Reduction of greenhouse gas emissions from closed activated sludge- to aerobic granular sludge-based biosystems via gas circulation.

Greenhouse gas (GHG) emissions from biological treatment units are challenging wastewater treatment plants (WWTPs) due to their wide applications and global warming. This study aimed to reduce GHG emissions (especially N2O) using a gas circulation strategy in a closed sequencing-batch reactor when the biological unit varies from activated sludge (AS) to aerobic granular sludge (AGS). Results show that gas circulation lowers pH to 6.3 ± 0.2, facilitating regular granules but elevating total N2O production. From AS to AGS, N2O emission factor increased (0.07-0.86 %) due to decreasing ammonia-oxidizing rates while the emissions of CO2 (0.3 ± 0.1 kg-CO2/kg-chemical oxygen demand) and CH4 remained in the closed biosystem. The gas circulation decreased N2O emission factor by 63 ± 15 % after granulation higher than 44 ± 34 % before granulation, which is implemented by heterotrophic denitrification. This study provides a feasible strategy to enhance heterotrophic N2O elimination in the biological WWTPs.

Photosynthetic oxygen-supported algal-bacterial aerobic granular sludge can facilitate carbon, nitrogen and phosphorus removal from wastewater: Focus on light intensity selection.

Photosynthetic O2 is a promising alternative for mechanical aeration, the major energy-intensive unit in wastewater treatment plants. This study aimed to investigate the effects of light intensity varied from 190 to 1400 µmol·s-1·m-2 on photosynthetic O2-supported algal-bacterial aerobic granular sludge (AGS) system. Results indicate photosynthetic O2 can implement aerobic phosphorus (P) uptake and ammonia oxidation under the test illumination range even at dissolved oxygen concentration < 0.5 mg/L. An obvious O2 accumulation occurred after 60-90% nutrients being removed under 330-1400 µmol·s-1·m-2, and highly efficient ammonia removal, P uptake, and dissolved inorganic carbon removal were achieved under 670-1400 µmol·s-1·m-2. On the other hand, photosynthesis as O2 supplier showed little effect on major ions except for K+. This study provides a better understanding of the roles of light intensity on photosynthetic O2-supported algal-bacterial AGS system, targeting a sustainable wastewater industry.

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.

Enhanced alginate-like exopolymers recovery from algal-bacterial aerobic granular sludge: Optimal cultivation condition and contribution of bacteria and microalgae during the transport/storage period.

Extracting alginate-like exopolymers (ALE) is a promising approach for valuable resources recovery from excess algal-bacterial aerobic granular sludge (AGS) to achieve circular bioeconomy and environmental sustainability in wastewater treatment plants (WWTPs). In this study, six batch cultivation tests were conducted to investigate the optimal cultivation duration or transport/storage period, light intensity, and temperature for algal-bacterial AGS after sampling and before further processing or ALE extraction. At a light intensity of 5 klux, the highest ALE content (36.33 mg/g-VSS) was detected at a low temperature of 10 °C, which increased by 300% from its original level after 6-h cultivation. Results from levofloxacin (LVX) exposure and dark condition imply that microalgae contributed more to ALE synthesis in the algal-bacterial granules. This work not only provides a better understanding of the mechanisms involved in ALE biosynthesis but also offers useful guidance for maintaining or improving ALE recovery after algal-bacterial biomass sampling.

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.

Characterization of Chicken α2A-Adrenoceptor: Molecular Cloning, Functional Analysis, and Its Involvement in Ovarian Follicular Development.

Adrenoceptors are suggested to mediate the functions of norepinephrine (NE) and epinephrine (EPI) in the central nervous system (CNS) and peripheral tissues in vertebrates. Compared to mammals, the functionality and expression of adrenoceptors have not been well characterized in birds. Here, we reported the structure, expression, and functionality of chicken functional α2A-adrenoceptor, named ADRA2A. The cloned chicken ADRA2A cDNA is 1335 bp in length, encoding the receptor with 444 amino acids (a.a.), which shows high amino acid sequence identity (63.4%) with its corresponding ortholog in humans. Using cell-based luciferase reporter assays and Western blot, we demonstrated that the ADRA2A could be activated by both NE and EPI through multiple signaling pathways, including MAPK/ERK signaling cascade. In addition, the mRNA expression of ADRA2A is found to be expressed abundantly in adult chicken tissues including thyroid, lung, ovary and adipose from the reported RNA-Seq data sets. Moreover, the mRNA expression of ADRA2A is also found to be highly expressed in the granulosa cells of 6-8 mm and F5 chicken ovarian follicles, which thus supports that ADRA2A signaling may play a role in ovarian follicular growth and differentiation. Taken together, our data provide the first proof that the α2A-adrenoceptor is functional in birds involving avian ovarian follicular development.

A comparative study on simultaneous recovery of phosphorus and alginate-like exopolymers from bacterial and algal-bacterial aerobic granular sludges: Effects of organic loading rate.

The effects of organic loading rate (OLR) on simultaneous phosphorus (P) and alginate-like exopolymers (ALE) recovery from bacterial aerobic granular sludge (AGS) and algal-bacterial AGS were examined and compared during 70 days' operation. With the increase of OLR (0.6-1.2 g COD/(L·day)), both AGS showed good settleability and granular strength with P bioavailability > 92% (Stage III). The moderate increase in OLR had a positive influence on simultaneous recovery of P and ALE. On day 60, the contents of ALE and guluronic acid/guluronic acid (GG) blocks reached the highest in algal-bacterial AGS, about 13.37 and 2.13 mg/g-volatile suspended solids (VSS), respectively. Meanwhile, about daily 0.55 kg of P is estimated to be recovered from the wastewater treatment plant with a treatment capacity of 10,000 m3/day. P mass balance analysis during ALE extraction from both AGS was conducive to further evaluation of P removal pathway and its application potentials.

Insight into aerobic phosphorus removal from wastewater in algal-bacterial aerobic granular sludge system.

This study aimed to figure out the main contributors to aerobic phosphorus (P) removal in the algal-bacterial aerobic granular sludge (AGS)-based wastewater treatment system. Kinetics study showed that aerobic P removal was controlled by macropore (contributing to 64-75% P removal) and micropore diffusion, and the different light intensity (0, 4.0, 12.3, and 24.4 klux) didn't exert significant (p > 0.05) influence on P removal. On the other hand, the increasing light intensity did promote microalgae metabolism, leading to the elevated wastewater pH (8.0-9.8). The resultant pH increase had a strongly negative relationship (R2 = 0.9723) with P uptake by polyphosphate-accumulating organisms, while promoted chemical Ca-P precipitation at a molar Ca/P ratio of 1.05. Results from this work could provide an in-depth understanding of microalgae-bacteria symbiotic interaction, which is helpful to better design and operate the algal-bacterial AGS systems.

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.

Neuropeptide S (NPS) and its receptor (NPSR1) in chickens: cloning, tissue expression, and functional analysis.

Neuropeptide S (NPS) and its receptor neuropeptide S receptor 1 (NPSR1) have been suggested to regulate many physiological processes in the central nervous system (CNS), such as arousal, anxiety, and food intake in mammals and birds, however, the functionality and tissue expression of this NPS-NPSR1 system remain unknown in birds. Here, we cloned NPS and NPSR1 cDNAs from the chicken brain and reported their functionality and tissue expression. The cloned chicken NPS is predicted to encode a mature NPS peptide of 20 amino acids, which shows a remarkable sequence identity (∼94%) among tetrapod species examined, while NPSR1 encodes a receptor of 373 amino acids conserved across vertebrates. Using cell-based luciferase reporter systems, we demonstrated that chicken NPS could potently activate NPSR1 expressed in vitro and thus stimulates multiple signaling pathways, including calcium mobilization, cyclic adenosine monophosphate/protein kinase A (cAMP/PKA), and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways, indicating that NPS actions could be mediated by NPSR1 in birds. Quantitative real-time PCR revealed that NPS and NPSR1 are widely expressed in chicken tissues, including the hypothalamus, and NPSR1 expression is likely controlled by a promoter upstream exon 1, which shows strong promoter activities in cultured DF-1 cells. Taken together, our data provide the first proof that the avian NPS-NPSR1 system is functional and helps to explore the conserved role of NPS and NPSR1 signaling in tetrapods.

Characterization of Four Orphan Receptors (GPR3, GPR6, GPR12 and GPR12L) in Chickens and Ducks and Regulation of GPR12 Expression in Ovarian Granulosa Cells by Progesterone.

The three structurally related orphan G protein-coupled receptors, GRP3, GPR6, and GPR12, are reported to be constitutively active and likely involved in the regulation of many physiological/pathological processes, such as neuronal outgrowth and oocyte meiotic arrest in mammals. However, the information regarding these orphan receptors in nonmammalian vertebrates is extremely limited. Here, we reported the structure, constitutive activity, and tissue expression of these receptors in two representative avian models: chickens and ducks. The cloned duck GPR3 and duck/chicken GPR6 and GPR12 are intron-less and encode receptors that show high amino acid (a.a.) sequence identities (66-88%) with their respective mammalian orthologs. Interestingly, a novel GPR12-like receptor (named GPR12L) sharing 66% a.a. identity to that in vertebrates was reported in the present study. Using dual-luciferase reporter assay and Western blot, we demonstrated that GPR3, GPR6, GPR12, and GPR12L are constitutively active and capable of stimulating the cAMP/PKA signaling pathway without ligand stimulation in birds (and zebrafish), indicating their conserved signaling property across vertebrates. RNA-seq data/qRT-PCR assays revealed that GPR6 and GPR12L expression is mainly restricted to the chicken brain, while GPR12 is highly expressed in chicken ovarian granulosa cells (GCs) and oocytes of 6 mm growing follicles and its expression in cultured GCs is upregulated by progesterone. Taken together, our data reveal the structure, function, and expression of GPR3, GPR6, GPR12, and GPR12L in birds, thus providing the first piece of evidence that GPR12 expression is upregulated by gonadal steroid (i.e., progesterone) in vertebrates.

Mobilization and transformation of arsenic from ternary complex OM-Fe(III)-As(V) in the presence of As(V)-reducing bacteria.

Organic matter (OM) was proved to have a high affinity for arsenic (As) in the presence of ferric iron (Fe(III)), the formed ternary complex OM-Fe(III)-As(V) were frequently studied before; however, the mobilization and transformation of As from OM-Fe(III)-As(V) in the presence of As(V)-reducing bacteria remains unclear. Two different strains (Desulfitobacterium sp. DJ-3, Exiguobacterium sp. DJ-4) were incubated with OM-Fe(III)-As(V) to assess the biotransformation of As and Fe. Results showed that Desulfitobacterium sp. DJ-3 could substantially stimulate the reduction and release of OM-Fe complexed As(V) and resulted in notable As(III) release (30 mg/L). The linear combination fitting result of k3-weighted As K-edge EXAFS spectra showed that 56% of OM-Fe-As(V) was transformed to OM-Fe-As(III) after 144 h. Besides, strain DJ-3 could also reduce OM complexed Fe(III), which lead to the decomposition of ternary complex and the release of 11.8 mg/g Fe(II), this microbial Fe(III) reduction process has resulted in 11% more As liberation from OM-Fe(III)-As(V) than without bacteria. In contrast, Exiguobacterium sp. DJ-4 could only reduce free As(V) but cannot stimulate As release from the complex. Our study provides the first evidence for microbial As reduction and release from ternary complex OM-Fe(III)-As(V), which could be of great importance in As geochemical circulation.

Variability of chromium bioaccessibility and speciation in vegetables: The influence of in vitro methods, gut microbiota and vegetable species.

There is limited research concentrating on the effects of gut microbiota on the bioaccessibility and speciation of chromium (Cr) in vegetables. In this study, the physiologically based extraction test (PBET) and the unified BARGE method (UBM), were combined with the simulator of human intestinal microbial ecosystems (SHIME) to determine the bioaccessibility and speciation of Cr from vegetables. The results showed that the Cr bioaccessibility was the highest in the gastric phase. The Cr bioaccessibility from the water spinach was the highest, and was 1.6-3.4 and 1.1-1.8 times that of leaf lettuce and celery, respectively. The Cr bioaccessibilities of the UBM method were slightly greater than those of the PBET method. Additionally, the gut microbiota increased the Cr bioaccessibility and reduced more toxic Cr(VI) to less toxic Cr(III) from vegetables. Therefore, our study reveals the possible health risks of consuming Cr-contaminated vegetables based on the bioaccessibility and speciation of Cr.

[Oxidation of Humic Acid Complexing As(â ¢) by As(â ¢)-Oxidizing Bacteria].

The interaction between soil arsenic and soil microorganisms has been identified as one of the important parts of the morphological transformation of soil arsenic. In order to investigate the interaction between Humic Acid complexing As(Ⅲ)[HA-As(Ⅲ)] and As(Ⅲ)-oxidizing bacteria (HN-2), the changes in arsenic speciation in the liquid phase and the solid phase, with different pH, were studied. The results indicated there was better As(Ⅲ) oxidation efficiency in the pH 7 reaction system. A part of As(Ⅲ) would be discharged from the HA-As(Ⅲ) solid phase during hours 0-10 in the reaction system, with or without HN-2, and meanwhile it was found that HN-2 oxidized As(Ⅲ) to As(Ⅴ) rapidly, while As(Ⅲ) was oxidized into As(Ⅴ) by HA gradually. As(Ⅲ) complexing HA can be transformed into free-As(Ⅲ), and then oxidized into free-As(Ⅴ) by HN-2 over hours 10-24 of the reaction. The system achieved the equilibrium state after 48 h. The results of the X-ray absorption near edge structure (XANES) further confirmed the conclusions above.

Nutritional status affects the bioaccessibility and speciation of arsenic from soils in a simulator of the human intestinal microbial ecosystem.

Arsenic (As) is a highly toxic contaminant in food and soil. In this study, we investigated the effects of four nutritional states (including a fed state with vitamin C, a fed state with protein powder, a fed state with glucose and a fasted state) on the variability of soil As bioaccessibility and biotransformation using the physiologically based extraction test (PBET) combined with a simulator of the human intestinal microbial ecosystem model (SHIME). The results indicated that the vitamin C and protein powder increased As bioaccessibility in gastric digests. In the colon phase, As bioaccessibility was observably enhanced by protein powder, and it varied under the vitamin C and glucose conditions. Additionally, the order of As methylation percentages in the four nutritional states was protein powder > vitamin C > fasted state > glucose (except S2); As bioaccessibility increased 1.3-13.7% and 15.8-35.4% in treatments of the vitamin C and protein powder, respectively. Meanwhile, large amounts of monomethylarsonic acid (MMAV) were observed in the colon digest in the protein powder condition. In contrast, As methylation was significantly decreased with the addition of glucose, with a decline of 25.9-45.5%. Additionally, glucose enhanced the reduction of As(V). Therefore, nutritional status is a crucial parameter for the prediction of bioaccessibility and speciation of As when assessing health risks from As following oral exposure.

In vitro study of soil arsenic release by human gut microbiota and its intestinal absorption by Caco-2 cells.

Arsenic (As) speciation is essential in assessing health risks from As-contaminated soil. Release of soil-bound arsenic, As transformation by human gut microbiota, and the subsequent intestinal absorption of soil As metabolites were evaluated. A colon microbial community in a dynamic human gut model and the intestinal epithelial cell line Caco-2 were cultured. Arsenic speciation analysis and absorption of different As species were undertaken. In this study, soil As release (3.7-581.2 mg kg-1) was observed in the colon. Arsenic in the colon digests was transformed more quickly than that in the soil solid phase. X-ray absorption near-edge spectroscopy (XANES) analysis showed that 44.2-97.6% of arsenite [As(III)] generated due to arsenate [As(V)] reduction was in the soil solid phase after the colon phase. We observed a high degree of cellular absorption of soil As metabolites, exhibiting that the intestinal absorption of monomethylarsonic acid and As(III) (33.6% and 30.2% resp.) was slightly higher than that of dimethylarsinic acid and As(V) (25.1% and 21.7% resp.). Our findings demonstrate that human gut microbiota can directly release soil-bound arsenic, particularly As-bearing amorphous Fe/Al-oxides. Determining As transformation and intestinal absorption simultaneously will result in an accurate risk assessment of human health with soil As exposures.

Comparison of arsenate reduction and release by three As(V)-reducing bacteria isolated from arsenic-contaminated soil of Inner Mongolia, China.

Arsenic (As) contamination has become a worldwide environmental problem: arsenite (As(Ⅲ)) especially has posed a major threat to human health. Here, we report the first three isolates of anaerobic As(Ⅴ)-reducing bacterial strains (strains JQ, DJ-3 and DJ-4) from a soil sample containing 48.7% of total As in the form of As(III) collected in Chifeng, Inner Mongolia, China. Strains JQ, DJ-3 and DJ-4 were phylogenetically closely related to Bacillus, Desulfitobacterium and Exiguobacterium, respectively. Among these strains, JQ and DJ-3 have the arsC gene, DJ-4 possesses the arrA gene. The three strains could all resist and reduce high concentrations of As(Ⅴ) under anoxic conditions. The order of resistance to As(Ⅴ) was DJ-3 > JQ > DJ-4. Strain DJ-3 not only possesses the strongest resistance to As(Ⅴ) but could also reduce 53% of the As(Ⅴ) to As(III) in the treatment of 60 mM As(Ⅴ) in 5 d. All three strains could release As from goethite; strain DJ-4 has the highest ability to promote the release of As (90.5%) from goethite. These results suggested that strains JQ, DJ-3 and DJ-4 may play an important role in the mobilization and transformation of As in soil.

Variability of arsenic bioaccessibility and metabolism in soils by human gut microbiota using different in vitro methods combined with SHIME.

Arsenic (As) speciation analysis is essential when evaluating the risks upon oral exposure to As-contaminated soils. In this study, we first investigated the variability in the As bioaccessibility and speciation using a combination of five common in vitro methods (SBRC, PBET, DIN, UBM and IVG) (gastric and small intestinal phases) and the SHIME model (colon phase). Our results indicate that the As bioaccessibility varies in the colon phase. An increase in the As bioaccessibility for SBRC and PBET, and a decrease for UBM and IVG were observed in the colon phase. In addition, we found different extents of methylation and large amounts of arsenite [As(III)] due to microbial reduction in the colon digests. The UBM-SHIME method displayed a higher methylation percentage of 13.5-82.1%, but a lower methylation percentage of 0.2-21.8% was observed in the SBRC-SHIME method. Besides, The MMA(V) levels in the colon digests were positively correlated with those of As(III) and DMA(V), so DMA(V) can be considered an indicator to evaluate the As metabolic speed of in vitro cultured human gut microbiota. Based on the standard reference soil of NIST 2710a, the As bioaccessibility in the colon phase of PBET-SHIME and SBRC-SHIME were the closest to the in vivo results. Combining in vitro methods and SHIME will remarkably affect the accurate assessment of potential risks to human health associated with oral exposure to soil As.

[Effects of Human Gut Microbiota on Bioaccessibility of Soil Cd, Cr and Ni Using SHIME Model].

The influence of human gut micobiota on bioaccessibilities of soil Cd, Cr, and Ni were investigated in this study. Five soil samples were collected from some sites of China, and the bioaccessibilities of soil Cd, Cr, and Ni in the gastric, small intestinal, and colon phases were determined using the PBET method (physiologically based extraction test) combined with SHIME model (simulator of human intestinal microbial ecosystem). The results showed that the bioaccessibilities of Cd, Cr, and Ni in the gastric phase were 4.3%-94.0%, 6.4%-21.6%, and 11.3%-47.3%, respectively. In the small intestinal phase, the bioaccessibilities of Cr and Ni were either congruent or slightly increased, while for Cd, the values were reduced by 1.4-1.6 folds except for soil 2. In the gastric and small intestinal phases, the mean bioaccessibility of Cd was higher but that of Cr was lower. In the colon phase, the bioaccessibilities of Cr and Ni were 1.3-2.4 and 1.0-2.1 times higher than those in the small intestinal phase. Furthermore, the bioaccessibility of Cd also increased except for soil 3 and 4. Human gut micobiota could induce Cd, Cr, and Ni release from soils and increase their bioaccessibilities, which may result in high risk to human health.