compared to the SBR.

Effects of scouring on membrane aerated biofilm reactor performance and microbial community composition.

This study investigated the effects of scouring on Membrane Aerated Biofilm Reactors (MABRs). Laboratory-scale MABRs were operated under conditions typical for municipal wastewater. Scouring was induced by bubbling dinitrogen gas through the reactor bulk at low and high frequencies (LF and HF). At low nitrogen surface loads, almost complete ammonium removal was observable while HF scouring resulted in less total nitrogen (TN) removal compared to LF scouring. High nitrogen surface loads combined with HF scouring resulted in a higher TN removal as LF scouring. HF scouring resulted in around four times more sludge production and less residual biofilm mass compared to LF scouring. 16S amplicon sequencing of the biofilm, detached biomass and flocs revealed major differences between the microbial community compositions of these fractions. These results indicate that by varying the scouring strategy is a potential control mechanism for MABR operation and can help to reach specific treatment targets.

Comprehensive mutation profiling from wastewater in southern Germany extends evidence of circulating SARS-CoV-2 diversity beyond mutations characteristic for Omicron.

Tracking SARS-CoV-2 variants in wastewater is primarily performed by detecting characteristic mutations of the variants. Unlike the Delta variant, the emergence of the Omicron variant and its sublineages as variants of concern has posed a challenge in using characteristic mutations for wastewater surveillance. In this study, we monitored the temporal and spatial variation of SARS-CoV-2 variants by including all the detected mutations and compared whether limiting the analyses to characteristic mutations for variants like Omicron impact the outcomes. We collected 24-hour composite samples from 15 wastewater treatment plants (WWTP) in Hesse and sequenced 164 wastewater samples with a targeted sequencing approach from September 2021 to March 2022. Our results show that comparing the number of all the mutations against the number of the characteristic mutations reveals a different outcome. A different temporal variation was observed for the ORF1a and S gene. As Omicron became dominant, we observed an increase in the overall number of mutations. Based on the characteristic mutations of the SARS-CoV-2 variants, a decreasing trend for the number of ORF1a and S gene mutations was noticed, though the number of known characteristic mutations in both genes is higher in Omicron than Delta.