Nondestructive and three-dimensional visualization by identifying elements using synchrotron radiation microscale X-ray CT reveals microbial and cavity distributions in anaerobic granular sludge.

We developed a nondestructive three-dimensional microbial visualization method utilizing synchrotron radiation X-ray microscale computed tomography to better understand the relationship between microorganisms and their surrounding habitats. The method was tested and optimized using a mixture of axenic Escherichia coli and Comamonas testosteroni. The osmium-thiocarbohydrazide-osmium method was used to stain all the microbial cells, and gold in situ hybridization was used to detect specific phylogenetic microbial groups. The stained samples were embedded in epoxy resin for microtomographic analysis. Differences in X-ray absorbances were calculated by subtracting the pre-L3-edge images from the post-L3-edge images to visualize the osmium and gold signals. Although we successfully detected cells stained with osmium, those labeled with gold were not detected, probably because of the insufficient density of gold atoms in the microbial cells. We then applied the developed technique to anaerobic granules and visualized the distribution of microbial cells and extracellular polymeric substances. Empty spaces were highlighted to determine the cavity distribution in granules. Numerous independent cavities of different sizes were identified in the granules. The developed method can be applied to various environmental samples for deeper insights into microbial life in their habitats. IMPORTANCE: Microorganisms inhabit diverse environments and often form biofilms. One factor that affects their community structure is the surrounding physical environment. The arrangement of residential space within the formed biofilm plays a crucial role in the supply and transportation of substances, as well as the discharge of metabolites. Conventional approaches, such as scanning electron microscopy and confocal laser scanning microscopy combined with fluorescence in situ hybridization, have limitations as they provide information primarily from the biofilm surface and cross-sections. In this study, we developed a method for detecting microorganisms in biofilms using synchrotron radiation X-ray microscale computer tomography. The developed method allows nondestructive three-dimensional observation of biofilms at a single-cell resolution (voxel size of approximately 200 nm), facilitating an understanding of the relationship between microorganisms and their physical habitats.

Improved Properties and Enhancement Strategies of Hydroxyapatite-Based Functional Granular Sludge for a High-Rate Partial Nitritation/Anammox System.

Retaining sufficient anammox bacteria (AnAOB) while keeping the anammox-based process stable is the focus of the study of anammox technology, especially in a one-stage partial nitritation/anammox (PNA) process. The use of hydroxyapatite (HAP) granules in an anammox-based process is innovative for its potential to improve the nitrogen removal rate and achieve simultaneous removal of phosphorus. In this study, the HAP-based granular sludge was employed using enhancement strategies for an excellent nitrogen removal performance in a one-stage PNA process. Compared to those of other granular sludge PNA systems, a remarkable sludge volume index of 7.8 mL/g and an extremely high mixed liquor volatile suspended solids of 15 g/L were achieved under a low hydraulic retention time of 2 h. Consequently, an unprecedented nitrogen removal rate as high as 4.8 kg N/m3/d at 25 °C was obtained under a nitrogen loading rate of 6 kg N/m3/d. After a long-term operation of 870 days, the enhancement strategies underlying the superior performance of the granular sludge were identified. These findings clearly demonstrate that the enhancement strategies are crucial for the superior operating performance of the PNA process, and they can promote the application of the anammox-based process.

Kinetic and elemental characterization of HAP-based high-rate partial nitritation/anammox system orienting stability and inorganic elemental requirements.

Anammox-based processes are attractive for biological nitrogen removal, and the combination of anammox and hydroxyapatite (HAP) is promising for the simultaneous removal of nitrogen and phosphorus from wastewater. However, the kinetics of one-stage partial nitritation/anammox (PNA) in which ammonia-oxidizing bacteria (AOB) and anammox bacteria (AnAOB) exist in a reactor are poorly understood. Moreover, inorganic elements are required to promote microbial cell synthesis and growth; therefore, monitoring of elements to prevent the limitation and inhibition of the process is critical. The minimum amounts of inorganic elements required for a one-stage PNA process and the elemental flow remain unknown. Therefore, in this study, kinetics, stoichiometry, and element flow in the long-term, high-rate, continuous, one-stage HAP-PNA process with microaerobic granular sludge at 25 °C were determined using process modeling, parameter estimation, and mass balance. The biomass elemental composition was determined to be CH2.2O0.89N0.18S0.0091, and the biomass yield (Yobs) was calculated to be 0.0805 g/g NH4+-N. Therefore, a stoichiometric reaction equation for the one-stage HAP-PNA system was also proposed. The maximum specific growth rate (µm) of AnAOB and AOB were 0.0360 and 0.0982 d-1 with doubling times of 19 and 7.1 d, respectively. Finally, the elemental requirements for stable and high-rate performance were determined using element flow analysis. These findings are essential for developing the anammox-based process in a stable and resource-efficient manner and determining engineering applicability.

Comparative study of high-performance mesophilic and thermophilic anaerobic membrane bioreactors in the co-digestion of sewage sludge and food waste: Methanogenic performance and energy recovery potential.

To support smart cities in terms of waste management and bioenergy recovery, the co-digestion of sewage sludge (SeS) and food waste (FW) was conducted by the anaerobic membrane bioreactor (AnMBR) under mesophilic and thermophilic conditions in this study. The biogas production rate of the thermophilic AnMBR (ThAnMBR) at the SeS to FW ratio of 0:100, 75:25, 50:50 and 100:0 was 2.84 ± 0.21, 2.51 ± 0.26, 1.54 ± 0.26 and 1.31 ± 0.08 L-biogas/L/d, inconspicuous compared with that of the mesophilic AnMBR (MeAnMBR) at 3.00 ± 0.25, 2.46 ± 0.30, 1.63 ± 0.23 and 1.30 ± 0.17 L-biogas/L/d, respectively. The higher hydrolysis ratio and the poorer rejection efficiencies of the membrane under thermophilic conditions, resulting that the permeate COD, carbohydrate and protein of the ThAnMBR was higher than that of the MeAnMBR. The lost COD that might be converted into biogas was discharged with the permeate in the ThAnMBR, which was partly responsible for the inconspicuous methanogenic performance. Furthermore, the results of energy recovery potential assessment showed that the energy return on investment (EROI) of the MeAnMBR was 4.54, 3.81, 2.69 and 2.22 at the four SeS ratios, which was higher than that of the ThAnMBR at 3.29, 2.97, 2.02 and 1.80, respectively, indicating the advantage of the MeAnMBR over the ThAnMBR in energy recovery potential. The outcomes of this study will help to choose a more favorable temperature to co-digest SeS and FW to support the construction of smart cities.

Enhanced digestion of sludge via co-digestion with food waste in a high-solid anaerobic membrane bioreactor: Performance evaluation and microbial response.

A 15 L high-solid mesophilic AnMBR was operated for the digestion of food waste, primary sludge and excess sludge. The digestion performance was evaluated from the perspective of methane generation, permeate quality and organic reduction. Furthermore, the change in the microbial community was investigated by 16S rRNA gene analysis. The results showed that the introduction of sludge decreased the H2S levels in biogas compared with the mono-digestion of food waste and the co-digestion with food waste increased biogas and methane production compared with the mono-digestion of sludge. A substitution ratio of 25 % became a turning point of permeate composition and reaction rates. The energy recovery ratios of the mesophilic AnMBR were over 75 % based on stoichiometric analysis. In reaction kinetics analysis, hydrolysis as the first step of anaerobic digestion was found to be most influenced by the composition of the substrate. Finally, the microbial community structures were stable under tested conditions while the evolutionary relationships within the dominant phyla were observed. In the archaea community, Methanosaeta was the dominant methanogen regardless sludge ratio in the substrate.

A review on upgrading of the anammox-based nitrogen removal processes: Performance, stability, and control strategies.

The anaerobic ammonia oxidation (anammox) process is a promising biological nitrogen removal technology. However, owing to the sensitivity and slow cell growth of anammox bacteria, long startup time and initially low nitrogen removal rate (NRR) are still limiting factors of practical applications of anammox process. Moreover, nitrogen removal efficiency (NRE) is often lower than 88 %. This review summarizes the most common methods for improving NRR by increasing microorganism concentration, and modifying reactor configuration. Recent integrated anammox-based systems were evaluated, including hydroxyapatite (HAP)-enhanced one-stage partial nitritation/anammox (PNA) process for a high NRR of over 2 kg N/m3/d at 25 °C, partial denitrification/anammox (PDA) process, and simultaneous partial nitrification, anammox, and denitrification process for a high NRE of up to 100 %. After discussing the challenges for the application of these systems critically, a combined system of anaerobic digestion, HAP-enhanced one-stage PNA and PDA is proposed in order to achieve a high NRR, high NRE, and phosphorus removal simultaneously.

Enhanced degradation and biogas production of waste activated sludge by a high-solid anaerobic membrane bioreactor together with in pipe thermal pretreatment process.

An integrated system combining in pipe thermal pretreatment with a high-solid anaerobic membrane bioreactor (AnMBR) was developed to promote the anaerobic digestion of waste activated sludge (WAS). Two different pretreatment methods investigated were the venturi nozzle treatment (VNT) and steam injector treatment (SIT), both at a low temperature of 70 °C. The biogas production after pretreatment was 23.5-30.5% higher than that of untreated WAS, and the VS based biogas yield was 0.46-0.47 L/g-VS when HRT was 15 days. The membrane operated smoothly when the average flux was 9.6 and 4.5 L/m2/h under an MLTS of 25 and 30 g/L, respectively. The calculations of the mass balance indicated that 44-45% COD was converted to methane with pretreatment and only 1% remained in the permeate. That is, high energy recovery and organic matter removal efficiency were achieved for the treatment of WAS using the high-solid AnMBR with in pipe thermal pretreatment.

Nitrogen removal by a Hydroxyapatite-enhanced Micro-granule type One-stage partial Nitritation/anammox process following anaerobic membrane bioreactor treating municipal wastewater.

Nitrogen removal from wastewater by the partial nitritation/anammox (PNA) technology is promising from both economic and environmental perspectives. However, this technology has not been popularized in the mainstream because of low biomass retention and the growth of the nitrite oxidizing bacteria. In this study, a one-stage PNA process with hydroxyapatite (HAP)-enhanced granules was used to treat effluent from a mainstream anaerobic membrane bioreactor. The HAP-enhanced reactor allowed an enriched high biomass of 6.9 ± 0.2 g/L at a low hydraulic retention time of 2 h. A nitrogen removal efficiency of 80 ± 6.0 %, a nitrogen removal rate of 0.36 ± 0.05 kg/m3/d and a COD removal efficiency of 54 ± 15 % were achieved stably, leading to a low total nitrogen concentration of 8.5 ± 2.7 mg/L and a low COD concentration of 19.7 ± 5.9 mg/L in the effluent. Anammox bacteria of Candidatus Kuenenia stuttgartiensis and ammonium oxidizing bacteria of Nitrosomonas were found to be the two most predominant bacteria.

Long term operation performance and membrane fouling mechanisms of anaerobic membrane bioreactor treating waste activated sludge at high solid concentration and high flux.

High solid anaerobic membrane bioreactor (HSAnMBR) is widely applied in biomass treatment and energy regeneration, while membrane operation performance and membrane fouling control remain critical issues. In this study, a HSAnMBR was utilized for waste activated sludge (WAS) treatment at organic loading rates of 3.69-3.72 gCOD/L·d and biogas yield was ranged in 0.38-0.39 L/gVSfed with the COD conversion efficiency of 40 %. The membrane operated stably when the average flux was 9.6, 4.5 and 1.2 L/m2/h at mixed liquor total solid of 25, 30 and 40 g/L with a filtration: relaxation of 4:1, 1:1 and 1:2, respectively. The distinctive characteristics of membrane fouling at high solid condition were that the polysaccharides and proteins had high fouling propensity and were the main composition of the foulant layer. Furthermore, phosphorus and magnesium were the predominant causes of inorganic fouling. The minerals precipitated on the membrane and were embedded into membrane pores, contributing to cake layer formation and pore blocking. This research provided a comprehensive analysis of the membrane operation characterization and fouling mechanisms of HSAnMBR, which was expected to push forward HSAnMBR applications to WAS treatment.

High nitrogen removal performance of anaerobically treated fish processing wastewater by one-stage partial nitritation and anammox process with hydroxyapatite (HAP)-based syntrophic granules and granule structure.

The one-stage partial nitritation and anammox process with the hydroxyapatite (HAP)-based syntrophic granules was studied for the ammonium nitrogen removal from the effluents of a self-agitated anaerobic baffled reactor treating the fish processing wastewater. When the ammonium in the influent was 1140 mg N·L-1, a high nitrogen removal rate and nitrogen removal efficiency of 1.51 ± 0.10 kg N·m-3·d-1 and 88.2% were obtained, respectively. Anammox bacteria of Candidatus Kuenenia stuttgartiensis and ammonium oxidizing bacteria of Nitrosomonas were the two most predominant bacteria, while nitrite oxidizing bacteria activity was low and could be neglected during the treatment. The inorganic element properties of the sludge were analyzed by several methods to confirm the existence of HAP granules. Optical microscopic observation and scanning electron microscopy analysis revealed the structure of the granular sludge.This study supports the feasibility and potential of this process for high-efficiency nitrogen removal from fish processing wastewater.

Methanogenic performance and microbial community during thermophilic digestion of food waste and sewage sludge in a high-solid anaerobic membrane bioreactor.

The methanogenic performance and microbial community of the thermophilic anaerobic mono-digestion and co-digestion of food waste and sewage sludge in a high-solid membrane bioreactor were investigated by a continuous experiment. The methane recovery rate of the system reached 98.0% and 89.0% when the substrate was pure food waste and 25% sewage sludge substitution, respectively. Kinetics characterization showed that hydrolysis was the rate-limiting step in both mono-digestion and co-digestion while methanogenic performance and microbial community were significantly affected by feed condition. The dominant archaea for methane generation shifted from Methanothermobacter thermophilus (72.82%) to Methanosarcina thermophila (96.25%) with sewage sludge gradually added from 0% to 100% in the substrate. The relationships between digestion performance, such as the accumulation of soluble proteins in the reactor, and functional microbial groups were also carefully analyzed. Finally, reasonable metabolic pathways for mono-digestion and co-digestion were summarized.

Bioenergy recovery from methanogenic co-digestion of food waste and sewage sludge by a high-solid anaerobic membrane bioreactor (AnMBR): mass balance and energy potential.

To support smart city in terms of municipal waste management and bioenergy recovery, a high-solid anaerobic membrane bioreactor (AnMBR) was developed for sewage sludge (SeS) and food waste (FW) treatment in this study. COD mass balance showed that 54.1%, 66.9%, 73.5%, 91.4% and 93.5% of the COD input was converted into methane at the FW ratio of 0, 25%, 50%, 75% and 100%, respectively. The corresponding net energy balance was 13.6, 14.1, 17.1, 22.9 and 27.4 kJ/g-VS, respectively. An important finding of this investigation was that, for the first time, the relationship between net energy balance and carbon to nitrogen (C/N) ratio was revealed and the established sigmoid-type function was shown to be capable of predicting energy balance at different C/N ratios regardless of the region. The outcomes of this study show the potential of high-solid AnMBRs in SeS and FW treatment for supporting smart cities in the future.

High solid mono-digestion and co-digestion performance of food waste and sewage sludge by a thermophilic anaerobic membrane bioreactor.

The performance of co-digestion of food waste (FW) and sewage sludge (sludge) by a thermophilic anaerobic membrane bioreactor (ThAnMBR) was firstly investigated. The long-term stable operation showed the feasibility of the utilization of ThAnMBR for mono- and co-digestion of FW and sludge at a high solid condition. Good permeate quality was obtained at all sludge ratios while the addition of sludge restricted the methane generation. For a sludge substitution with a 25% TS-based substrate, the biogas yield of 0.812 L/g-VSfed was at 91% and 158% that of the mono-digestion of FW and sludge, respectively. Membrane performance indicated that the ThAnMBR operated stably at a high flux of 5 LMH under the high solid (~27 g/L) condition. Furthermore, membrane filtration with a 0.1 µm pore size of hollow fiber not only completely removed suspended solids but also rejected about 70% of soluble COD, 80% of soluble carbohydrates and 17% of soluble proteins.

Advanced methanogenic performance and fouling mechanism investigation of a high-solid anaerobic membrane bioreactor (AnMBR) for the co-digestion of food waste and sewage sludge.

Disposal of the increasingly huge amounts of sewage sludge (SeS) has become an impending problem worldwide. To solve this problem, a high-solid anaerobic membrane bioreactor (AnMBR) was used for the anaerobic co-digestion (AcoD) of SeS and food waste (FW). This study investigated the effects of SeS ratio on the methanogenic performance of the AcoD with a gradual increase value from 0 to 25%, 50%, 75% and 100% (total solids based). The results showed that the highest methanogenic performance was achieved at mono FW digestion. As for the co-digestion, the optimal FW/SeS ratio for methanogenic performance was 75%:25% among all the mixing ratios. The COD based biogas yield and methane yield were 0.498 L-biogas/g-CODfed and 0.295 L-CH4/g-CODfed at this optimal mixing ratio, which were 67.7% and 67.6% higher than those of the mono SeS digestion, respectively. The upgraded values were attributed to the improved hydrolysis ratio (by 8.14%) and the balanced carbon-to-nitrogen (C/N) ratio by co-digestion with FW, which synergistically stimulated methanogenesis ratio by 81.0%. The continuous membrane filtration property was investigated and four typical trans-membrane pressure (TMP) variation curves at different fouling degrees were determined. The membrane could sustainably operate at a flux of 6 L/m2/h (LMH) at the mixed liquor total solids (MLTS) concentration of 25 - 30 g/L. The combination of continuous membrane filtration property, particle size distribution of the mixed liquor and the external forces analysis was firstly applied to unravel the membrane fouling mechanism of a high-solid AnMBR. The result of this study will contribute to the establishment of an efficient FW and SeS treatment strategy.