Adsorption of Binary Mixture of Highly Positively Charged PTMA5M and Partially Negatively Charged PAA onto PSL Particles Studied by Means of Brownian Motion Particle Tracking and Electrophoresis.

Effects of ionic strength on the adsorption of highly charged polycationic ((2-dimethylamino)ethyl methacrylate) methyl chloride quaternary salt (PTMA5M) individually as well as in a binary mixture with polyanionic acrylic acid (PAA) onto polystyrene sulfate latex (PSL) particles with negative charges were investigated by means of Brownian movement particle tracking and measurement of electrophoretic mobility. In addition, the adsorption mechanism was confirmed by Fourier transform infrared (FT-IR) and energy-dispersive X-ray (EDX) spectroscopic methods. The hydrodynamic thickness of the adsorbed polyelectrolyte layer (δH) and electrophoretic mobility (EPM) of particles as a function of concentration ratios of the two polyelectrolytes were measured to clarify the effect of negatively charged molecules on the structure of the positively charged adsorbed layer at various ionic strengths. Extremely thick δH was confirmed for the case of excess dosage of polycations. The δH decreased more significantly with the addition of PAA than increasing ionic strength. Interestingly, in the presence of PAA, the adsorbed layer thickness increased more at lower ionic strength than at higher ionic strength. In addition, the initial δH decreases remarkably after a time lapse of 1 h. Contrary to the decrease of the δH, almost all EPM either remained unchanged or increased over time depending on the concentration ratio of the two polyelectrolytes. Constant charge density, desorption of polyanions, and then reconformation of the adsorbed polycationic layer are proposed model components.

Effect of shear flow on the hydrodynamic drag force of a spherical particle near a wall evaluated using optical tweezers and microfluidics.

The hydrodynamic drag force on a spherical particle in shear flow near-wall is investigated using optical tweezers and microfluidics. Simple shear flow is applied using a microfluidic channel at different volumetric flow rates. The hydrodynamic drag force exerted on the particle is detected from the displacement of the trapped particle. The effect of the wall is obtained from the force balance of the trapping and hydrodynamic drag force employing the exact solution of the theoretical model using the lubrication theory for a sphere near the wall. Here, we report the experimentally obtained hydrodynamic drag force coefficient under the influence of shear flow. The drag correction factor increases with decreasing distance from the wall due to the effect of the wall surface. We found that the calculated hydrodynamic drag force coefficient is in quantitative comparison with the theoretical prediction for a shear flow past a sphere near-wall. This study provides a straightforward investigation of the effect of the shear flow on the hydrodynamic drag force coefficient on a particle near the wall. Furthermore, these pieces of information can be used in various applications, particularly in optimizing microfluidic designs for mixing and separations of particles or exploiting the formation of the concentration gradient of particles perpendicular to flow directions caused by the non-linear hydrodynamic interactions.

Initial-Stage Dynamics of Flocculation of Cationic Colloidal Particles Induced by Negatively Charged Polyelectrolytes, Polyelectrolyte Complexes, and Microgels Studied Using Standardized Colloid Mixing.

The initial-stage dynamics of flocculation of positively charged latex particles induced by polyelectrolytes (PEs) and polyelectrolyte complexes (PECs), composed of linear polyacrylic acid (PAA) and a PAA-based hydrophilic microgel (PAA#) with a small amount of a linear polycation, was comparatively analyzed by applying the standardized colloidal mixing procedure. Based on the rate of flocculation, this method allows us to investigate the dynamics of flocculation immediately after the onset. In addition to confirming the prediction made regarding the initial rate of flocculation with linear polyanions-which was mostly similar to that observed in negatively charged colloids with positively charged PEs-we have confirmed two important new results regarding the microgel: (1) the increase of the initial rate is less markedly affected by the microgel concentration than by the linear polymer concentration, which can be explained by the fact that the three-dimensional (3D) cross-linked structure of the microgel that does not deform as easily as the linear structure upon touching the colloidal surface; and (2) there is a remarkable increase of the initial rate due to the contribution of instant aggregation of the negatively charged microgel induced by the polycation adsorption. These results suggest the significance of state and formation dynamics of PECs prior to reaching the surface of targeted colloidal particles for the intension of effective flocculation. These aspects are not treated so far in the dynamic process of flocculation.

Vascularized and functional human liver from an iPSC-derived organ bud transplant.

A critical shortage of donor organs for treating end-stage organ failure highlights the urgent need for generating organs from human induced pluripotent stem cells (iPSCs). Despite many reports describing functional cell differentiation, no studies have succeeded in generating a three-dimensional vascularized organ such as liver. Here we show the generation of vascularized and functional human liver from human iPSCs by transplantation of liver buds created in vitro (iPSC-LBs). Specified hepatic cells (immature endodermal cells destined to track the hepatic cell fate) self-organized into three-dimensional iPSC-LBs by recapitulating organogenetic interactions between endothelial and mesenchymal cells. Immunostaining and gene-expression analyses revealed a resemblance between in vitro grown iPSC-LBs and in vivo liver buds. Human vasculatures in iPSC-LB transplants became functional by connecting to the host vessels within 48 hours. The formation of functional vasculatures stimulated the maturation of iPSC-LBs into tissue resembling the adult liver. Highly metabolic iPSC-derived tissue performed liver-specific functions such as protein production and human-specific drug metabolism without recipient liver replacement. Furthermore, mesenteric transplantation of iPSC-LBs rescued the drug-induced lethal liver failure model. To our knowledge, this is the first report demonstrating the generation of a functional human organ from pluripotent stem cells. Although efforts must ensue to translate these techniques to treatments for patients, this proof-of-concept demonstration of organ-bud transplantation provides a promising new approach to study regenerative medicine.

Growth of Floc Structure and Subsequence Compaction into Smaller Granules through Breakup and Rearrangement of Aluminum Flocs in a Constant Laminar Shear Flow.

We have constructed an outer-cylinder-rotating Couette device for high-speed shear flow in laminar flow conditions and visualized the structure formation and subsequent rearrangement of PACl (flocculant made of aluminum hydroxide gel) and kaolinite flocs by visible light imaging. In a previous report, we analyzed the case of relatively low shear rate (G-value = 29 1/s) and confirmed that the flocculation process could be separated into two stages: a floc growth stage and a breakup/rearrangement stage. Once the large bulky flocs that reached the maximum size appeared, they rearranged and densified through structural fracture and rearrangement. In this report, this process was further investigated by conducting experiments under two different high shear rates (58 and 78 1/s) at which breakup and rearrangement became more pronounced, and three different aluminum kaolinite ratios (ALT ratios) that were over and under the optimum dosage (neutralization point by Zeta potential). Visualization results confirmed that, during the growth stage, the flocculation rate could be approximated by a scaling relationship between floc size and elapsed time, which depended on the ALT ratio. After reaching the maximum size, the floc rapidly became compact and dense following adsorption of the gel, incorporating fine fragments from erosion breakup. The over and under dosages created a lot of fragments of erosion breakup, but less so in the optimum dosage. In the optimum ALT ratio, fragments did not remain because they were incorporated into the flocs and densified, and the floc size was thought to be maintained. The floc circularity distribution peaked at around 0.6 and 1, suggesting that the flocs were spherical in shape due to erosion breakup.

Adsorption kinetics of polyacrylamide-based polyelectrolyte onto a single silica particle studied using microfluidics and optical tweezers.

HYPOTHESIS: Polyelectrolyte adsorption is considered important in tuning the surface property and the fate of particles; however, often studied on macroscopic surfaces. To gain insights into how polyelectrolytes are adsorbed onto a single particle, it is imperative to utilize techniques capable of elucidating adsorption kinetics on a single-particle level in a controlled flow field. EXPERIMENTS: The polyelectrolyte adsorption kinetics was investigated by electrophoretic mobility measurements combined with the kinetics study onto a single-particle viewpoint using microfluidics and optical tweezers. We directly evaluated the thickness, δH, of adsorbed polyelectrolyte onto a negatively-charged silica particle to deduce the adsorbed polyelectrolyte's conformation. The effect of charge density and salt concentrations were studied. FINDINGS: All polyelectrolytes exhibited dependence of δH on salt concentration. The attractive interactions control the cationic polyelectrolytes adsorption process. The δH depends on charge density indicating more loops and tail confirmation for the weakly-charged polyelectrolytes. The anionic polyelectrolytes showed a dependence of the initial rate and saturation value of δH on salt concentrations, attributed to the repulsion between charged segments and the silica surfaces. Here, we present new insights into the polyelectrolyte adsorption kinetics, particularly the influence of electrostatic interaction from the single-particle perspective, inaccessible to conventional bulk measurements.

Revealing calcium ion behavior during anaerobic phosphorus release process in aerobic granular sludge system.

Calcium ions (Ca2+) are important for biological phosphorus (P) removal from wastewater, but its behavior has not been well documented during the anaerobic P release process. This study is aimed to explore the mechanisms of Ca2+ release in bacterial aerobic granular sludge (AGS) system. During the non-aeration (anaerobic) phase, nearly 40 % increase in Ca2+ concentration was detected at the bottom of AGS reactor where decrease in pH and increase in Mg2+ concentration occurred. The pH decrease due to anaerobic P release caused CaCO3 dissolution inside the granules, leading to Ca2+ release. In addition, the increased Mg2+ ions from hydrolysis of polyphosphates were detected to reversibly exchange with Ca2+ in granules at a molar ΔCa/ΔMg ratio of 0.51-0.65. Results from this work revealed that dissolution of CaCO3 and ions exchange between Ca2+ and Mg2+ were the two major contributors to Ca2+ release during anaerobic P release process.

Highly efficient carbon assimilation and nitrogen/phosphorus removal facilitated by photosynthetic O2 from algal-bacterial aerobic granular sludge under controlled DO/pH operation.

Reducing CO2 emission and energy consumption is crucial for the sustainable management of wastewater treatment plants (WWTPs). In this study, an algal-bacterial aerobic granular sludge (AGS) system was developed for efficient carbon (C) assimilation and nitrogen (N)/phosphorus (P) removal without the need for mechanical aeration. The photosynthetic O2 production by phototrophic organisms maintained the dissolved oxygen (DO) level at 3-4 mg/L in the bulk liquid, and an LED light control system reduced 10-30% of light energy consumption. Results showed that the biomass assimilated 52% of input dissolved total carbon (DTC), and the produced O2 simultaneously facilitated aerobic nitrification and P uptake with the coexisting phototrophs serving as a C fixer and O2 supplier. This resulted in a stably high total N removal of 81 ± 7% and an N assimilation rate of 7.55 mg/(g-MLVSS∙d) with enhanced microbial assimilation and simultaneous nitrification/denitrification. Good P removal of 92-98% was maintained during the test period at a molar ∆P/∆C ratio of 0.36 ± 0.03 and high P release and uptake rates of 10.84 ± 0.41 and 7.18 ± 0.24 mg/(g- MLVSS∙h), respectively. Photosynthetic O2 was more advantageous for N and P removal than mechanical aeration. This proposed system can contribute to a better design and sustainable operation of WWTPs using algal-bacterial AGS.

[A case of papillary cystadenoma of the epididymis mimicking a testicular tumor].

A 68-year-old male presented with painless left scrotal enlargement of one year duration. Ultrasound, computed tomography (CT) and magnetic resonance imaging showed a multilocular cystic mass, 10×7.5× 8.5 cm in size, in the left scrotum. The intracystic fluid was partially hemorrhagic. A solid part of the tumor, seen at the base of the scrotum, was partially calcified and was enhanced by contrast medium. The left testis could not be identified by imaging studies. Although CT imaging showed a simple cyst in the right kidney, no other lesions in the kidneys, adrenal glands, pancreas or the central nervous system were detected. Serum tumor marker values for testicular cancer were within the normal range. Under the pre-operative diagnosis of a left testicular tumor, left high orchiectomy was performed. Grossly the specimen consisted of a multilocular cystic tumor, 12.5×8.5×8.5 cm in size, with a 2.7 cm tan-colored solid component within the wall of the cyst. The left testis was atrophic, 1.3 cm in size, and demonstated no continuity with the solid part of the tumor. Histologically, the solid component of the tumor showed tubular and papillary growth of cuboidal and columnar tumor cells with clear cytoplasm. Histopathological diagnosis of papillary cystadenoma of the epididymis (PCE) was made. Von Hippel-Lidau disease was ruled out by subsequent genetic analysis. After follow up for 18 months, there was no sign of recurrence. To our knowledge, this is the 33rd and the largest case of PCE reported in Japan.

Shielding behavior of electrokinetic properties of polystyrene latex particle by the adsorption of neutral poly(ethylene oxide).

To understand the shielding of electrokinetics of colloidal particles by polymer coating, we measured the electrophoretic mobility of negatively charged polystyrene sulfate latex (PSL) adsorbed with electrostatically neutral polyethylene oxide (PEO) chains with various molecular weights under different ionic strengths. We confirmed that substantial adsorbed neutral polymer on the particle surface would decrease the absolute value of electrophoretic mobility. Even though the polymer layer is sufficiently thicker compared to the thickness of electric double layer (EDL), the electrophoretic mobility (EPM) never vanishes, which indicates the incompleteness of electrokinetic shielding by an adsorbed neutral polymer. To relate such interesting phenomena, a simple mathematical model has been proposed to evaluate the electrophoretic mobility, assuming the presence of a scaling structure of adsorbed permeable polymer layer does not influence the Poisson-Boltzmann distribution of ions in the electric double layer (EDL). An analytical expression of electrophoretic mobility under Debye-Hu¨ckel approximation has been derived using the method of Ohshima-Kondo theory, which successfully justifies the experimentally obtained data.

Achieving stably enhanced biological phosphorus removal from aerobic granular sludge system via phosphorus rich liquid extraction during anaerobic period.

In order to sustainably manage wastewater treatment plants and the environment, enhanced biological phosphorus (P) removal (EBPR) was proposed to achieve P recovery through extracting P-rich liquid (i.e., Phostrip) from the bottom of aerobic granular sludge (AGS)-based sequencing batch reactors (SBRs) under no mixing during the anaerobic phase. Results showed both tested bacterial AGS (BAGS) and algal-bacterial AGS (A-BAGS) systems stably produced low effluent P (<0.05 mg-P/L) with little impact on their organics and NH4+-N removals (>99%). The collected P-rich liquids (55-83 mg-P/L) from both systems showed great potential for P recovery of about 83.85 ± 0.57 % (BAGS) or 83.99 ± 0.77% (A-BAGS), which were contributed by the influent P (>95%) and P reserves in granules based on P balance analysis. This study suggests that the AGS-based SBRs coupling the Phostrip holds great potentials for P recovery profit and further reduction in energy consumption.

A review on recovery of extracellular biopolymers from flocculent and granular activated sludges: Cognition, key influencing factors, applications, and challenges.

A reasonable recovery of excess sludge may shift the waste into wealth. Recently an increasing attention has been paid to the recycling of extracellular biopolymers from conventional and advanced biological wastewater treatment systems such as flocculent activated sludge (AS), bacterial aerobic granular sludge (AGS), and algal-bacterial AGS processes. This review provides the first overview of current research developments and future directions in the recovery and utilization of high value-added biopolymers from the three types of sludge. It details the discussion on the recent evolvement of cognition or updated knowledge on functional extracellular biopolymers, as well as a comprehensive summary of the operating conditions and wastewater parameters influencing the yield, quality, and functionality of alginate-like exopolymer (ALE). In addition, recent attempts for potential practical applications of extracellular biopolymers are discussed, suggesting research priorities for overcoming identification challenges and future prospects.

Organic anion transporter 3 mediates the efflux transport of an amphipathic organic anion, dehydroepiandrosterone sulfate, across the blood-brain barrier in mice.

The present study investigated the efflux transport systems of organic anions across the blood-brain barrier (BBB) using dehydroepiandrosterone sulfate (DHEAS) as a probe. The elimination of DHEAS from the brain after microinjection into the cerebral cortex was characterized in wild-type mice and mice with deficiency of well characterized organic anion transporters, organic anion-transporting polypeptide 1a4 (Oatp1a4)/Slco1a4 and organic anion transporter 3 (Oat3)/Slc22a8, at the BBB. The saturable efflux of DHEAS from the brain was completely inhibited by probenecid, benzylpenicillin, and estrone-3-sulfate and moderately inhibited by taurocholate and p-aminohippurate (50-57%). Uptake of DHEAS and estrone-3-sulfate was greater in murine Oat3 cRNA-injected oocytes than that in water-injected oocytes. Efflux of these compounds from the brain was significantly delayed in Oat3(-/-) mice compared with that in wild-type mice, indicating that indeed Oat3 is functionally important in vivo. Furthermore, probenecid and taurocholate inhibited DHEAS efflux completely in Oat3(-/-) mice. Contrary to the past report in rats that suggested involvement of Oatp1a4, specific uptake of DHEAS and estrone-3-sulfate by murine Oatp1a4 was not detected in vitro, and efflux of both compounds from the brain was not altered in Oatp1a4(-/-) mice. There was no significant difference in the uptake of DHEAS by brain slices prepared from wild-type, Oatp1a4(-/-), and Oat3(-/-) mice. Taken together, these results suggest that Oat3 plays a significant role in the efflux of steroid conjugates across the BBB in mice and that the BBB also expresses other unknown organic anion transporters for the efflux of DHEAS. Transport mechanisms of organic anions at the BBB are far more diverse than they were assumed to be.

Novel insight into enhanced recoverability of acidic inhibition to anaerobic digestion with nano-bubble water supplementation.

Nano-bubble water (NBW) has been proven to be effective in promoting organics utilization and CH4 production during anaerobic digestion (AD) process, suggesting its potential in improving the stability of the AD process and thereby alleviating acidic inhibition. In this work, the effect of NBW on digestion stability and CH4 production was investigated to evaluate the ability of NBW on AD recovery from acidic inhibition. Results showed that NBW supplementation increased the total alkalinity (TA) and partial alkalinity (PA), and reduced the ratio of VFA/TA, thus maintained the stability of the AD process. Generation/consumption of VFAs was also enhanced with NBW supplementation under acidic inhibition with pH values of 5.5, 6.0 and 6.5. The cumulative CH4 production was 246-257 mL/g-VS in NBW groups, which was 12.1-17.2% higher than the control. Moreover, with NBW supplementation, the maximum CH4 production rate was raised according to the modeling results.

Alleviation of ammonia inhibition via nano-bubble water supplementation during anaerobic digestion of ammonia-rich swine manure: Buffering capacity promotion and methane production enhancement.

Anaerobic digestion (AD) of ammonia-rich swine manure (SM) with nano-bubble water (NBW) supplementation was studied in this work with the expectation of ammonia inhibition alleviation, buffering capacity promotion, and methane production enhancement. Results indicated that cumulative methane yield was elevated by 12.3-38.7% in NBW groups. Besides, the reduced methane production rate and elongated lag phase under ammonia inhibition were increased and shortened by NBW supplementation, respectively. The rapid increase of total alkalinity (TA) and partial alkalinity (PA) could be observed with NBW supplementation, as well as the rapid decline of VFA/TA, thus improved buffering capacity and alleviated ammonia inhibition. Moreover, higher level of extracellular hydrolases and coenzyme F420 could be detected in NBW groups. In conclusion, NBW with higher mobility and zeta potential (absolute value) could be a promising strategy for the alleviation of ammonia suppression during the AD of SM.

Fabrication, Microstructure and Colloidal Stability of Humic Acids Loaded Fe3O4/APTES Nanosorbents for Environmental Applications.

Nowadays, numerous researches are being performed to formulate nontoxic multifunctional magnetic materials possessing both high colloidal stability and magnetization, but there is a demand in the prediction of chemical and colloidal stability in water solutions. Herein, a series of silica-coated magnetite nanoparticles (MNPs) has been synthesized via the sol-gel method with and without establishing an inert atmosphere, and then it was tested in terms of humic acids (HA) loading applied as a multifunctional coating agent. The influence of ambient conditions on the microstructure, colloidal stability and HA loading of different silica-coated MNPs has been established. The XRD patterns show that the content of stoichiometric Fe3O4 decreases from 78.8% to 42.4% at inert and ambient atmosphere synthesis, respectively. The most striking observation was the shift of the MNPs isoelectric point from pH ~7 to 3, with an increasing HA reaching up to the reversal of the zeta potential sign as it was covered completely by HA molecules. The zeta potential data of MNPs can be used to predict the loading capacity for HA polyanions. The data help to understand the way for materials' development with the complexation ability of humic acids and with the insolubility of silica gel to pave the way to develop a novel, efficient and magnetically separable adsorbent for contaminant removal.

Ionic response of algal-bacterial granular sludge system during biological phosphorus removal from wastewater.

Biological phosphorus removal (BPR) from wastewater can be generally realized through alternative non-aeration and aeration operation to create anaerobic and aerobic conditions respectively for P release and uptake/accumulation by polyphosphate accumulating organisms (PAOs), with P removal finally achieved by controlled discharge of P-rich sludge. In this study, the response of algal-bacterial aerobic granular sludge (AB-AGS) during BPR to main ions including Ac- (acetate), Cl-, SO42-, NH4+, K+, Mg2+, Ca2+ and Na+ in wastewater was investigated with conventional bacterial AGS (B-AGS) as control and acetate as the sole carbon source. Results show that BPR process mainly involved the changes of Ac-, K+, Mg2+, and Ca2+ rather than Cl-, SO42-, NH4+ and Na+. The mole ratio of ΔP/ΔAc kept almost unchanged during the non-aeration (P release) phase in both B-AGS and AB-AGS systems (ΔPB-AGS/ΔAcB-AGS > ΔPAB-AGS/ΔAcAB-AGS), and it was negatively influenced by the light in AB-AGS systems, in which 62% of acetate was not utilized for P release at the high illuminance of 81 k lux. During the entire non-aeration/aeration period, both ΔK/ΔP and ΔMg/ΔP remained constant, while ΔKAB-AGS/ΔPAB-AGS > ΔKB-AGS/ΔPB-AGS and ΔMgAB-AGS/ΔPAB-AGS ≈ ΔMgB-AGS/ΔPB-AGS. The presence of algae seemed not beneficial for PAOs to remove P, while more K+ and P uptake by algae in AB-AGS suggest its great potential for manufacturing biofertilizer.

Morphology and breaking of latex particle deposits at a cylindrical collector in a microfluidic chamber.

We report an analysis for the morphology and breaking behavior of deposits of spherical latex particles (1 and 3.6 µm in diameter) at a cylindrical collector in a microfluidic channel fabricated by soft-lithography. In-situ observation of particle deposition over a large range of flow rate conditions evidence the relationship between deposit morphology and mode of particle transport toward the collector. For low Péclet number (Pe), particle deposits are nearly uniform all over the collector surface except at the rear where particles do not attach. Upon increase of Pe, deposits gradually adopt a columnar morphology at the collector stagnation point. These results are qualitatively consistent with previously reported Monte Carlo simulations of deposits formation in stagnation point flow systems. However, these simulations fail to quantitatively predict the observed deposition at the rear of the collector for sufficiently high flow rate. Additional deposit breaking experiments together with numerical evaluations of particle flux around the collector suggest that such "anomalous" deposition at large Pe is significantly governed by concomitant detachment of deposited particles at the stagnation point and the presence of recirculation flow at the collector rear. Finally, kinetics of deposition are discussed in connection with particle size-dependence of deposit breaking features.

Effect of nano-bubble water on high solid anaerobic digestion of pig manure: Focus on digestion stability, methanogenesis performance and related mechanisms.

In this study, high solid anaerobic digestion of pig manure (PM) under nano-bubble water (NBW) addition was investigated with focus on digestion stability, methanogenesis performance and related mechanisms. Volatile fatty acids (VFAs) inhibition occurred when total solids (TS) was about 8% without NBW addition, which was alleviated with improved digestion stability under NBW addition, facilitating the process of high solids anaerobic digestion (HSAD). The cumulative CH4 yield, on the other hand, was 201-230 mL/g-VS in the NBW reactors at TS of 3-6%, about 20.3-25.0% higher than the control reactors. At the same time, with higher water mobility and zeta potential, NBW was found to promote the consumption of soluble proteins/carbohydrates during the above AD process.

Coupling biogas recirculation with FeCl3 addition in anaerobic digestion system for simultaneous biogas upgrading, phosphorus conservation and sludge conditioning.

The high costs involved in sewage sludge treatment and disposal in wastewater treatment plants (WWTPs) not only bring about improper sludge disposal and thus environmental pollutions, but also limit the investment on construction of WWTPs, especially in rural areas or low-income regions. This comparative study examined the effect of biogas recirculation coupled with chemical addition in a semi-continuous anaerobic digester for sludge treatment, which was proven to achieve biogas upgrading, phosphorus conservation and sludge conditioning simultaneously, largely reducing the sludge treatment cost. Results show that FeCl3 addition coupling biogas recirculation can improve sludge dewaterability by 94% in comparison to 75% by equivalent MgCl2 addition, and 97% phosphorus in digestate can be conserved in solid with formation of vivianite-like crystals. Biogas recirculation can enhance CH4 yield and content by 13% and 11%, respectively, likely attributable to the increased relative abundances of both hydrogenotrophic Methanomicrobiales and acetoclastic Methanosarcinales.