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Ethanol-type sludge fermentation has recently attracted much attention because it can enhance direct interspecies electron transfer and thus improve the anaerobic digestion of waste activated sludge (WAS). In this paper, the enhancement of short-term ethanol-type fermentation of WAS via adding Saccharomyces was investigated. The experimental results show that the maximum ethanol production of 1030.8 ± 20.6 mg/L was achieved, with the optimum fermentation conditions of a pH of 5.1, temperature of 26.0 ℃ and time of 8.0 hr. Although the content of volatile fatty acid (VFA) increased within 10 hr, it is one order of magnitude lower than the content of ethanol, indicating that the VFA generation did not affect the efficient production of ethanol. The analyses of changes in the microbial community during the fermentation process demonstrate that the greatest Saccharomyces activity occurred in the first 8 hr and it can play an important role in ethanol production even at a very low relative abundance. Meanwhile, most typical acid-producing bacteria were inhibited, but the hydrogenotrophic methanogens (i.e., Methanobacterium) were enriched to a certain extent. Further statistical analyses reveal that the Rhodobacter, Thermomonas, Terrimonas and Saccharomyces are responsible for ethanol production during the fermentation. However, these findings not only provide a reference for the development of enhancing ethanol-type fermentation of sludge, but also are expected to provide a new way of thinking for the efficient bioenergy and resource recovery from sludge.

The objective of this study was to evaluate the impacts of leachate co-treatment on a full-scale municipal WWTP by comparing plant performance at varying levels of leachate contributions and hydraulic loadings. Leachate BOD:COD ratio was 0.08 ± 0.07 and indicated a stabilized, old matrix and concentrations of zinc, iron, aluminum, chloride and sulfate were 0.174, 38, 1.47, 1803 and 119.1 mg/L, respectively. The average volumetric leachate ratio (VLR%) was approximately 0.01% corresponding to a daily volume of 30 m3 but reaching a maximum of 270 m3 (VLR% = 0.1%) and fluctuating on a daily-basis. A cluster analysis revealed 5 VLR% groupings that were used for subsequent analyses: no leachate, 0

Presence of methanotrophs in diverse environmental habitats helps to reduce emissions of greenhouse gas like methane. Isolation and culture of undiscovered wealth of methanotrophic organisms can help in exploitation of these organisms in value added products. The present study focuses on the enrichment of methanotroph dominated mixed microbial community by use of three stage strategy of revival, proliferation, and segregation. During the enrichment process amplicon sequencing of 16 s rRNA V3-V4 region showed relative abundance of mixed culture comprising single methanotrophic species of Methylocystis genus (88.92%) along with only three other species. Methylocystis dominant mixed culture (MMI-11) was observed to produce polyhydroxyalkanoates (PHA). During studies to identify favourable culture conditions, nitrate was found to be preferred nitrogen source for growth and PHA production. Cell growth ability to produce PHA was also evaluated at 14 L fermentor by supplying gas using continuous bubbling and through pressurization in the headspace. The mixed methanotrophic culture was found to accumulate maximum of 22.20% polyhydroxybutyrate (PHB) under nitrate limited condition. The molecular weight of PHB was found to be 2.221 × 105 g mol-1 with polydispersity of 1.82.

Plant-derived natural active products have attracted increasing attention for use in flavors and perfumes. These compounds also have applications in insect pest control because of their environment-friendly properties. Holy basil (Ocimum sanctum), a famous herb used in Ayurveda in India, is a natural source of medical healing agents and insecticidal repellents. Despite the available genomic sequences and genome-wide bioinformatic analysis of terpene synthase genes, the functionality of the sesquiterpene genes involved in the unique fragrance and insecticidal activities of Holy basil are largely unknown. In this study, we systematically screened the sesquiterpenoid biosynthesis genes in this plant using a precursor-providing yeast system. The enzymes that synthesize β-caryophyllene and its close isomer α-humulene were successfully identified. The enzymatic product of OsaTPS07 was characterized by in vivo mining, in vitro reaction, and NMR detection. This product was revealed as (-)-eremophilene. We created a mutant yeast strain that can achieve a high-yield titer by adjusting the gene copy number and FPP precursor enhancement. An optimized two-stage fed-batch fermentation method achieved high biosynthetic capacity, with a titer of 34.6 g/L cyclic sesquiterpene bioproduction in a 15-L bioreactor. Further insect-repelling assays demonstrated that (-)-eremophilene repelled the insect pest, fall leafworm, suggesting the potential of (-)-eremophilene as an alternative to synthetic chemicals for agricultural pest control. This study highlights the potential of our microbial platform for the bulk mining of plant-derived ingredients and provides an impressive cornerstone for their industrial utilization.

Bioponics has the potential to recover nutrients from organic waste streams, such as chicken manure and digestate with high volatile fatty acid (VFA) contents through crop production. Acetic acid, a dominant VFA, was supplemented weekly (0, 500, 1000, and 1500 mg/L) in a chicken manure-based bioponic system, and its effect on the performance of bioponics (e.g., plant yield and nitrogen and phosphorus availabilities) was examined. Microbial communities were analyzed using 16S rRNA gene sequencing, and the functional gene abundances were predicted using PICRUSt. Although acetic acid negatively affected plant yield, no significant difference (p 0.05) was noted in the average nitrogen or phosphorus concentration. In terms of nutrient recovery, the bioponic systems still functioned well, although higher concentrations of acetic acid decreased plant yield and altered the bacterial communities in plant roots and chicken manure sediments. These data suggest that an acetic acid concentration of 500 mg/L or a longer loading interval is recommended for the effective operation of chicken manure and digestate-based bioponics.

Herein, the effect of inorganic carbon (IC) limitation on the nitrogen removal performance of the single-stage reactor containing nitritation and anammox gel carriers was investigated. As a result of a continuous feeding test, the effluent ammonium concentration increased as the IC concentration decreased, indicating the deterioration of nitritation activity, not anammox. Furthermore, the sensitivity of IC to anammox and nitritation activity was investigated in anammox and nitritation reactors, respectively. Consequently, the relationship between the effluent IC concentration and nitritation rate was well described using the Michaelis-Menten equation. The apparent Km value of nitritation was calculated as 4.4 mg-C L-1. In anammox reactor, it was calculated as 1.7 mg-C L-1. These results revealed that the affinity of nitritation gel carriers to IC was lower than that of anammox, supporting that nitritation activity was easily deactivated by decrease in the IC concentration rather than anammox. Microbial community analysis revealed that Nitrosomonas europaea and Candidatus Jettenia asiatica were the dominant species of ammonium-oxidizing and anammox bacteria.

La Trobe University Faculty of Science, Technology and Engineering Murray Darling Freshwater Research CentreMDFRC item.The bacterial community of an aerobic:anaerobic non-P removing SBR biomass fed a mixture of acetateand glucose was analysed using several 16S rRNA based methods. Populations responsible for anaerobicglucose and acetate assimilation were determined with fluorescent in situ hybridization (FISH) in combinationwith microautoradiography (FISH/MAR). At ‘steady state’ this community consisted of α-Proteobacteria(26%) and γ-Proteobacteria (14%), mainly appearing as large cocci in tetrads (i.e. typical ‘GBacteria’).Large numbers of low G+C bacteria (22%), and high G+C Gram-positive bacteria (29%) seenas small cocci in clusters or in sheets were also detected after FISH. DGGE fingerprinting of PCR amplified16S rDNA fragments and subsequent cloning and sequencing of several of the major bands led tothe identification of some of these populations. They included an organism 98% similar in its 16S rRNAsequence to Micropruina glycogenica, and ca. 76% of the high G+C bacteria responded to a probe MIC184, designed against it. The rest responded to the KSB 531 probe designed against a high G+C clone sequence,sbr-gs28 reported in other similar systems. FISH analyses showed that both these high G+C populationswere almost totally dominated by small clustered cocci. Only ca. 2% of cells were β-Proteobacteria.None of the α- and γ-Proteobacterial ‘G-bacteria’ responded to FISH probes designed for the ‘GBacteria’Amaricoccus spp. or Defluvicoccus vanus. FISH/MAR revealed that not all the α-Proteobacterial‘G-Bacteria’ could take up acetate or glucose anaerobically. Almost all of the γ-Proteobacterial ‘G-Bacteria’assimilated acetate anaerobically but not glucose, the low G+C clustered cocci only took up glucose,whereas the high G+C bacteria including M. glycogenica and the sbr-gs28 clone assimilated both acetateand glucose. All bacteria other than the low G+C small cocci and a few of the α-Proteobacteria accumulatedPHB. The low G+C bacteria showing anaerobic glucose assimilation ability were considered responsiblefor the lactic acid produced anaerobically by this SBR biomass, and M. glycogenica for its highglycogen content.

Ergothioneine is a naturally occurring antioxidant that has shown potential in ameliorating neurodegenerative and cardiovascular diseases. In this study, we investigated the potential of the Crabtree-negative, oleaginous yeast Yarrowia lipolytica as an alternative host for ergothioneine production. We expressed the biosynthetic enzymes EGT1 from Neurospora crassa and EGT2 from Claviceps purpurea to obtain 158 mg/L of ergothioneine in small-scale cultivation, with an additional copy of each gene improving the titer to 205 mg/L. The effect of phosphate limitation on ergothioneine production was studied, and finally, a phosphate-limited fed-batch fermentation in 1 L bioreactors yielded 1.63 ± 0.04 g/L ergothioneine in 220 hours, corresponding to an overall volumetric productivity of 7.41 mg L-1 h-1 , showing that Yarrowia lipolytica is a promising host for ergothioneine production.

High-solid anaerobic digestion of the very small particle fraction of mechanically-sorted organic fraction of municipal solid waste (OFMSW) was examined in mesophilic digestion tests in a conventional laboratory (0.013 m3) and a pilot (0.300 m3) reactor. The non-biodegradable and recalcitrant molecules together with the low protein and starch contents of the small-particles of OFMSW limited the methane generation potential of substrate. In the conventional AD system, methane yields remained low at 0.139 m3kgVS-1 due to formation of a non-reacting layer on digestate surface, which restricted utilization of the available in OFMSW digestible organics. The absence of surface solid crust in the pilot unit favoured consumption of a greater proportion of volatile solids of the OFMSW. Dry AD was remarkably stable over the entire period and negligibly effected by the toxic H2S yields. Methane generation (0.167 m3kgVS-1) was increased 1.2-fold compared to the conventional system due to a better mixing of substrate and microorganisms achieved inside the pilot reactor, which led to an increase of the digested volatile organics. Digestate presented low stability and high heavy metal content, both of which restrain its implementation as soil conditioner or fertilizer in agriculture. A secondary co-digestion treatment may be required for the neutralization of digestate.

Ammoniacal nitrogen and long chain fatty acids (LCFA) are common inhibitors of the anaerobic digestion process. However, the interaction between these inhibitors has received little attention. Understanding the interaction between these inhibitors is important to optimise the operation of anaerobic digesters treating slaughterhouse waste or using fat, oil and grease (FOG) as co-substrate among others. To study the interaction between ammoniacal nitrogen and LCFA inhibition, 20 different conditions were trialled in mesophilic batch tests. Experimental conditions included 5 mixtures between slaughterhouse wastewater and LCFA (100:0, 75:25, 50:50, 20:80, 0:100 on a VS basis), each one tested at 4 different ammoniacal nitrogen concentrations (0, 1, 3, 6 gNadded·L-1). Experimental and modelling results showed that ammoniacal nitrogen inhibition was less severe in LCFA-rich mixtures, indicating that LCFA mitigated ammoniacal nitrogen inhibition to a certain extent. However, the positive interaction between inhibitors did not only depend on the LCFA concentration. A protective LCFA coat that limited the diffusion of free ammonia into the cell and/or provided a localised lower pH in the vicinity of the microbial cell could explain the experimental results. However, ammoniacal nitrogen and LCFA inhibition comprise up to 6 different but interrelated inhibitors (i.e. NH3, NH4+, LCFA, VFA, H2 and pH) and therefore the specific mechanism could not be elucidated. Nonetheless, these results suggest that LCFA do not exacerbate TAN-related inhibition and that LCFA-rich substrates can be utilised as co-substrates in mesophilic N-rich digesters.

The expanding use of chitosan in sewage and sludge treatment processes raises concerns about its potential environmental impacts. However, investigations of the impacts of chitosan on sewage sludge anaerobic digestion where chitosan is present at substantial levels are sparse. This study therefore aims to fill this knowledge gap through both long-term and batch tests. The results showed that 4 g/kg total suspended solid (TSS) chitosan had no acute effects on methane production, but chitosan at 8-32 g/kg TSS inhibited methane production by 7.2-30.3%. Mass balance and metabolism of organic analyses indicated that chitosan restrained the transfer of organic substrates from solid phase to liquid phase, macromolecules to micromolecules, and finally to methane. Further exploration revealed that chitosan suppressed the secretion of extracellular polymeric substances of anaerobes by occupying the connection sites of indigenous carbohydrates and increased the mass transfer resistance between anaerobes and substrates, which thereby lowered the metabolic activities of anaerobes. Although chitosan could be partly degraded by anaerobes, it is much more persistent to be degraded compared with indigenous organics in sludge. Microbial community and key enzyme encoding gene analyses further revealed that the inhibition of chitosan to CO2-dependent methanogenesis was much severer than that to acetate-dependent methanogenesis.

Partial nitritation is necessary for the implementation of the mainstream anammox (anaerobic ammonium oxidation) process in wastewater treatment plants. However, the difficulty in outcompeting nitrite-oxidizing bacteria (NOB) at mainstream conditions hinders the performance of partial nitritation. The present work aimed to develop a high-rate partial nitritation process for low-ammonium wastewater treatment at low temperatures by seeding aerobic granules. Experimental results suggested that both stratified structure of nitrifiers developed in the granules and sufficient residual ammonium concentration (18-35 mg N L-1) in the bulk liquid contributed to efficient NOB repression. With the hydraulic retention time progressively shortened from 1.0 to 0.17 h, the influent nitrogen loading rate of the partial nitritation process reached 6.8 ± 0.4 kg N m-3 d-1 even at 10-15 °C. The high concentration (7.5 gVSS L-1) and activity (0.48 g N g-1 VSS d-1 at 11 °C) of granular sludge made the reactor possess an overcapacity evaluated by the ratio between the actual ammonium oxidation rate of the granules and their maximum potential. The overcapacity helped the reactor to face the adverse effect of decreasing temperatures. Overall, this work indicated the great potential of applying aerobic granules to achieve high-rate partial nitritation at mainstream conditions. Moreover, anammox bacteria with a relative abundance of 2.8% was also identified in the partial nitritation granules at the end of this study, suggesting that the granules provided a habitable niche for anammox bacteria growth. Note that these results cannot fully relate to the treatment of real domestic/municipal wastewater, they are a source of important information increasing the knowledge about low temperature partial nitrification.

Transitions in microbial community structure in response to increasing ammonia concentrations were determined by monitoring mesophilic anaerobic digesters seeded with a predominantly acetoclastic methanogenic community from a sewage sludge digester. Ammonia concentration was raised by switching the feed to source segregated domestic food waste and applying two organic loading rates (OLR) and hydraulic retention times (HRT) in paired digesters. One of each pair was dosed with trace elements (TE) known to be essential to the transition, with the other unsupplemented digester acting as a control. Samples taken during the trial were used to determine the metabolic pathway to methanogenesis using 14C labelled acetate. Partitioning of 14C between the product gases was interpreted via an equation to indicate the proportion produced by acetoclastic and hydrogenotrophic routes. Archaeal and selected bacterial groups were identified by 16S rRNA sequencing, to determine relative abundance and diversity. Acclimatisation for digesters with TE was relatively smooth, but OLR and HRT influenced both metabolic route and community structure. The 14C ratio could be used quantitatively and, when interpreted alongside archaeal community structure, showed that at longer HRT and lower loading Methanobacteriaceae were dominant and hydrogenotrophic activity accounted for 77% of methane production. At the higher OLR and shorter HRT, Methanosarcinaceae were dominant with the 14C ratio indicating simultaneous production of methane by acetoclastic and hydrogenotrophic pathways: the first reported observation of this in digestion under mesophilic conditions. Digesters without TE supplementation showed similar initial changes but, as expected failed to complete the transition to stable operation.

Mainstream anaerobic wastewater treatment has received increasing attention for the recovery of methane-rich biogas from biodegradable organics, but subsequent mainstream nitrogen and dissolved methane removal at low temperatures remains a critical challenge in practical applications. In this study, granular sludge coupling n-DAMO with Anammox was employed for mainstream nitrogen removal, and the dissolved methane removal potential of granular sludge at low temperatures was investigated. A stable nitrogen removal rate (0.94 kg N m-3 d-1 at 20 °C) was achieved with a high-level effluent quality (

Microbial superhost strains should provide an ideal platform for the efficient homologous or heterologous phenotypic expression of biosynthetic gene clusters (BGCs) of new and novel bioactive molecules. Our aim in the current study was to perform a comparative study at the bioprocess and metabolite levels of the previously designed superhost strain Streptomyces coelicolor M1152 and its derivative strain S. coelicolor M1581 heterologously expressing chloramphenicol BGC. Parent strain M1152 was characterized by a higher specific growth rate, specific CO2 evolution rate, and a higher specific l-glutamate consumption rate as compared with M1581. Intracellular primary central metabolites (nucleoside/sugar phosphates, amino acids, organic acids, and CoAs) were quantified using four targeted LC-MS/MS-based methods. The metabolite pathways in the nonantibiotic producing S. coelicolor host strain were flooded with carbon from both carbon sources, whereas in antibiotic-producing strain, the carbon of l-glutamate seems to be draining out through excreting synthesized antibiotic. The 13 C-isotope-labeling experiments revealed the bidirectionality in the glycolytic pathway and reversibility in the non-oxidative part of PPP even with continuous uptake of d-glucose. The change in the primary metabolites due to the insertion of BGC disclosed a clear linkage between the primary and secondary metabolites.

Polyphosphate-accumulating organisms (PAOs), which can store high levels of phosphate (Pi) in the form of polyphosphate (polyP), are employed to engineer enhanced biological P removal (EBPR) from wastewaters. Co-localization of Mg and K in polyP granules of PAOs has been reported, and higher abundance of Mg-polyP granules relative to other metal complexes was correlated positively with EBPR performance stability. However, the underlying mechanism remains unknown. Here, we obtained molecular structural information of hydrated polyP complexes with four physiologically relevant metal cations (Na+, K+, Ca2+, and Mg2+) using computational and experimental techniques. Molecular dynamics simulations revealed that Mg-polyP and K-polyP complexes were the most and least stable of the complexes, respectively, suggesting that the co-occurrence of these complexes facilitates variable polyP bioavailability. The relative thermodynamic stability reflected the strength of metal chelation whereby the coordination distance between the polyP ligand O and the metal was 1.71-2.01 A for Mg2+ but this distance was 2.64-2.70 A for K+. Pair distribution function analysis of X-ray scattering data obtained with a Mg-polyP solution corroborated the theoretical Mg-polyP coordination geometry. These findings implied a possible mechanistic role of metal complexation in the P cycling traits of PAOs in engineered and natural systems.

Biosynthesis of bacterial cellulose (BC) in cylindrical oxygen permeable molds allows the production of hollow tubular structures of increasing interest for biomedical applications (artificial blood vessels, ureters, urethra, trachea, esophagus, etc.). In the current contribution a simple set-up is used to obtain BC tubes of predefined dimensions; and the effects of fermentation time on the water holding capacity, nanofibrils network architecture, specific surface area, chemical purity, thermal stability, mechanical properties, and cell adhesion, proliferation and migration of BC tubes are systematically analysed for the first time. The results reported highlight the role of culture time on key properties of the BC tubes produced, with significant differences arising from the denser and more compact fibril arrangements generated at longer fermentation intervals.

The combination of photodynamic therapy (PDT) and chemodynamic therapy (CDT) have attracted a great deal of interest, but tumor hypoxia and glutathione (GSH) overproduction still limit their further applications. Herein, an intelligent reactive oxygen species (ROS) nanogenerator Ce6/GOx@ZIF-8/PDA@MnO2 (denoted as CGZPM; Ce6, GOx, ZIF-8, PDA, MnO2 are chlorin e6, glucose oxidase, zeolitic imidazolate framework-8, polydopamine and manganese dioxide respectively) with O2-generating and GSH-/glucose-depleting abilities was constructed by a facile and green one-pot method. After intake by tumor cells, the outer MnO2 was rapidly degraded by the acidic pH, and the overexpression of hydrogen peroxide (H2O2) and GSH with abundant Mn2+ and O2 produced would eventually achieve multifunctionality. The Mn2+ acted as an ideal Fenton-like agent and magnetic resonance (MR) imaging contrast agent, while the O2 promoted the PDT via hypoxia relief and facilitated the intratumoral glucose oxidation by GOx for starvation therapy (ST). Benefiting from the GOx-based glycolysis process, sufficient H2O2 was generated to improve the CDT efficacy through Mn2+-mediated Fenton-like reaction. Notably, MnO2 and PDA could decrease the tumor antioxidant activity by consuming GSH, resulting in remarkably enhanced PDT/CDT. Such a novel cascade bioreactor with tumor microenvironment (TME)-modulating capability opens new opportunities for ROS-based and combinational treatment paradigms.

Large quantities of rejects from coal processing plants are currently disposed of as waste piles or in ponds and rivers, resulting in environmental concerns including pollution of rivers and ground and surface water contamination. This work investigates for the first time, a two-stage microbial process for converting coal processing wastes to methane, involving (1) fungal solubilisation of coal rejects and (2) microbial methanation of the solubilised products. Phanerochaete chrysosporium, Trichoderma viride and Neurospora discreta were screened for their ability to solubilise coal rejects. N. discreta was found to be the most suitable candidate based on the extent of bio-solubilisation, laccase activity, and reversed-phase high performance liquid chromatography (RP-HPLC) analysis. Bio-methanation of fungal-solubilised coal rejects was carried out in mesophilic anaerobic reactors with no additional carbon source, using inoculum from an anaerobic food digester. Coal rejects solubilised by N. discreta produced 3 to 6-fold higher methane compared to rejects solubilised by the other two fungi. No methane was produced from untreated coal rejects, demonstrating the importance of the fungal solubilisation stage. A total of 4.1 mmol methane was generated per gram of carbon in 15 days from N. discreta-solubilised coal rejects. This process offers a timely, environment-friendly, and sustainable solution for treatment of coal rejects and the generation of value-added products such as methane and volatile fatty acids.

Anaerobic digestion of sewage sludge (SS) is one of the effective ways to reduce the waste generated from human life activities. To date, there are many reports to improve or repress methane production during the anaerobic digestion of SS. In the anaerobic digestion process, many microorganisms work positively or negatively, and as a result of their microbe-to-microbe interaction and regulation, methane production increases or decreases. In other words, understanding the complex control mechanism among the microorganisms and identifying the strains that are key to increase or decrease methane production are important for promoting the advanced production of bioenergy and beneficial compounds. In this mini-review, the literature on methane production in anaerobic digestion has been summarized based on the results of antibiotic addition, quorum sensing control, and inorganic substance addition. By optimizing the activity of microbial groups in SS, methane or acetate can be highly produced. KEY POINTS: • Bactericidal agents such as an antibiotic alter microbial community for enhanced CH4 production. • Bacterial interaction via quorum sensing is one of the key points for biofilm and methane production. • Anaerobic digestion can be altered in the presence of several inorganic materials.

Biofilm process is a promising wastewater treatment technology and biofilm carrier (biocarrier) is regarded as the core of this process. However, the traditional commercial biocarriers have their inherent drawbacks, therefore, the development of new-type biocarrier to enhance wastewater treatment efficiency is significantly important to biofilm-based reactors. In this study, based on radical suspension polymerization, a novel kind of magnetic porous carriers (PMCs) was prepared by modifying the porous polymer carriers (PPCs) with inorganic particles, and then applied in a fluidized bed bioreactor (FBBR) with a low packing ratio of 10 % (v/v) to synthetic wastewater treatment. The results showed that this novel biocarrier possesses paramagnetism with saturation magnetization of 1.01emu/g, low density (1.26 g/cm3), excellent hydrophilicity (surface water contact angle approaching zero) and rough surface. Besides, compared with the PPCs, the developed PMCs have larger pores (up to 50 μm or more), in which the larger-sized microbes are able to colonize. Moreover, as compared to the PPCs-based FBBR, the PMCs-based reactor achieved shorter time (7 days) for biofilm formaiton and significantly enhanced NH3-N removal efficiency ( nearly 20 % increase at the level of influent NH3-N concentration about 100 mg/L). High-throughput sequencing (HTS) results indicated that this new biocarrier could promote biodiversity and improve the abundance of Nitrosomonadales (the functional bacteria for ammonia removal in the bio-system), thus enhancing the ammonification process. Therefore, the developed PMCs could be preferable biocarriers for biofilm formation and provide an alternative to the traditional suspended biocarrier, demonstrating a promising potential, even at a lower filling ratio, to enhance the pollutants removal performance.

The dephenolization of palm oil mill effluent (POME) with oil palm fiber-immobilized Trametes hirsuta AK 04 was conducted in a temporary immersion bioreactor to reduce the inhibitory effects of phenolics in anaerobic digestion. Longer immersion times provided greater removal of phenolics due to a higher release of manganese peroxidase. The most effective dephenolization was observed at 6 h immersed and 2 h non-immersed time (immersion ratio 6/8) with maximum removal of 85% from 1277 mg L−1 of phenolics in 4 days. The immobilized fungus maintained its high activity during multiple repeated batch treatments. The pretreated POME of 2 h showed higher methane yields compared with the untreated POME substrate. The methane yields increased with increasing pretreatment time and dephenolization levels. The results suggested that an increased abundance of methanogens was associated with the detoxification of phenolics. The fungal biomass contained crude protein, amino acids, and essential phenolics, which can be used as animal feed supplements.

A moderately halophilic bacterium isolated from the water samples collected from a salt field, Salinivibrio sp. TGB10 was found capable of producing poly-3-hydroxybutytate (PHB) from various sugars. Cell dry weight (CDW) of 8.82 g/L and PHB titer of 6.84 g/L were obtained using glucose as the carbon source after 24 h of cultivation in shake flasks. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was synthesized when propionate was provided as secondary carbon source. Salinivibrio sp. TGB10 exhibited favorable tolerance to propionate. The use of 8 g/L propionate and 20 g/L glucose as combinational substrates yielded 1.45 g/L PHBV with a 3-hydroxyvalerate monomer content of 72.02 mol% in flask cultures. In bioreactor study, CDW of 33.45 g/L and PHBV titer of 27.36 g/L were obtained after 108 h of fed-batch cultivation. The results indicated that Salinivibrio sp. TGB10 is a promising halophilic bacterium for the production of PHBV with various polymer compositions.

The methane and digestate production from biowaste (BW, 95% food waste and 5% garden waste based on fresh mass) and grease trap sludge (GTS) co-digestion at the Grossache-Nord WWTP (Austria) as a basis for a cost-benefit analysis was determined using two approaches: The first one was to determine the specific methane yields (SMY) and total solids (TS) removals (%) of the used substrates in biomethane potential (BMP) tests. In the second, the full-scale process data from a supervisory control and data acquisition (SCADA) system were analyzed. From these data, the SMY of the sewage sludge (SS) was calculated for a period without co-digestion and applied to the study period. Thus, it was possible to calculate the methane and digestate production from the co-substrates. Both approaches produced different co-substrate SMYs and TS degradation results. In the approach using the BMP, the SMY was 518 m3/t TSadded and the TS degradation was 77%. For the full-scale method, these values were found to be 620 m3/t TSadded and 66%, respectively. However, the cost-benefit analysis of both approaches indicated that electricity generation from co-digestion can cover the associated costs. The benefit to cost ratio was 1.14 and 1.08 for the BMP and full-scale approach, respectively. The application of the respective approach depends on the availability and quality of full-scale process SCADA data.

Presence of higher concentration of ammonia (> 0.5 mg/L) as well as nitrite (> 0.2 mg/L) in aquaculture environment create difficulties for fish survival. The existing methods for removal of these pollutants are time-consuming. A stable biofilm-based system for ammonia removal from aquaculture wastewater was developed in the current study to overcome the limitations of conventional treatment processes. In order to do so, initially the bacterial candidate was well characterized and tested for rapid biofilm development. The ammonia bioremediating Bacillus albus (ASSF01), from the activated sludge of shrimp farm, with a generation time of 67 min 12 s in suspension culture, was a structured biofilm former. The staining based measurement showed biofilm initiation from the very first hour of incubation. This finding was further validated using scanning electron microscopy (SEM), profilometry, and ellipsometry with Brewster angular microscopy (BAM). Hurst exponent (H) calculation using the profilometer and ellipsometer data yielded a value of H of above 0.9 and 0.62, indicating positive correlation or persistence behavior, hence a stable biofilm former. Each method of biofilm measurement, in spite of variation in complexity and sensitivity, was equally effective for biofilm progression analysis. The generation time of ASSF01 upon immobilization was 15 min and 36 s, ensuring rapid development of stable system. Response surface methodology (RSM)-based optimization of aquaculture wastewater treatment by the isolate in a biofilm reactor at ambient temperature revealed the optimum influent concentration of ammonia (3.2 mg/L), nitrate (6.89 mg/L), and phosphate (1.17 mg/L) to be reduced to discharge level (as per aquaculture requirement) with 14 h of hydraulic retention time. This study demonstrates the potential of the isolate as an efficient bioremediant for treating ammonia-containing aquaculture wastewater in a single unit biofilm reactor, ensuring rapid stabilization, environmental protection, and aquaculture sustenance.

With the development of agriculture, a huge amount of vegetable waste (VW) is produced every year, posing a large considerable environmental problem that cannot be ignored. Anaerobic digestion (AD), as an eco-friendly, efficient, and sustainable biomass conversion technology, may be used to address the pollution caused by VW. The compositional components of various VWs are different, which will affect their biomethane potential and directly determine whether they are suitable substrates for AD. Thus, this study involved a systematic analysis of the composition and biomethane potential of 20 typical VWs. The results showed that the methane yields of the VWs were different (207.5-346.3 mL/g VS) owing to the differences in composition. More importantly, a correlation between the contents of organic components and methane production was established, and then used to predict methane production by VW rapidly. In addition, first-order model, modified Gompertz, and Cone models were used to describe the biochemical methanogenesis mechanism of these VWs. The results of this study can provide a reference for fundamental research on the AD of VW as well as serve a convenient and precise method to predict methane production by different VWs through analyzing compositional components, which will be beneficial for pollution prevention and the comprehensive utilization of VW in the future.

The objective of this research was to evaluate the anaerobic co-digestion of guinea pig manure (CY) with Andean agricultural residues such as amaranth (AM), quinoa (QU) and wheat (TR) in batch biodigesters under mesophilic conditions (37⁰C) for 40 days. As microbial inoculum, sewage treatment sludge was used in two inoculum/substrate ratios (ISR of 1 and 2). In terms of methane production, the best results occurred in the treatments that contained AM and QU as co-substrate and an ISR of 2. Thus, the highest methane production occurred in the CY:AM biodigesters (25:75) and CY:QU (25:75) with 341.86 mlCH4/g VS and 341.05 mlCH4/g VS, respectively. On the other hand, the results showed that methane production with an ISR of 2 was more feasible for guinea pig waste, where the methane fraction of the biogas generated was in a range of 57 and 69%. The kinetics of methane production from these raw materials was studied using five kinetic models: modified Gompertz, logistic equation, transfer, cone, and Richards. The cone model was the one that best adjusted the experimental values ​​with those observed with an r2 of 0.999 and an RMSE of 1.16 mlCH4/g VS. Finally, the highest biodegradability was obtained in the CY-AM biodigesters (25:75) with 67.92%.

Plant cell wall hydrolysates contain not only sugars but also substantial amounts of acetate, a fermentation inhibitor that hinders bioconversion of lignocellulose. Despite the toxic and non-consumable nature of acetate during glucose metabolism, we demonstrate that acetate can be rapidly co-consumed with xylose by engineered Saccharomyces cerevisiae. The co-consumption leads to a metabolic re-configuration that boosts the synthesis of acetyl-CoA derived bioproducts, including triacetic acid lactone (TAL) and vitamin A, in engineered strains. Notably, by co-feeding xylose and acetate, an enginered strain produces 23.91 g/L TAL with a productivity of 0.29 g/L/h in bioreactor fermentation. This strain also completely converts a hemicellulose hydrolysate of switchgrass into 3.55 g/L TAL. These findings establish a versatile strategy that not only transforms an inhibitor into a valuable substrate but also expands the capacity of acetyl-CoA supply in S. cerevisiae for efficient bioconversion of cellulosic biomass., Cellulosic hydrolysates contain substantial amounts of acetate, which is toxic to fermenting microorganisms. Here, the authors engineer Baker’s yeast to co-consume xylose and acetate for triacetic acid lactone production from a hemicellulose hydrolysate of switchgrass.

Energy efficiency (EE) depends mainly on the lower heating values (LHVs) of hydrogen and methane selected from the thermodynamics tables under ideal conditions. However, for practical applications, the heating value should be calculated by considering some environmental factors under real conditions. Accordingly, this study compared EE using the ideal LHV with the EE using the real LHV in a two-phase anaerobic digestion reactor treating synthetic wastewater. Additionally, the process performance and the stability were studied. The results showed that the EE value calculated using LHVideal was, on average, 35% higher than that evaluated using LHVreal; these differences are relevant to the estimation of real energy and also for practical applications. At the same time, the index buffer intermediate alkalinity/partial alkalinity was shown to be more accurate than the pH value to analyze the stability of the process. With regards to chemical oxygen demand, the removal efficiency in the methanogenic phase decreased drastically when utilizing 100% of the acidogenic phase. Future considerations for the optimization of each phase are highlighted. HIGHLIGHTS Lower heating value should be adjusted to estimate the energy efficiency.; Different approaches are considered to evaluate the energy gain and the energetic efficiency in a anaerobic digestion process in two phases.; The IA/PA ratio is recommended as a process indicator if compared only with the pH value.

Chemical pretreatment of lignocellulosic biomass is a critical step in the conversion of lignocellulose to biofuels and biochemical. The main drawback of this pretreatment process is the formation of inhibitors which exhibit combined toxicity to microorganisms and result to low product concentrations and yields. In this study, the selection of microbial consortia by enrichment on hydrolysate of H2SO4-pretreated corn stover (pre-CS) without detoxification has been investigated as an efficient way to develop new strategies for lignocellulose utilization. The analysis of cattle stomach-dervied microbial consortia domesticated to degrade hydrolysate of pre-CS to produce lactic acid (LA) at different temperatures was investigated. Bacterial 16S rRNA gene amplicon sequencing analyses indicated that the three microbial consortia were taxonomically distinct and Enterococcus became dominant at high temperature. The highest glucose consumption rate was observed at 45 °C, while the three microbial consortia showed similar consumption rates of xylose and arabinose. The selected microbial consortia DUT37, DUT45 and DUT47 showed preferable resistances to inhibitors in hydrolysate of pre-CS and abilities of xylose utilization. A batch simultaneous saccharification and fermentation (SSF) process was developed by microbial consortium DUT47 at 47 °C to produce LA from pre-CS under non-detoxified and non-sterile conditions. The LA concentration and yield were 43.73 g/L and 0.50 g/g-corn stover (CS), respectively. Microbial consortium DUT47 has been shown to be suitable for LA production from H2SO4-pretreated corn stover without detoxification due to its thermophilic growth characteristics, robust tolerance of inhibitors, and the simultaneous utilization of glucose and xylose.

Raw syngas contains tar contaminants including toluene and naphthalene, which inhibit its conversion to methane. Cell encasement in a hydrophilic reverse membrane bioreactor (RMBR) could protect the cells from hydrophobic contaminants. This study aimed to investigate the inhibition of toluene and naphthalene and the effect of using RMBR. In this work, toluene and naphthalene were added at concentrations of 0.5-1.0 and 0.1-0.2 g/L in batch operation. In continuous operation, concentration of 0-6.44 g/L for toluene and 0-1.28 g/L for naphthalene were studied. The results showed that no inhibition was observed in batch operation for toluene and naphthalene at concentrations up to 1 and 0.2 g/L, respectively. In continuous operation of free cell bioreactors (FCBRs), inhibition of toluene and naphthalene started at 2.05 and 0.63 g/L, respectively. When they were present simultaneously, inhibition of toluene and naphthalene occurred at concentrations of 3.14 and 0.63 g/L, respectively. In continuous RMBRs, no inhibition for toluene and less inhibition for naphthalene were observed, resulting in higher methane production from RMBR than that of FCBR. These results indicated that RMBR system gave a better protection effect against inhibitors compared with FCBR.

Sewage from residents and industries is collected and transported to wastewater treatment plants (WWTPs) with sewer networks. The operation of WWTPs results in emissions of greenhouse gases, such as methane (CH4), mostly due to sludge anaerobic digestion. Amounts of emissions depend on the source of influent, i.e. municipal and industrial wastewater as well as sewer systems (gravity and rising). Wastewater is the fifth-largest source of anthropogenic CH4 emissions in the world and represents 7-9% of total global CH4 emissions into the atmosphere. Global wastewater CH4 emission grew by approximately 20% from 2005 to 2020 and is expected to grow by 8% between 2020 and 2030, which makes wastewater an important CH4 emitter worldwide. This review initially considers the emission of CH4 from WWTPs and sewer networks. In the second part, biotechniques available for biodegradation of low CH4 concentrations (

Shortcut nitrification is crucial for application of autotrophic nitrogen removal which is beneficial for treating carbon-limited wastewater. In this experiment, rapid start-up of autotrophic shortcut nitrification system was studied in a small sequencing batch reactor (SBR) built in laboratory with intermittent aeration operation mode. The influent was artificially simulated inorganic domestic wastewater (the ammonium nitrogen concentration was 35.19-57.54 mg/L), the pH value was 7.6-7.8, the hydraulic loading was 1L, the operating temperature was 24.3-28.3 °C, and the dissolved oxygen (DO) was 2-4 mg/L and 0.5-0.9 mg/L at the stage of complete nitrification sludge domestication and shortcut nitrification sludge domestication. High-throughput sequencing technology was used to analyse the composition and changes of microbial populations in sludge. The experimental results showed that on the 24th day of the experiment, shortcut nitrification was started successfully, the accumulation rate of nitrite was 81.63% and the removal efficiency of ammonium nitrogen was 99.25%; the richness of the main denitrifying bacteria phylum Proteobacteria increased from 30.21% to 42.85%; the richness of Nitrosomonas (ammonia oxidizing bacteria, AOB) increased from 0.37% to 22.43%, and at the species level, AOB was the salt-tolerant bacteria Nitrosomonas. europaea; the richness of Nitrospira (nitrite oxidizing bacteria, NOB) decreased from 2.59% to 0.47%.

In extent of this study, ammonium removal from wastewater through biological nitrification process was performed in upflow biofilm reactors. The effects of hydraulic retention time (HRT) and nitrogen loading rate (NLR) on the nitrification process were investigated. For the nitrification process, the optimum HRT and NLR were determined to be 80 hr and 0.044 kg/ m3.day, respectively. It is observed that the efficiency increased from 53% to 96% along with the increase in HRT from 22 hr to 80 hr and the decrease in NLR from 0.165 kg/m3.day to 0.044 kg/m3.day.The substrate consumption kinetics were studied in the attached growth reactor, and the Monod kinetic model, first-order kinetic model, modified Stover-Kincannon and Grau second-order kinetic models were examined. For the substrate consumption kinetic study, experimental studies were performed at 125, 150, 175, 200, 225 mg NH4-N/L substrate concentrations and 62 hr at HRT during the nitrification process. As a result of the considering kinetic studies, it was determined that the kinetic study was suitable for the modified Stover- Kincannon kinetic model that had the highest coefficient of regression by 0.997 and when the effluent NH4-N concentrations and NH4-N removal efficiencies calculated using kinetic models were examined, it was observed that the results closest to the experimental results (4.5, 10.1, 19.7, 26.2 and 42.3 mg NH4-N/L) were obtained through the modified Stover-Kincannon model (4.16, 10.71, 18.92, 28.12 and 39.51 mg NH4-N/L)., This study was supported by Cumhuriyet University Scientific Research Projects (CÜBAP) Commission within the scope of project M-494.

Negative pressure wound therapy (NPWT) has become the prevailing standard of care for treating complex soft tissue wounds and is now being considered for use in alternative applications including improving skin graft take. While it is generally agreed that negative pressure leads to improved wound healing, universal consensus on its optimal application is not supported in the literature. We describe the design and validation of a bioreactor to determine the prospective benefits of NPWT on skin grafts and engineered skin substitutes (ESS). Clinically relevant pressures were applied, and the native human skin was able to withstand greater negative pressures than the engineered substitutes. Both skin types were cultured under static, flow-only, and -75 mm Hg conditions for 3 days. While it remained intact, there was damage to the epidermal-dermal junction in the ESS after application of negative pressure. The normal skin remained viable under all culture conditions. The engineered skin underwent apoptosis in the flow-only group; however, the application of negative pressure reduced apoptosis. Vascular endothelial growth factor levels were significantly higher in the normal flow-only group, 152.0 ± 75.1 pg/mg protein, than the other culture conditions, 81.6 ± 35.5 pg/mg for the static and 103.6 ± pg/mg for the negative pressure conditions. The engineered skin had a similar trend but the differences were not significant. This bioreactor design can be used to evaluate the impacts of NPWT on the anatomy and physiology of skin to improve outcomes in wounds after grafting with normal or engineered skin.

Background This study focuses on the processes occurring during the acidogenic step of anaerobic digestion, especially resulting from nutritional interactions between dark fermentation (DF) bacteria and lactic acid bacteria (LAB). Previously, we have confirmed that DF microbial communities (MCs) that fed on molasses are able to convert lactate and acetate to butyrate. The aims of the study were to recognize the biodiversity of DF-MCs able and unable to convert lactate and acetate to butyrate and to define the conditions for the transformation. Results MCs sampled from a DF bioreactor were grown anaerobically in mesophilic conditions on different media containing molasses or sucrose and/or lactate and acetate in five independent static batch experiments. The taxonomic composition (based on 16S_rRNA profiling) of each experimental MC was analysed in reference to its metabolites and pH of the digestive liquids. In the samples where the fermented media contained carbohydrates, the two main tendencies were observed: (i) a low pH (pH ≤ 4), lactate and ethanol as the main fermentation products, MCs dominated with Lactobacillus, Bifidobacterium, Leuconostoc and Fructobacillus was characterized by low biodiversity; (ii) pH in the range 5.0–6.0, butyrate dominated among the fermentation products, the MCs composed mainly of Clostridium (especially Clostridium_sensu_stricto_12), Lactobacillus, Bifidobacterium and Prevotella. The biodiversity increased with the ability to convert acetate and lactate to butyrate. The MC processing exclusively lactate and acetate showed the highest biodiversity and was dominated by Clostridium (especially Clostridium_sensu_stricto_12). LAB were reduced; other genera such as Terrisporobacter, Lachnoclostridium, Paraclostridium or Sutterella were found. Butyrate was the main metabolite and pH was 7. Shotgun metagenomic analysis of the selected butyrate-producing MCs independently on the substrate revealed C.tyrobutyricum as the dominant Clostridium species. Functional analysis confirmed the presence of genes encoding key enzymes of the fermentation routes. Conclusions Batch tests revealed the dynamics of metabolic activity and composition of DF-MCs dependent on fermentation conditions. The balance between LAB and the butyrate producers and the pH values were shown to be the most relevant for the process of lactate and acetate conversion to butyrate. To close the knowledge gaps is to find signalling factors responsible for the metabolic shift of the DF-MCs towards lactate fermentation.

Biogas (methane) as a source of renewable energy, was produced in the anaerobic co-digestion of blackwater (BW, municipal toilet wastewater) and organic kitchen waste (KW). The impact on methane production of various BW to KW mixing ratios, with and without the addition of granular activated carbon (GAC), were studied under thermophilic (55 °C) temperatures. GAC is reported to enhance methane production in such digestions through direct interspecies electron transfer. The results showed that the co-digestion of BW and KW under the 1:2 VS ratio significantly improved the biomethane potential (BMP). In the absence of GAC, an optimal BW:KW ratio was found to be 1:2, achieving a BMP of 0.76 g CH4-COD/g feed-COD. With GAC addition, the BMP increased to 0.81 g CH4-COD/g feed-COD, the lag phase in the digestion was significantly reduced, and the methane production rate increased. Microbial communities in the BW-KW anaerobic digestion were analyzed with and without the addition of GAC. Methanothermobacter and Methanosarcina were predominant archaea in BW-KW digests, with and without GAC amendment, while a third methanogen, Methanomassiliicoccus, was enriched with the addition of GAC to the digest. Further, through SEM image, the enrichment of pili-like stucture was observed in GAC surface.

The increasing rate of food waste (FW) generation globally, makes it an attractive resource for renewable energy through anaerobic digestion (AD). The biogas recovered from AD can be upgraded by the methanation of internally produced carbon dioxide, CO2 with externally sourced hydrogen gas, H2 (biomethanation). In this work, H2 was added to AD reactors processing FW in three successive phases, with digestate from preceding phases recycled in succession with the addition of fresh inoculum to enhance acclimation. The concentration of H2 was increased for succeeding phases: 5%, 10% and 15% of the reactor headspace in Phase 1 (EH1), Phase 2 (EH2) and Phase 3 (EH3), respectively. The H2 utilisation rate and biomethane yields increased as acclimation progressed from EH1 through EH3. Biomethane yield from the controls: EH1_Control, EH2_Control and EH3_Control were 417.6, 435.4 and 453.3 NmL-CH4/gVSadded accounting for 64.8, 73.9 and 77.8% of the biogas respectively. And the biomethane yield from the test reactors EH1_Test, EH2_Test and EH3_Test were 468.3, 483.6, and 499.0 NmL-CH4/gVSadded, accounting for 77.2, 78.1 and 81.0% of the biogas respectively. A progressive in-situ biomethanation could lead to biomethane production that meets higher fuel standards for gas-to-grid (GtG) injections and vehicle fuel – i.e. >95% CH4. This would increase the energy yield and carbon savings compared to conventional biogas upgrade methods. For example, biogas upgrade for GtG by in-situ biomethanation could yield 7.3 MWh/tFW energy and 1343 kg-CO2e carbon savings, which is better than physicochemical upgrade options (i.e., 4.6–4.8 MWh/tFW energy yield and 846–883 kg-CO2e carbon savings).

Proteobacteria became the predominant phylum in the denitrifying anaerobic methane oxidation system of goat rumen fluid. Pseudomonas aeruginosa NWAFUP1 individually perform the whole denitrifying anaerobic methane oxidizing process. P . aeruginosa NWAFUP1 supplementation reduced methane emissions and alleviated the nitrite accumulation and propionic acid synthesis inhibition induced by nitrate supplementation., Summary After the occurrence of nitrate‐dependent anaerobic methane oxidation (AMO) in rumen fluid culture was proved, the organisms that perform the denitrifying anaerobic methane oxidizing (DAMO) process in the rumen of dairy goat were investigated by establishing two enrichment culture systems, which were supplied with methane as the sole carbon source and NaNO3 or NaNO2 as the electron acceptor. Several Operational Taxonomic Units (OTU) belonging to Proteobacteria became dominant in the two enrichment systems. The identified Pseudomonas aeruginosa, which was isolated from the NaNO2 enrichment system, could individually perform a whole denitrifying anaerobic methane oxidizing process. Further in vitro rumen fermentation showed that supplementation with the isolated P. aeruginosa could reduce methane emissions, alleviate the nitrite accumulation and prevent the decrease in propionic acid product caused by nitrate supplementation.

Methanotrophic microorganisms play a critical role in controlling the flux of methane from natural sediments into the atmosphere. Methanotrophs have been shown to couple the oxidation of methane to the reduction of diverse electron acceptors (e.g., oxygen, sulfate, nitrate, and metal oxides), either independently or in consortia with other microbial partners. Although several studies have reported the phenomenon of methane oxidation linked to selenate reduction, neither the microorganisms involved nor the underlying trophic interaction has been clearly identified. Here, we provide the first detailed evidence for interspecies electron transfer between bacterial populations in a bioreactor community where the reduction of selenate is linked to methane oxidation. Metagenomic and metaproteomic analyses of the community revealed a novel species of Methylocystis as the most abundant methanotroph, which actively expressed proteins for oxygen-dependent methane oxidation and fermentation pathways, but lacked the genetic potential for selenate reduction. Pseudoxanthomonas, Piscinibacter, and Rhodocyclaceae populations appeared to be responsible for the observed selenate reduction using proteins initially annotated as periplasmic nitrate reductases, with fermentation by-products released by the methanotrophs as electron donors. The ability for the annotated nitrate reductases to reduce selenate was confirmed by gene knockout studies in an isolate of Pseudoxanthomonas. Overall, this study provides novel insights into the metabolic flexibility of the aerobic methanotrophs that likely allows them to thrive across natural oxygen gradients, and highlights the potential role for similar microbial consortia in linking methane and other biogeochemical cycles in environments where oxygen is limited.

A pilot-scale biocover was constructed at a sanitary landfill and the mitigation of methane and odor compounds was compared between the summer and non-summer seasons. The average inlet methane concentrations were 22.0%, 16.3%, and 31.3%, and the outlet concentrations were 0.1%, 0.1%, and 0.2% during winter, spring, and summer, respectively. The odor removal efficiency was 98.0% during summer, compared to 96.6% and 99.6% during winter and spring, respectively. No deterioration in methane and odor removal performance was observed even when the internal temperature of the biocover increased to more than 40°C at midday during summer. During summer, the packing material simultaneously degraded methane and dimethyl sulfide (DMS) under both moderately thermophilic (40-50°C) and mesophilic conditions (30°C). Hyphomicrobium and Brevibacillus, which can degrade methane and DMS at 40°C and 50°C, were isolated. The diversity of the bacterial community in the biocover during summer did not decrease significantly compared to other seasons. The thermophilic environment of the biocover during summer promoted the growth of thermotolerant and thermophilic bacterial populations. In particular, the major methane-oxidizing species were Methylocaldum spp. during summer and Methylobacter spp. during the nonsummer seasons. The performance of the biocover remained stable under moderately thermophilic conditions due to the replacement of the main species and the maintenance of bacterial diversity. The information obtained in this study could be used to design biological processes for methane and odor removal during summer and/or in subtropical countries.

Bacterial extracellular polymeric substances (EPS) are promising materials that have a role in enhancing growth, metabolite production, and harvesting efficiency. However, the validity of the EPS effectiveness in scale-up cultivation of microalgae is still unknown. Therefore, in order to verify whether the bacterial metabolites work in the scale-up fermentation of microalgae, we conducted a bioreactor fermentation following the addition of bacterial EPS derived from the marine bacterium, Pseudoalteromonas sp., to Euglena gracilis. Various culture strategies (i.e., batch, glucose fed-batch, and glucose and EPS fed-batch) were conducted to maximize metabolite production of E. gracilis in scale-up cultivation. Consequently, biomass and paramylon concentrations in the continuous glucose and EPS-treated culture were enhanced by 3.0-fold and 4.2-fold (36.1 ± 1.4 g L-1 and 25.6 ± 0.1 g L-1), respectively, compared to the non-treated control (12.0 ± 0.3 g L-1 and 6.1 ± 0.1 g L-1). Also, the supplementation led to the enhanced concentrations of α-tocopherols and total fatty acids by 3.7-fold and 2.8-fold, respectively. The harvesting efficiency was enhanced in EPS-supplemented cultivation due to the flocculation of E. gracilis. To the best of our knowledge, this is the first study that verifies the effect of bacterial EPS in scale-up cultivation of microalgae. Also, our results showed the highest paramylon productivity than any other previous reports. The results obtained in this study showed that the scale-up cultivation of E. gracilis using bacterial EPS has the potential to be used as a platform to guide further increases in scale and in the industrial environment. KEY POINTS: Effect of EPS on Euglena gracilis fermentation was tested in bioreactor scale. EPS supplement was effective for the paramylon, α-tocopherol, and lipid production. EPS supplement induced the flocculation of E. gracilis.

The impact of pharmaceutical and personal care products (PPCPs) on the performance of biological wastewater treatment plants (WWTPs) has been widely studied using whole-community approaches. These contaminants affect the capacity of microbial communities to transform nutrients; however, most have neither honed their examination on the nitrifying communities directly nor considered the impact on individual populations. In this study, six PPCPs commonly found in WWTPs, including a stimulant (caffeine), an antimicrobial agent (triclosan), an insect repellent ingredient (N,N-diethyl-m-toluamide (DEET)) and antibiotics (ampicillin, colistin and ofloxacin), were selected to assess their short-term toxic effect on enriched nitrifying cultures: Nitrosomonas sp. and Nitrobacter sp. The results showed that triclosan exhibited the greatest inhibition on nitrification with EC50 of 89.1 μg L−1. From the selected antibiotics, colistin significantly affected the overall nitrification with the lowest EC50 of 1 mg L−1, and a more pronounced inhibitory effect on ammonia-oxidizing bacteria (AOB) compared to nitrite-oxidizing bacteria (NOB). The EC50 of ampicillin and ofloxacin was 23.7 and 12.7 mg L−1, respectively. Additionally, experimental data suggested that nitrifying bacteria were insensitive to the presence of caffeine. In the case of DEET, moderate inhibition of nitrification (−1. These findings contribute to the understanding of the response of nitrifying communities in presence of PPCPs, which play an essential role in biological nitrification in WWTPs. Knowing specific community responses helps develop mitigation measures to improve system resilience.

Members of the genus Dechloromonas are often abundant in enhanced biological phosphorus removal (EBPR) systems and are recognized putative polyphosphate accumulating organisms (PAOs), but their role in phosphate removal is still unclear. Here, we used 16S rRNA gene sequencing and fluorescence in situ hybridization (FISH) to investigate the abundance and distribution of Dechloromonas spp. in Danish and global wastewater treatment plants. The two most abundant species worldwide revealed in situ dynamics of important intracellular storage polymers, measured by FISH-Raman in activated sludge from four full-scale EBPR plants and from a lab-scale reactor fed with different substrates. Moreover, seven distinct Dechloromonas species were determined from a set of ten high-quality metagenome-assembled genomes (MAGs) from Danish EBPR plants, each encoding the potential for polyphosphate (poly-P), glycogen, and polyhydroxyalkanoates (PHA) accumulation. The two species exhibited an in situ phenotype in complete accordance with the metabolic information retrieved by the MAGs, with dynamic levels of poly-P, glycogen, and PHA during feast-famine anaerobic–aerobic cycling, legitimately placing these microorganisms among the important PAOs. They are potentially involved in denitrification showing niche partitioning within the genus and with other important PAOs. As no isolates are available for the two species, we propose the names Candidatus Dechloromonas phosphoritropha and Candidatus Dechloromonas phosphorivorans.

Anaerobic digestion (AD) is a biological treatment process to stabilize organic solids and produce biogas. If present, sulfate is reduced to sulfide by anaerobic sulfate-reducing bacteria and the sulfide can be toxic to anaerobic microorganisms. Here, the effect of high initial sulfate concentration on AD of wastewater sludge was investigated using lab-scale batch experiments. Additionally, a systematic mathematical modeling approach was applied for insight into the experimental results. Cumulative biogas and methane production decreased with increasing initial sulfate doses (0-3.300 mg S L-1 ). The correlation between the sulfate dose and methane production was consistent with theoretical predictions and model results, indicating no toxic effect of sulfide on methane production. The carbon dioxide content in the biogas decreased linearly with the increasing sulfate dose, which is consistent with the model-predicted behavior of the bicarbonate and hydrogen sulfide buffering system. The examined high sulfate concentrations resulted in no clear negative effects on the COD removal or VSS destruction of the wastewater sludge, indicating negligible inhibition by sulfide toxicity. Even considering the possibility of ferrous sulfide precipitation and the low model estimates of residual sulfide concentration the residual sulfide concentration was higher than reported concentrations that trigger process inhibition. PRACTITIONER POINTS: The effect of sulfate loading on anaerobic digestion of waste activated sludge was characterized. The stoichiometry of sulfate reduction allows accurate prediction of CH4 loss. High sulfate levels (up to 3300 mg/L as S) did not affect COD/VSS removal. Sulfide formation increases effluent COD; often misinterpreted as sulfide toxicity. Correcting COD for sulfide's contributions is crucial for results interpretation.

The partial nitritation/anammox process (PN/A) could be a promising alternative for nitrogen removal from high-strength wastewater. There is, however, a lack of information about suitable aeration and temperature for PN/A in single-stage reactors for high-strength wastewater, such as food waste (FW) digestate treatment. To this end, a laboratory-scale (10 L) partial nitritation/anammox sequencing batch reactor was operated for more than 230 days under four different intermittent aeration strategies and temperature variations (35°C and ambient temperature - 26-29°C) to investigate the feasibility of nitrogen removal from real FW digestate. High ammonium ( NH 4 + -N) and total nitrogen (TN) removal median efficiencies of 81 and 63%, respectively (corresponding to median NH 4 + -N and TN loads removed of 76 and 67 g.m-3.d-1), were achieved when the aeration strategy comprised by 7 min/14 min off and an airflow rate of 0.050 L.min-1.Lreactor-1 was applied. Nitrogen removal efficiencies were not affected by temperature variations in southeastern Brazil. COD, chloride and organic nitrogen (520, 239 and 102.8 mg.L-1, respectively) did not prevent PN/A. Changes of the bacterial community in response to aeration strategies were observed. Candidatus Brocadia dominated most of the time being more resistant to aeration and temperature changes than Candidatus Jettenia. This study demonstrated that optimizations of anoxic periods and airflow rate support PN/A with high nitrogen removal from FW digestate.

In our previous work, we reported a novel approach for increasing lipid production in an oleaginous fungus Mucor circinelloides by overexpression of mitochondrial malate transporter protein. This transporter plays a vital role in fatty acid biosynthesis during malate and citrate transport systems in oleaginous fungi. In this study, the controlling metabolic supplementation strategy was used to improve the lipid production by overexpression of malate transporter protein in M. circinelloides strain coded as Mc-MT-2. The effects of different metabolic intermediates on lipid production in batch fermentation by Mc-MT-2 were investigated. The optimal lipid production was obtained at 0.8% malic acid after 24 h of fermentation. Furthermore, in fed-batch bioreactors containing glucose as a carbon source supplemented with malic acid, the highest cell growth, and lipid production were achieved. The resulting strain showed the fungal dry biomass of 16 g/L, a lipid content of 32%, lipid yield of 5.12 g/L in a controlled bench-top bioreactor, with 1.60-, 1.60- and 2.56-fold improvement, respectively, compared with the batch control without supplementation of malic acid. Our findings revealed that the addition of malic acid during fermentation might play an important role in lipid accumulation in the recombinant M. circinelloides Mc-MT-2. This study provides valuable insights for enhanced microbial lipid production through metabolic supplementation strategy in large scale and industrial applications.

Streptokinase is a well-established cost-effective therapeutic molecule for thrombo-embolic complications. In the current study, a tag-free variant of streptokinase with a native N-terminus (N-rSK) was developed using the Pichia expression system. A three-copy clone was screened that secreted 1062 mg/L of N-rSK in the complex medium at shake flask level. The biologically active (67,552.61 IU/mg) N-rSK recovered by anion exchange chromatography was predicted to contain 15.43% α-helices, 26.43% β-sheets. The fermentation run in a complex medium yielded a poor quality product due to excessive N-rSK degradation. Therefore, modified basal salt medium was also employed during fermentation operations to reduce the proteolytic processing of the recombinant product. The concomitant feeding of 1 g/L/h soya flour hydrolysate with methanol during the protein synthesis phase reduced the proteolysis and yielded 2.29 g/L of N-rSK. The fermentation medium was also supplemented with urea during growth and induction phases. The combined feeding approach of nitrogen-rich soya flour hydrolysate and urea during bioreactor operations showed significant improvement in protein stability and resulted in a 4-fold increase in N-rSK concentration to a level of 4.03 g/L over shake flask. Under optimized conditions, the volumetric productivity and specific product yield were 52.33 mg/L/h and 33.24 mg/g DCW, respectively.

Substrate concentrations greatly influence bacterial growth and metabolism. However, optimal nitrogen concentrations for anammox bacteria in nitrogen-limited environments remain unclear. Here, we observed enhanced nitrogen metabolism and anabolism of anammox bacteria at low nitrogen conditions. Efficient nitrogen removal was achieved at ammonium and nitrite influent concentration of 30 mg/L under HRT of 1 hr, with an average nitrogen removal rate (NRR) of 0.73 kg N/(m3 ·day) in I-ABR composed of four compartments. The highest anammox activity of 6.25 mmol N/ (gVSS·hr) was observed in the fourth compartment (C4) with the lowest substrate levels (ammonium and nitrite of 11.6 mg/L and 7 mg/L). This could be resulted from the highest expression level of genes involved in nitrogen metabolism in C4, which was 1.49-1.67 times higher than that in other compartments. Besides, the second compartment (C2) exhibited the most active anabolism at ammonium and nitrite of 17 mg/L and 13 mg/L, respectively, which contributed to the most active amino acid synthesis and thus the highest EPS (1.35 times higher) in C2. This enhanced amino acid auxotrophy between anammox bacteria with heterotrophs, and consequently, heterotrophs thrived and competed for nitrite. These results hint at the potential application of anammox process in micro-polluted water. PRACTITIONER POINTS: High nitrogen removal and efficient biomass retention at low nitrogen concentrations under short HRT was achieved in I-ABR. Optimal concentrations for anammox nitrogen removal and anabolism were discussed under low nitrogen concentrations. More active anabolism contributed to enhanced amino acid synthesis and thus higher EPS contents. Low substrate levels led to enhanced expression of genes involved in nitrogen metabolism and thus high anammox activity.

Solid-state cultivation (SSC) is microbial growth on solid supports under limited water conditions. Citric acid, one of the products obtained by SSC, is a microbial aerobic metabolic product with various industrial applications. Several wastes from agro-industries are used in SSC, such as sugarcane bagasse and vinasse. As xylanolytic enzymes of inoculum breakdown the lignocellulosic material (bagasse), mixed fungal cultures or co-cultures are used in these SSC. Thus, this study aims to evaluate the effect of inoculum (Aspergillus niger and Trichoderma reesei consortium) in the production of citric acid from sugarcane bagasse impregnated with vinasse using bench packed bed reactors (PBR). The results show the importance of T. reesei in inoculum with A. niger at a ratio of 50:50 and 25:75, suggesting the use of solid support due to the complementation of the hydrolytic enzymes. The highest concentration of approximately 1000 mg L− 1 of citric acid yield for 100 mm of bed height in 48 and 72 h, with the maximum yield from glucose to citric acid (2.2 mg citric acid mg glucose−1). kLa indicates that maintaining solid moisture and liquid film thickness is important to keep the oxygen transfer in SSC.