Quadrupling the capacity of post aerobic digestion treating anaerobically digested sludge using a moving-bed biofilm (MBBR) configuration.

Wastewater treatment plants produce large amounts of sludge requiring stabilization before safe disposal. Traditional biological stabilization approaches are cost-effective but generally require either an extended retention time (10-40 days), or elevated temperatures (40-80 °C) for effective pathogens inactivation. This study overcomes these limitations via a novel acidic aerobic digestion process, leveraging an acid-tolerant ammonia-oxidizing bacterium (AOB) Candidatus Nitrosoglobus. To retain this novel but slowly growing AOB, we proposed the first-ever application of a classical wastewater configuration-moving bed biofilm reactor (MBBR)-for sludge treatment. The AOB in biofilm maintains acidic pH and high nitrite levels in sludge, generating free nitrous acid in situ to expedite sludge stabilization. This process was tested in two laboratory-scale aerobic digesters processing full-scale anaerobically digested sludge. At an ambient temperature of 20 °C, pathogens were reduced to levels well below the threshold specified for the highest stabilization level (Class A), within a retention time of 3.5 days. A high volatile solids reduction of 27.4 ± 5.2% was achieved. Through drastically accelerating stabilization and enhancing reduction, this process substantially saves capital and operational costs for sludge disposal.

Acidophilic partial nitrification (pH<6) facilitates ultra-efficient short-flow nitrogen transformation: Experimental validation and genomic insights.

Partial nitrification (PN) represents an energy-efficient bioprocess; however, it often confronts challenges such as unstable nitrite accumulation, nitrite oxidizing bacteria shocks, and slow reaction rate. This study established an acidophilic PN with self-sustained pH as low as 5.36. Over 120-day monitoring, nitrite accumulation rate (NAR) was stabilized at more than 97.9 %, and an ultra-high ammonia oxidation rate of 0.81 kg/m3·d was achieved. Notably, least NAR of 77.8 % persisted even under accidental nitrite oxidizing bacteria invasion, aeration delay, alkalinity fluctuations, and cooling shocks. During processing, despite detrimental effects on bacterial diversity, there was a discernible increase in acid-tolerant bacteria responsible for nitrification. Candidatus Nitrosoglobus, gradually dominated in nitrifying guild (2.15 %), with the substantially reduction or disappearance of typical nitrifying microorganisms. Acidobacteriota, a key player in nitrogen cycling of soil, significantly increased from 0.45 % to 9.98 %, and its associated nitrogen metabolism genes showed a substantial 215 % rise. AmoB emerged as the most critical functional gene influencing acidophilic PN, exhibiting significantly higher unit gene expression than other nitrification genes. Blockade tricarboxylic acid cycle, DNA damage, and presence of free nitrous acid exert substantial effects on nitrite-oxidizing bacteria (NOB), serving as internal driving forces for acidophilic PN. This highlights the reliable potential for shortening nitrogen transformation process.

Superior mainstream partial nitritation in an acidic membrane-aerated biofilm reactor.

Shortcut nitrogen removal holds significant economic appeal for mainstream wastewater treatment. Nevertheless, it is too difficult to achieve the stable suppression of nitrite-oxidizing bacteria (NOB), and simultaneously maintain the activity of ammonia-oxidizing bacteria (AOB). This study proposes to overcome this challenge by employing the novel acid-tolerant AOB, namely "Candidatus Nitrosoglobus", in a membrane-aerated biofilm reactor (MABR). Superior partial nitritation was demonstrated in low-strength wastewater from two aspects. First, the long-term operation (256 days) under the acidic pH range of 5.0 to 5.2 showed the successful NOB washout by the in situ free nitrous acid (FNA) of approximately 1 mg N/L. This was evidenced by the stable nitrite accumulation ratio (NAR) close to 100 % and the disappearance of NOB shown by 16S rRNA gene amplicon sequencing and fluorescence in situ hybridization. Second, oxygen was sufficiently supplied in the MABR, leading to an unprecedentedly high ammonia oxidation rate (AOR) at 2.4 ± 0.1 kg N/(m3 d) at a short hydraulic retention time (HRT) of a mere 30 min. Due to the counter diffusion of substrates, the present acidic MABR displayed a significantly higher apparent oxygen affinity (0.36 ± 0.03 mg O2/L), a marginally lower apparent ammonia affinity (14.9 ± 1.9 mg N/L), and a heightened sensitivity to FNA and pH variations, compared with counterparts determined by flocculant acid-tolerant AOB. Beyond supporting the potential application of shortcut nitrogen removal in mainstream wastewater, this study also offers the attractive prospect of intensifying wastewater treatment by markedly reducing the HRT of the aerobic unit.

Ammonium-based bioleaching of toxic metals from sewage sludge in a continuous bioreactor.

The broader reuse of sewage sludge as a soil fertilizer or conditioner is impeded by the presence of toxic metals. Bioleaching, a process that leverages microbial metabolisms and metabolites for metal extraction, is viewed as an economically and environmentally feasible approach for metal removal. This study presents an innovative bioleaching process based on microbial oxidation of ammonia released from sludge hydrolysis, mediated by a novel acid tolerant ammonia-oxidizing bacteria (AOB), Ca. Nitrosoglobus. Over a span of 1024 days, a laboratory-scale bioleaching reactor processing anaerobically digested (AD) sludge achieved an in-situ pH of 2.5 ± 0.3. This acidic environment facilitated efficient leaching of toxic metals from AD sludge, upgrading its quality from Grade C to Grade A (qualified for unrestricted use), according to both stabilization and contaminants criteria. The improved quality of AD sludge could potentially reduce sludge disposal expenses and enable a broader reuse of biosolids. Furthermore, this study revealed a pH-dependent total ammonia affinity of Ca. Nitrosoglobus, with a higher affinity constant at pH 3.5 (67.3 ± 20.7 mg N/L) compared to pH 4.5-7.5 (7.6 - 9.6 mg N/L). This finding indicates that by optimizing ammonium concentrations, the efficiency of this novel ammonium-based bioleaching process could be significantly increased.

Bioleaching of toxic metals from anaerobically digested sludge without external chemical addition.

Anaerobically digested (AD) sludge is widely applied to agricultural land as fertilizer. However, heavy metals in AD sludge potentially pose a significant threat to environment. This study reports a novel bioleaching approach, with no need for externally added chemicals. Sludge acidification was achieved using the protons produced from microbial oxidation of the inherent ammonium in AD sludge. An acid-tolerant microbial consortium, dominated by ammonia-oxidizing bacteria from the genus Candidatus Nitrosoglobus (i.e. relative abundance of 72.5 ± 2.3% based on 16S rRNA gene sequencing), was enriched after 120 days incubation in a laboratory sequencing batch reactor. The consortium oxidizes ammonium even at pH 2.5, at approximately 30% of its maximum rate, measured at pH 5.5. Inoculating the consortium at a solid ratio of 1:20, caused the pH of the AD sludge to decrease from 7.5 to 2.0 over five days under aerobic conditions. As a result, metals in the AD sludge were efficiently extracted into the liquid phase. In particular, two of the most abundant toxic metals, Cu and Zn, were solubilized with high efficiencies of 88 ± 4% and 96 ± 3%, respectively. Overall, the results of this study enable the economical and safe reuse of excess sludge generated during biological wastewater treatment.

Feasibility of methane bioconversion to methanol by acid-tolerant ammonia-oxidizing bacteria.

Bioconversion of biogas to value-added liquids has received increasing attention over the years. However, many biological processes are restricted under acidic conditions owing to the excessive carbon dioxide (CO2, 30-40% v/v) in biogas. Here, using an enriched culture dominated by acid-tolerant ammonia-oxidizing bacteria (AOB) 'Candidatus Nitrosoglobus', this study examined the feasibility of producing methanol from methane in the CO2-acidified environment (i.e. pH of 5.0). Within the tested dissolved methane range (0.1-0.9 mM), methane oxidation by the acid-tolerant AOB culture followed first-order kinetics, with the same rate constant (i.e. 0.43 (L/(g VSS‧h)) between pH 7.0 and 5.0. The acidic methane oxidation showed robustness against high dissolved concentrations of CO2 (up to 4.06 mM) and hydrogen sulfide (H2S up to 0.11 mM), which led to a high methanol yield of about 30-40%. As such, the raw biogas containing toxic CO2 and H2S can directly serve for methanol production by this acid-tolerant AOB culture, economizing a conventionally costly biogas upgradation process. Afterwards, two batch reactors fed with methane and oxygen intermittently both obtained a final concentration of 1.5 mM CH3OH (equal to 72 mg chemical oxygen demand/L) in the liquid, suggesting it is a useful carbon source to enhance denitrification in wastewater treatment systems. In addition, ammonia availability was identified to be critical for a higher rate of this AOB-mediated methanol production. Overall, our results for the first time demonstrated the capability of a novel acid-tolerant AOB culture to oxidize methane, and also illustrated the technical feasibility to utilize raw biogas for methanol production at acidic conditions.

Stoichiometric and kinetic characterization of an acid-tolerant ammonia oxidizer 'Candidatus Nitrosoglobus'.

Recently, acidic (i.e. pH<5) nitrification in activated-sludge is attracting attention because it enables stable nitritation (NH4+ â†’ NO2-), and enhances sludge reduction and stabilization. However, the key acid-tolerant ammonia oxidizers involved are poorly understood. In this study, we performed stoichiometric and kinetic characterization of a new acid-tolerant ammonia-oxidizing bacterium (AOB) belonging to gamma-proteobacterium, Candidatus Nitrosoglobus. Ca. Nitrosoglobus was cultivated in activated-sludge in a laboratory membrane bioreactor over 200 days, with a relative abundance of 55.1 ± 0.5% (indicated by 16S rRNA gene amplicon sequencing) at the time of the characterization experiments. Among all known nitrifiers, Ca. Nitrosoglobus bears the highest resistance to nitrite, low pH, and free nitrous acid (FNA). These traits define Ca. Nitrosoglobus as an adversity-strategist that tends to prosper in acidic activated-sludge, where the low pH (< 5.0) and high levels of FNA (at parts per million levels) sustained and inhibited all other nitrifiers. In contrast, in the conventional pH-neutral activated-sludge process, Ca. Nitrosoglobus is less competitive with canonical AOB (e.g. Nitrosomonas) due to the relatively slow specific growth rate and low affinities to both oxygen and total ammonia. These results advance our understanding of acid-tolerant ammonia oxidizers, and support further development of the acidic activated-sludge process in which Ca. Nitrosoglobus can play a critical role.

Acidic aerobic digestion of anaerobically-digested sludge enabled by a novel ammonia-oxidizing bacterium.

Anaerobic digestion is a commonly used process for the reduction and stabilization of wasted activated sludge generated in wastewater treatment plants. However, anaerobically-digested (AD) sludge is still a problematic waste stream due to its large volume and often poor quality. In this study, two aerobic digesters were set up to treat anaerobically-digested sludge, with one digester operated in self-generated acidic condition as the experimental reactor, and one at neutral pH as the control reactor. The acidic condition in the experimental reactor was driven by an inoculated special ammonia-oxidizing bacterium, 'Candidatus Nitrosoglobus', which can tolerate low pH. As a result of ammonium oxidation by Ca. Nitrosoglobus, the pH decreased to 4.8 ± 0.2 and nitrite accumulated to and stayed at 200.0 ± 17.2 mg N L-1, from which free nitrous acid (FNA) at 8.5 ± 1.8 mg HNO2N L-1 formed in-situ. As a combined effect of low pH and high concentration of FNA, the experimental reactor reduced the total solids (TS), volatile solids (VS) and non-volatile solids (NVS) in the AD sludge by 25.2 ± 7.0%, 29.8 ± 4.3%, and 22.6 ± 5.5%, respectively. In contrast, the control reactor without Ca. Nitrosoglobus inoculation (operated at a near-neutral pH of 6.8 ± 0.3 and no FNA formation) only reduced VS in the AD sludge by 10.4 ± 4.3%, along with negligible NVS reduction. Additionally, the acidic aerobic digestion in the experimental reactor significantly stabilized AD sludge, decreasing the specific oxygen uptake rate (SOUR) to 0.5 ± 0.1 mg O2 g-1VS h-1 and the most probable number (MPN) of Faecal Coliforms to 2.4 ± 0.1 log(MPN g-1TS), both of which meet USEPA standards for Class A biosolids. In comparison, the control reactor produced biosolids at Class B level only, with an SOUR of 1.8 ± 0.2 mg O2 g-1VS h-1 and a Faecal Coliforms MPN of 3.6 ± 0.1 log(MPN g-1TS). By reducing the volume and improving the quality of the AD sludge, the acidic aerobic digestion of AD sludge enabled by Ca. Nitrosoglobus has the potential to significantly save the sludge disposal costs in wastewater treatment.