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In the present study the non-conventional yeast Wickerhamomyces anomalus X19, was evaluated as biocatalyst for direct, low cost bioethanol production, using natural date palm sap (DPS) as substrate. Initial experiments were conducted with synthetic media in order to characterize the strain in terms of its nitrogen requirements, the effect of initial substrate concentration on the ethanol yields and the main kinetic constants during alcoholic fermentation. Subsequently, the effect of aerobic or anaerobic conditions was assessed with DPS as substrate, in batch mode and the possible improvement of ethanol yields was further investigated via fed batch experiments. It was shown that W. anomalus X19 was not inhibited by low pH (<3) and high initial sugar concentrations (up to 160 g/L), leading in all cases to ethanol yields that exceeded 80% of the theoretically estimated values. The highest ethanol concentration from DPS fermentation was achieved in fed batch experiments, reaching 73.11 g/L and the ethanol yield was 0.46 g/g sugars, which corresponded to more than 90% of the theoretical maximum. • Date palm sap was assessed as renewable EtOH production feedstock via W. anomalus. • Batch and fed batch fermentations reached to high yields without pH adjustment. • EtOH max at fed-batch mode exceeded 73 g/L leading to 91% fermentation efficiency. [ABSTRACT FROM AUTHOR]

The biotransformation of the pre-dried and shredded organic fraction of kitchen waste to ethanol was investigated, via co-cultures of the yeasts Saccharomyces cerevisiae and Pichia stipitis (Scheffersomyces stipitis ). Preliminary experiments with synthetic media were performed, in order to investigate the effect of different operational parameters on the ethanol production efficiency of the co-culture. The control of the pH and the supplementation with organic nitrogen were shown to be key factors for the optimization of the process. Subsequently, the ethanol production efficiency from the waste was assessed via simultaneous saccharification and fermentation experiments. Different loadings of cellulolytic enzymes and mixtures of cellulolytic with amylolytic enzymatic blends were tested in order to enhance the substrate conversion efficiency. It was further shown that for solids loading up to 40% waste on dry mass basis, corresponding to 170 g.L −1 initial concentration of carbohydrates, no substrate inhibition occurred, and ethanol concentration up to 45 g.L −1 was achieved. [ABSTRACT FROM AUTHOR]

The accumulation efficiency and the properties of polyhydroxyalkanoates (PHAs) produced from acidified waste glycerol (AWG) and its derivatives via an enriched microbial consortium derived from soil, were investigated in this study. AWG consisted mainly from short chain fatty acids, 1,3 propanediol and residual glycerol, which were also evaluated individually as substrates. Accumulation capacity and yields were estimated after solvent extraction and purification and PHAs were further analyzed in terms of their chemical structure, thermal properties, molecular masses and mechanical properties. The lowest accumulation capacity was noticed for non-acidified waste glycerol as carbon source which led to the generation of P(3HB), whereas for the other carbon sources co -polymers of 3HB with 3HV or 3HHx were produced. Average molecular mass weights were quite high in all cases reaching ~1.8 × 10 6 Da. The thermal properties and the mechanical behavior of PHAs were shown to be highly affected by their monomeric composition, whereas it was also concluded that DSC and DMA results were in good agreement. [ABSTRACT FROM AUTHOR]

A continuous attached growth process for the production of biohydrogen from crude glycerol was developed. The process consisted of an anaerobic up-flow column bioreactor (UFCB), packed with cylindrical ceramic beads, which constituted the support matrix for the attachment of bacterial cells. The effect of crude glycerol concentration, pH and hydraulic retention time on glycerol conversion, hydrogen yield and metabolite distribution was investigated. It was shown that the most critical parameter for the efficient bioconversion was the pH of the influent, whereas the hydrogen yield increased with an increase in feed glycerol concentration and a decrease in the hydraulic retention time. The main soluble metabolite detected was 1,3-propanediol in all cases, followed by butyric and hexanoic acids. The latter is reported to be produced from glycerol for the first time. Acidification of the waste reached 38.5%, and the maximum H 2 productivity was 107.3 ± 0.7 L/kg waste glycerol at optimal conditions. [ABSTRACT FROM AUTHOR]

Abstract: This study investigated the impact of olive mill wastewater (OMW) as a pollutant of the marine environment, via the detection of stress indice alterations in mussels Mytilus galloprovincialis. Due to the absence of data concerning the levels of OMW in the receiving waters, mortality test (96h) was first performed in order to estimate the range of OMW concentration where no mortality occurs. OMW concentrations ranging from 0.01 to 0.1% (v/v) showed no increased mortality and thus were used for the determination of pre-pathological alterations in tissues of mussels. In particular, mussels exposed to either 0.1 or 0.01% (v/v) OMW for 5 days showed significant alterations of stress indices in their tissues. Specifically, decreased neutral red retention (NRR) assay time values, inhibition of acetylcholinesterase (AChE) activity, as well as a significant increase of micronucleus (MN) frequency and DNA damage were detected in haemolymph/haemocytes and gills, compared with values measured in tissues of control mussels. The results of the present study showed that OMW disposal into the marine environment could induce pre-pathological alterations in marine organisms, before severe disturbances, such as disease, mortality, or population changes occur. [ABSTRACT FROM AUTHOR]

As an easily obtained organic waste, by-product acetic acid could be an appropriate co-substrate with blue algae wastes (increase C/N ratio of substrates) for co-fermentation of PHA production. However, there are still acrylic acid and other chemicals in by-product acetic acid, which could cause severe inhibition for fermenting microorganisms during PHA production process. The current study represented that alkali pretreatment (pH level of 12) is a more favorable method compared with thermal pretreatment (80 ℃ for 30 min) for breaking cell walls of blue algae. It seemed that there was no synergistic effect of the combination of thermal and alkali pretreatment methods (temperature of 80 ℃ and pH level of 12). Optimal parameters during electro-fenton process for removal of inhibitors in by-product acetic acid were under current of 0.5 A, pH level of 3 and reaction time of 120 min. Both the highest dry weight of PHA and PHA concentration were achieved by applying blue algae and by-product acetic acid (after pretreatment) as co-substrates (mixed ratio of 3:1, stirring speed of 200 r/min, 24 h), indicating that using by-product acetic acid (after pretreatment) as co-substrate could increase C/N ratio and promote PHA production successfully. The current study could offer new insights for improving PHA production by co-fermentation. [Display omitted] • Alkali pretreatment is a more favorable method compared with thermal pretreatment. • There was no synergistic effect of the combination of thermal and alkali pretreatment. • Optimal parameters for removal of inhibitors in by-product acetic acid were determined. • By-product acetic acid (after pretreatment) as co-substrate could promote PHA production. [ABSTRACT FROM AUTHOR]

In the current study, a mixed microbial culture (MMC) of polyhydroxyalkanoates (PHAs) producers was developed under nutrient stress and was assessed as biocatalyst for the production of high-yielding PHAs from fermented (acidified) discarded fruit juices (DFJ). The structure of the MMC was analyzed periodically to determine its microbial dynamics, revealing that Zoogloae sp. dominated throughout the operation of the system. The efficiency of PHAs production from the MMC was further optimized in batch mode by altering the ratio of C to N, the ratio of carbon sources (propionate and butyrate), and the initial pH, and subsequently different fermentation mixtures of acidified DFJ were assessed as substrates at optimal conditions. Upon solvent extraction, the properties of recovered PHAs were analyzed, showing that in all cases P(3HB-co-3HV) was produced, with T m ranging from 90.5 to 168.8 °C, and maximum obtained yields 54.61 ± 4.31 % and 43.27 ± 2.13 %, from synthetic substrates and DFJ, respectively. Overall, it was shown that the developed MMC can be efficiently applied as biocatalyst for the exploitation of sugary wastewaters, such as DFJ, towards bio-based and biodegradable plastics bearing the required properties to substitute fossil plastics, into the concept of a circular economy. • A PHAs producing consortium dominated by Zoogloae was developed under nutrient stress. • The accumulation capacity of the consortium excided 50 % g PHAs/g dry biomass. • Random copolymers of HB and HV were synthesized from organic acids and acidified juices. • The HV content of the copolymers was linked to the propionate content of the substrate. [ABSTRACT FROM AUTHOR]

The scope of the current study was to investigate the efficiency of a two-stage anaerobic-aerobic process for the simultaneous treatment and valorization of selective wastewater streams from a confectionary industry. The specific wastewater (confectionary industry wastewater, CIW) was a mixture of the rinsing eluting during washing of the cauldrons in which jellies and syrups were produced, and contained mainly readily fermentable sugars, being thus of high organic load. The first stage of the process was the dark fermentation (DF) of the CIW in continuous, attached-biomass systems, in which the effect on hydrogen yields and distribution of metabolites were studied for different packing materials (ceramic or plastic), hydraulic retention times, HRTs (12 h–30 h) and feed substrate concentration (20 g COD/L- 50 g COD/L). In the second stage, the effectiveness of the aerobic treatment of the DF effluents was evaluated in terms of the reduction of the organic load and the production of polyhydroxyalkanoates (PHAs) through an enriched mixed microbial culture (MMC). The MMC was developed in a continuous draw and fill system, in which the accumulation potential of PHAs was studied. It was shown that the hydrogen production rates decreased for increasing substrate concentration and HRTs, with a maximum of 12.70 ± 0.35 m3 H 2 /m3 initial CIW achieved for the lowest HRT and feed concentration and using ceramic beads as packing material. Butyrate, acetate and lactate were the main metabolites generated in all cases, in different ratios. The distribution of metabolites during DF was shown to highly affect the efficiency of the second process in terms of both the reduction of organic load and the PHAs yields. The highest removal of organic load achieved after 48 h of aerobic treatment was 84.0 ± 0.9 %, whereas the maximum PHAs yield was 21.46 ± 0.13 kg PHAs/m3 initial CIW. • The simultaneous treatment and valorization of confectionary wastewater was assessed via an anaerobic-aerobic process. • The operational optimization of the anaerobic stage and the biocatalysts of the aerobic stage are equally important. • The hydrogen yields and distribution of the metabolites are mainly affected by the feeding strategy. • Up to 12.70 ± 0.35 m3 H 2 /m3 initial wastewater and 21.46 ± 0.13 kg PHAs/m3 initial wastewater were produced. • Efficient reduction up to ∼90% of its organic load was achieved by the two stage process. [ABSTRACT FROM AUTHOR]

The effect of different pretreatment approaches based on alkali (NaOH)/hydrogen peroxide (H2O2) on willow sawdust (WS) biomass, in terms of delignification efficiency, structural changes of lignocellulose and subsequent fermentation toward ethanol, was investigated. Bioethanol production was carried out using the conventional yeast Saccharomyces cerevisiae, as well as three non-conventional yeasts strains, i.e., Pichia stipitis, Pachysolen tannophilus, Wickerhamomyces anomalus X19, separately and in co-cultures. The experimental results showed that a two-stage pretreatment approach (NaOH (0.5% w/v) for 24 h and H2O2 (0.5% v/v) for 24 h) led to higher delignification (38.3 ± 0.1%) and saccharification efficiency (31.7 ± 0.3%) and higher ethanol concentration and yield. Monocultures of S. cerevisiae or W. anomalus X19 and co-cultures with P. stipitis exhibited ethanol yields in the range of 11.67 ± 0.21 to 13.81 ± 0.20 g/100 g total solids (TS). When WS was subjected to H2O2 (0.5% v/v) alone for 24 h, the lowest ethanol yields were observed for all yeast strains, due to the minor impact of this treatment on the main chemical and structural WS characteristics. In order to decide which is the best pretreatment approach, a detailed techno-economical assessment is needed, which will take into account the ethanol yields and the minimum processing cost. [ABSTRACT FROM AUTHOR]

In the present study the bioconversion of dried household food waste (FORBI) to energy carriers was investigated aiming to its sustainable management and valorization. FORBI was either directly fermented towards ethanol and hydrogen or was previously subjected to extraction with water resulting to a liquid fraction (extract) rich in sugars and a solid residue, which were then fermented separately. Subsequently, the effluents were assessed as substrates for methane production via anaerobic digestion (AD). Mono-cultures and co-cultures of C5 and C6 yeasts were used for the alcoholic fermentation whereas for the production of hydrogen, mixed acidogenic consortia were used. Taking into account the optimum yields of biofuels, the amount of recoverable energy was estimated based for each different approach. The maximum ethanol yield was 0.16 g ethanol per kg of FORBI and it was achieved for separate fermentation of liquid and solid fractions of the waste. The highest hydrogen yield that was observed was 210.44 L ± 4.02 H 2 /kg TS FORBI for 1% solids loading and supplementation with cellulolytic enzymes. Direct AD of either the whole FORBI or its individual fractions led to lower overall energy recovery, compared to that obtained when fermentation and subsequent AD were applied. The recoverable energy was estimated for the different exploitation approaches of the waste. The maximum achieved recoverable energy was 21.49 ± 0.57 MJ/kg. Unlabelled Image • FORBI (food waste) was bio converted to H 2 , ethanol and methane in 2 stage systems. • Water extraction for separate fermentation of sugars/solids enhanced ethanol yield. • Extraction process did not affect final hydrogen yields. • Energy gain was higher from 2 stage approach than direct AD of FORBI or fractions. [ABSTRACT FROM AUTHOR]

• The potential use of potato peels waste for bioethanol production was tested. • A newly isolated strain of W. anomalus was used for ethanol production. • Different methods were tested for saccharification. • The possibility of using lab-scale produced enzymes was also explored. • Different process schemes were evaluated for fermentation. The present study focuses on the exploration of the potential use of potato peels waste (PPW) as feedstock for bioethanol production, using a newly isolated yeast strain, Wickerhamomyces anomalus, via different saccharification and fermentation schemes. The saccharification of PPW was performed via thermal and chemical (acid, alkali) pretreatment, as well as via enzymatic hydrolysis through the use of commercial enzymes (cellulase and amylase) or enzymes produced at lab scale (alpha-amylase from Bacillus sp. Gb67), either separately or in mixtures. The results indicated that the enzymatic treatment by commercial enzymes led to a higher saccharification efficiency (72.38%) and ethanol yield (0.49 g/g consumed sugars) corresponding to 96% of the maximum theoretical. In addition, acid pretreatment was found to be beneficial for the process, leading also to high hydrolysis and ethanol yields, indicating that PPW is a very promising feedstock for bio-ethanol production by W. anomalus under different process schemes. [ABSTRACT FROM AUTHOR]

A combined Lewis-Brønsted acid ethanolysis of sugars was thoroughly investigated with the aim of producing ethyl levulinate (EL) in a single step. Ethanolysis carried out at 453 K for 4 h using H 2 SO 4 (1 wt%) and AlCl 3 ·6H 2 O (30 mol % with respect to sugars) produced a yield of 60 mol % of EL respect to glucose and starch. Such optimised conditions were positively applied directly on different food waste, preliminarily characterised and found to be mainly composed by simple (10–15%) and relatively complex sugars (20–60%), besides proteins (6–10%) and lipids (4–10%), even in their wet form. The catalytic system resulted robust enough to the point that the copresence of proteins, lignin, lipids and mineral salts not only did not negatively affect the overall reactivity, but resulted efficiently converted into soluble species, and specifically, into other liquid biofuels of different nature. [ABSTRACT FROM AUTHOR]

The treatment and biotechnological exploitation of expired fruit juices (EFJ) towards biohydrogen, polyhydroxyalkanoates (PHAs) and lipids production was investigated. The EFJ were initially fermented via an acidogenic consortium, during which the effect of the organic loading rate (OLR, 20–40 g sugars/L.d) on the sugars uptake and the yields of metabolites i.e. hydrogen, volatile fatty acids (VFAs) and lactate, was studied. The increase of the OLR resulted to lower bioconversion of sugars and metabolic shift to lactate production, whereas the highest hydrogen yield, 10.83 ± 0.41 L H 2 /m3 EFJ, was obtained for the OLR 30 g sugars/L.d. The acidogenic effluents were then utilized for the production of PHAs and microbial lipids, via bacterial heterotrophic and cyanobacterial consortia, respectively. The PHAs produced were in all cases copolymers, and the highest yield, 20.69 ± 1.01 kg PHAs/m3 EFJ was achieved from the effluent with the highest VFAs content. Microbial lipids were also successfully produced from all effluents. The results revealed that low VFAs and high sugars concentration, allowed cyanobacterial growth of up to 373.5 mg /L.d and maximum lipid content of up to 11.7 % d.w. (5.47 ± 0.49 kg biolipids/m3 EFJ), as well as significant substrate degradation (dissolved oxygen demand - d-COD, total nitrogen-ΤΝ, orthophosphates-PO 4 3-, total sugars up to about 93 %, 95 %, 67 % and 96 %, respectively). • The simultaneous treatment and valorization of expired fruit juices (EFJ) was investigated. • The organic loading during continuous dark fermentation of EFJ affected products' yields. • Effluents with high amount of volatile fatty acids yielded to high PHAs. • Effluents with high amount of residual sugars yielded to high microbial lipids. • The utilization strategy of the effluents may vary based on their composition. [ABSTRACT FROM AUTHOR]

In this study fungal pretreatment of willow sawdust (WSD) via the white rot fungi Leiotrametes menziesii and Abortiporus biennis was studied and the effect on fractionation of lignocellulosic biomass and biochemical methane potential (BMP), was evaluated. Scanning electron microscopy (SEM) and IR spectroscopy were used to investigate the changes in the structural characteristics of the pretreated WSD. Fungal pretreatment results revealed that A. biennis is more attractive, since it resulted in higher lignin degradation and lower holocellulose uptake. Samples of the 14th and 30th d of cultivation (i.e. the middle and the end of the pretreatment experiment) with both fungi were used for BMP tests and the effect of pretreatment duration was also evaluated. BMP increase by 31 and 43% was obtained due to the cultivation of WSD with A. biennis , for 14 and 30 d, respectively. In addition, combination of biological (after 30 d of cultivation) with alkaline (NaOH 20 g/100 gTS) pretreatment was performed, in order to assess the effect of the chemical agent on biologically pretreated WSD, in terms of lignocellulosic content and BMP. Combination of alkaline with fungal pretreatment led to high lignin degradation for both fungi, while the cellulose and hemicellulose removal efficiencies were higher for combined alkaline and L. menziesii pretreatment. The maximum BMP was observed for the combined alkaline and A. biennis pretreatment and was 12.5 and 50.1% higher than the respective alkaline and fungal pretreatment alone and 115% higher than the respective BMP of raw WSD. [ABSTRACT FROM AUTHOR]

PHAs are a form of cellular storage polymers with diverse structural and material properties, and their biodegradable and renewable nature makes them a potential green alternative to fossil fuel-based plastics. PHAs are obtained through extraction via various mechanical, physical and chemical processes after their intracellular synthesis. Most studies have until now focused on pure cultures, while information on mixed microbial cultures (MMC) remains limited. In this study, ultrasonic (US) disruption and alkaline digestion by NaOH were applied individually and in combination to obtain PHAs products from an acclimated MMC using phenol as the carbon source. Various parameters were tested, including ultrasonic sound energy density, NaOH concentration, treatment time and temperature, and biomass density. US alone caused limited cell lysis and resulted in high energy consumption and low efficiency. NaOH of 0.05–0.2 M was more efficient in cell disruption, but led to PHAs degradation under elevated temperature and prolonged treatment. Combining US and NaOH significantly improved the overall process efficiency, which could reduce energy consumption by 2/3rds with only minimal PHAs degradation. The most significant factor was identified to be NaOH dosage and treatment time, with US sound energy density playing a minor role. Under the semi-optimized condition (0.2 M NaOH, 1300 W L-1, 10 min), over 70% recovery and 80% purity were achieved from a 3 g L−1 MMC slurry of approximately 50% PHAs fraction. The material and thermal properties of the products were analyzed, and the polymers obtained from US + NaOH treatments showed comparable or higher molecular weight to previously reported results. The products also exhibited good thermal stability and rheological properties, compared to the commercial standard. In conclusion, the combined US and NaOH method has the potential in real application as an efficient process to obtain high quality PHAs from MMC, and cost-effectiveness can be further optimized. [Display omitted] • Ultrasonic (US) and alkaline digestion used to recover PHB from a mixed culture. • US disruption had limited effect of cell lysis and high energy consumption. • NaOH digestion was more efficient but led to PHA degradation under high temperature. • US + NaOH showed synergetic effect, reduced treatment time and energy consumption. • US + NaOH can be cost-effective after optimization, showing potential of scaling-up. [ABSTRACT FROM AUTHOR]

The present study evaluates a battery of marine species-based bioassays against chemically characterized municipal wastewater samples (raw and WWTP treated). We estimated Dunaliella tertiolecta growth rate inhibition ( 24–96 h IC 50 values) , Artemia franciscana immobilization (24h LC 50 values), mussel hemocytes viability and lipid peroxidation enhancement (in terms of neutral red retention assay/NRRT and malondialdehyde/MDA content, respectively) in influent- and WWTP effluent-treated species. We found algal growth arrest and stimulation respectively, almost similar 24hLC 50 values in Artemia sp., and significantly higher adverse effects (in terms of NRRT and MDA levels) in influent-treated mussel hemocytes. Furthermore, the estimation of hatchability, yolk-sac larvae mortality (24–120 hLC 50 ) and spinal deformities (SD) in sea bream Sparus aurata showed slight variations over time, with the lowest LC 50 and SD 50 (representing spinal deformities at 50% of yolk-sac larvae) values to be observed in influent-treated larvae at 120 h. Data interpretation (both chemical and biological) revealed that toxic endpoints, such as NRRT 50 , 96hIC 50 Dun, 120hLC 50 Sparus and 120hSD 50 Sparus, significantly related to WWTP removal efficiency and further mediated by the presence of dominant compounds, such as As and Cr, could be used for identifying main components of toxicity in wastewaters. [ABSTRACT FROM AUTHOR]

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Given that the social and economic sustainability of rural areas is highly based on the protection of natural resources, biodiversity and human health, simple-operated and cost-effective wastewater treatment systems, like artificial constructed wetlands (CWs), are widely proposed for minimizing the environmental and human impact of both water and soil pollution. Considering that the optimization of wastewater treatment processes is vital for the reduction of effluents toxic potential, there is imperative need to establish appropriate management strategies for ensuring CW performance and operational efficiency. To this end, the present study aimed to assess the operational efficiency of a horizontal free water surface CW (HFWS-CW) located in a world heritage area of Western Greece, via a twelve-month duration Toxicity Identification Evaluation (TIE)-like approach, including both chemical and biological tracking tools. Conventional chemical tracking, by means of pH, conductivity, total COD, and nitrogen-derived components, like nitrates and ammonia–nitrogen, were monthly recorded in both influents and effluents to monitor whether water quality standards are maintained, and to assess potent CW operational deficiencies occurring over time. In parallel, Whole Effluent Toxicity (WET) bioassays were thoroughly applied, using freshwater algae and higher plant species (producers), crustaceans and rotifers (consumers), as well as human lymphocytes (in terms of Cytokinesis Block Micronucleus assay) to evaluate the acute and short-term toxic and hazardous potential of both influents and effluents. The integrated analysis of abiotic (physicochemical parameters) and biotic (toxic endpoints) parameters, as well as the existence of "cause-effect" interrelations among them, revealed that CW operational deficiencies, mainly based on poorly removal rates, could undermine the risk posed by treated sewage. Those findings reinforce the usage of WET testing, thus giving rise to the importance of applying appropriate water management strategies and optimization actions, like oxygen enrichment of surface and bottom of HFWS-CW basins, expansion of the available land, the enhancement of bed depth and seasonal harvesting of plants, for ensuring sewage quality, in favor of water resources protection and sustainable growth in rural areas. [Display omitted] • Constructed wetland (CW) performance was recorded via a TIE-like approach. • Whole effluent toxicity (WET) testing and chemical tracking were performed. • Chemical tracking in poorly operated CWs could undermine the risk of treated sewage. • TIE-like approach, including biological tracking, improves the CW performance. • Integrating abiotic and WET data could favor CW optimization and water management. [ABSTRACT FROM AUTHOR]

[Display omitted] • Simultaneous ammonia mitigation and biogas upgrading using an integrated BES was accomplished. • Ammonia utilization and hydrogen production reached 73.67% and 202.87 mL under an applied voltage 0.4 V. • The highest methane production and content were attained with in situ H 2 injection at the beginning. • Both microbial communities and enzymatic abundances related to methanogenesis were amplified. Biohydrogen using migrated ammonia as nitrogen source, and biogas upgrading with hydrogen collected at biocathode in an integrated bioelectrochemical system (BES) were investigated, during the anaerobic digestion of Taihu blue algae. Under an applied voltage of 0.4 V, biohydrogen (202.87 mL) reached 2.34 and 2.90 times than groups with applied voltage of 0 V and 0.8 V, respectively. Moreover, biohydrogen of the group with 1000 mg/L initial ammonia addition (524.16 mL) reached 1.53 times than that the of the control. With 0.25 bar of H 2 injected at the beginning (R1), highest methane production (286.62) mL and content (75.73%) were obtained. Comparing to other groups, not only microbial genus responsible for both aceticlastic and hydrogenotrophic methanogens of the group R1 were apparently enriched, but key enzymes related to methane production also acquired better abundances. Therefore, it's promising to conduct the ammonia alleviating, hydrogen producing and biogas upgrading simultaneously using BES. [ABSTRACT FROM AUTHOR]