Adaptation of a microbial consortium to pelagic Sargassum modifies its taxonomic and functional profile that improves biomethane potential.

In recent years, pelagic Sargassum has invaded the Caribbean coasts, and anaerobic digestion has been proposed as a sustainable management option. However, the complex composition of these macroalgae acts as a barrier to microbial degradation, thereby limiting methane production. Microbial adaptation is a promising strategy to improve substrate utilization and stress tolerance. This study aimed to investigate the adaptation of a microbial consortium to enhance methane production from the pelagic Sargassum. Microbial adaptation was performed in a fed-batch mode for 100 days by progressive feeding of Sargassum. The evolution of the microbial community was analyzed by high-throughput sequencing of 16S rRNA amplicons. Additionally, 16S rRNA data were used to predict functional profiles using the iVikodak platform. The results showed that, after adaptation, the consortium was dominated by the bacterial phyla Bacteroidota, Firmicutes, and Atribacterota, as well as methanogens of the families Methanotrichaceae and Methanoregulaceae. The abundance of predicted genes related to different metabolic functions was affected during the adaptation stage when Sargassum concentration was increased. At the end of the adaptation stage, the abundance of the predicted genes increased again. The adapted microbial consortium demonstrated a 60% increase in both biomethane potential and biodegradability index. This work offers valuable insights into the development of treatment technologies and the effective management of pelagic Sargassum in coastal regions, emphasizing the importance of microbial adaptation in this context.

Effect of sodium formate and lactic acid bacteria treated rye silage on methane yield and energy balance in Hanwoo steers.

This study was performed to evaluate the effects of rye silage treated with sodium formate (Na-Fa) and lactic acid bacteria (LAB) inoculants on the ruminal fermentation characteristics, methane yield and energy balance in Hanwoo steers. Forage rye was harvested in May 2019 and ensiled without additives (control) or with either a LAB inoculant or Na-Fa. The LAB (Lactobacillus plantarum) were inoculated at 1.5 × 1010 CFU/g fresh matter, and the inoculant was sprayed onto the forage rye during wrapping at a rate of 4 L/ton of fresh rye forage. Sixteen percent of the Na-Fa solution was sprayed at a rate of approximately 6.6 L/ton. Hanwoo steers (body weight 275 ± 8.4 kg (n = 3, group 1); average body weight 360 ± 32.1 kg (n = 3, group 2)) were allocated into two pens equipped with individual feeding gates and used in duplicated 3 × 3 Latin square design. The experimental diet was fed twice daily (09:00 and 18:00) during the experimental period. Each period comprised 10 days for adaptation to the pen and 9 days for measurements in a direct respiratory chamber. The body weights of the steers were measured at the beginning and at the end of the experiment. Feces and urine were collected for 5 days after 1 day of adaptation to the chamber, methane production was measured for 2 days, and ruminal fluid was collected on the final day. In the LAB group, the ratio of acetic acid in the rumen fluid was significantly lower (p = 0.044) and the ratio of propionic acid in the rumen fluid was significantly higher (p = 0.017). Methane production per DDMI of the Na-FA treatment group was lower than that of the other groups (p = 0.052), and methane production per DNDFI of the LAB treatment group was higher than that of the other groups (p = 0.056). The use of an acid-based additive in silage production has a positive effect on net energy and has the potential to reduce enteric methane emissions in ruminants.

Effective grease separator management is the key to enhancing bioenergy recovery of fat, oil, and grease (FOG) and contributing to a circular bio-economy.

Management of fat, oil and grease (FOG) is crucial for the recovery of renewable resources and the protection of sewer systems. This study aims to identify the potential quantities and qualities of FOG that can be acquired through optimised grease separator (GS) management approaches in hotels and restaurants during seasonal tourism. A technical survey of 20 GS from hotels and restaurants in the federal state of Tyrol, Austria was conducted. The findings revealed that 55 % of the GS were in poor condition, often due to infrequent maintenance and limited operator's knowledge. The FOG layer quality and quantity was monitored over three years and physicochemical parameters including total residue, volatile solids, total organic carbon, lipid content, and biomethane yield, were analysed. An optimised management approach, which involved up to 4 GS emptying per season, revealed a significant increase in FOG quantity for the majority of the inspected establishments, with an overall doubling of the acquired FOG volume. Based on these results, the energy potential of GS is presented in three potential management scenarios. The energy recovered from GS increased by 246 %. This highlights the importance of proper GS management in the hospitality sector, which can play a critical role in promoting environmental sustainability and renewable energy production.

Ethanol-type anaerobic digestion enhanced methanogenic performance by stimulating direct interspecies electron transfer and interspecies hydrogen transfer.

Ethanol pre-fermentation of food waste effectively alleviates acidification; however, its effects on interspecies electron transfer remain unknown. This study configured the feed according to COD ratios of ethanol: sodium acetate: sodium propionate: sodium butyrate of 5:2:1.5:1.5 (ethanol-type anaerobic digestion) and 0:5:2.5:2.5 (control), and conducted semi-continuous anaerobic digestion (AD) experiments. The results showed that ethanol-type AD increased maximum tolerable organic loading rate (OLR) to 6.0 gCOD/L/d, and increased the methane production by 1.2-14.8 times compared to the control at OLRs of 1.0-5.0 gCOD/L/d. The abundance of the pilA gene, which was associated with direct interspecies electron transfer (DIET), increased by 5.6 times during ethanol-type AD. Hydrogenase genes related to interspecies hydrogen transfer (IHT), including hydA-B, hoxH-Y, hnd, ech, and ehb, were upregulated during ethanol-type AD. Ethanol-type AD improved methanogenic performance and enhanced microbial metabolism by stimulating DIET and IHT.

Effects of polypropylene microplastics on digestion performance, microbial community, and antibiotic resistance during microbial anaerobic digestion.

As an emerging pollutant, microplastics (MPs) have attracted increasing attention worldwide. The effects of polypropylene (PP) MPs on digestion performance, behaviors of dominant microbial communities, antibiotic resistance genes (ARGs) and mobile genetic elements in microbial anaerobic digesters were investigated. The results showed that the addition of PP-MPs to digesters led to an increase in methane production of 10.8% when 300 particles/g TSS of PP-MPs was introduced compared with that in digester not treated with PP-MPs. This increase was attributed to the enrichment of acetogens such as Syntrophobacter (42.0%), Syntrophorhabdus (27.0%), and Syntrophomonas (10.6%), and methanogens including Methanobacterium and Methanosaeta. tetX was highly enriched due to PP-MP exposure, whereas parC exhibited the greatest increase (35.5% - 222.7%). Horizontal gene transfer via ISCR1 and intI1 genes might play an important role in the spread of ARGs. Overall, these findings provide comprehensive insight into the ecological dynamics of PP-MPs during microbial anaerobic digestion.

Linking performance to dynamic migration of biofilm ecosystem reveals the role of voltage in the start-up of hybrid microbial electrolysis cell-anaerobic digestion.

Applied voltage is a crucial parameter in hybrid microbial electrolysis cells-anaerobic digestion (MEC-AD) systems for enhancing methane production from waste activated sludge (WAS). This study explored the impact of applied voltage on the initial biofilm formation on electrodes during the MEC-AD startup using raw WAS (Rr) and heat-pretreated WAS (Rh). The findings indicated that the maximum methane productivity for Rr and Rh were 3.4 ± 0.5 and 3.4 ± 0.2 mL/gVSS/d, respectively, increasing 1.5 times and 2.6 times over the productivity at 0 V. The biomass on electrode biofilms for Rr and Rh at 0.8 V increased by 70 % and 100 % compared to 0 V. The core functional microorganisms in the cathode biofilm were Methanobacterium and Syntrophomonas, and Geobacter in the anode biofilm, enhancing methane production through syntrophism and direct interspecies electron transfer, respectively. These results offer academic insights into optimizing AD functional electrode biofilms by applying voltage.

Model based analysis of trace metal dosing strategies to improve methane yield in anaerobic digestion systems.

Favourable effects of trace metals (TMs) on regulating anaerobic digestion (AD) performance are extensively utilised to improve methane yield. This study discusses a model-based approach to find out the best TM dosing strategies. The model has been applied to compare continuous, preloading, pulse dosing and in-situ loading. Simulations were also carried out to comprehend appropriate dosing form, dosing time and quantity of metals to be dosed. Model results show that the best way to dose TMs is repeated pulse dosing at low concentration levels in the optimum range with high frequency. Best dosing strategy for the system in this study was found to be 5 µM pulse loading at 5 days intervals as it gave maximum methane production and low effluent metal loss. Preferable dosing form depends on reactor configuration and this has been verified after model calibration with experimental data. Easily dissociable metal chlorides are ideal for continuous reactors.

Non-conductive silicon-containing materials improve methane production by pure cultures of methanogens.

Conductive materials (CM) enhance methanogenesis, but there is no clear correlation between conductivity and faster methane production (MP) rates. We investigated if MP by pure cultures of methanogens (Methanobacterium formicicum, Methanospirillum hungatei, Methanothrix harundinacea and Methanosarcina barkeri) is affected by CM (activated carbon (AC), magnetite), and other sustainable alternatives (sand and glass beads, without conductivity, and zeolites (Zeo)). The significant impact of the materials was on M. formicicum as MP was significantly accelerated by non-CM (e.g., sand reduced the lag phase (LP) duration by 48 %), Zeo and AC (LP reduction in 71% and 75 %, respectively). Conductivity was not correlated with LP reduction. Instead, silicon content in the materials was inversely correlated with the time required for complete MP, and silicon per se stimulated M. formicicum's activity. These findings highlight the potential of using non-CM silicon-containing materials in anaerobic digesters to accelerate methanogenesis.

An in-situ biochar-enhanced anaerobic membrane bioreactor for swine wastewater treatment under various organic loading rates.

A biochar-assisted anaerobic membrane bioreactor (BC-AnMBR) was conducted to evaluate the performance in treating swine wastewater with different organic loading rates (OLR) ranging from 0.38 to 1.13 kg-COD/(m3.d). Results indicated that adding spent coffee grounds biochar (SCG-BC) improved the organic removal efficiency compared to the conventional AnMBR, with an overall COD removal rate of > 95.01%. Meanwhile, methane production of up to 0.22 LCH4/gCOD with an improvement of 45.45% was achieved under a high OLR of 1.13 kg-COD/(m3.d). Furthermore, the transmembrane pressure (TMP) in the BC-AnMBR system was stable at 4.5 kPa, and no irreversible membrane fouling occurred within 125 days. Microbial community analysis revealed that the addition of SCG-BC increased the relative abundance of autotrophic methanogenic archaea, particularly Methanosarcina (from 0.11% to 11.16%) and Methanothrix (from 16.34% to 24.05%). More importantly, Desulfobacterota and Firmicutes phylum with direct interspecific electron transfer (DIET) capabilities were also enriched with autotrophic methanogens. Analysis of the electron transfer pathway showed that the concentration of c-type cytochromes increased by 38.60% in the presence of SCG-BC, and thus facilitated the establishment of DIET and maintained high activity of the electron transfer system even at high OLR. In short, the BC-AnMBR system performs well under various OLR conditions and is stable in the recovery energy system for swine wastewater.

Optimizing anaerobic digestion: Benefits of mild temperature transition from thermophilic to mesophilic conditions.

Anaerobic digestion (AD) plays a significant role in renewable energy recovery. Upgrading AD from thermophilic (50-57 °C) to mesophilic (30-38 °C) conditions to enhance process stability and reduce energy input remains challenging due to the high sensitivity of thermophilic microbiomes to temperature fluctuations. Here we compare the effects of two decreasing-temperature modes from 55 to 35 °C on cell viability, microbial dynamics, and interspecies interactions. A sharp transition (ST) is a one-step transition by 20 °C d-1, while a mild transition (MT) is a stepwise transition by 1 °C d-1. We find a greater decrease in methane production with ST (88.8%) compared to MT (38.9%) during the transition period. ST mode overproduced reactive oxygen species by 1.6-fold, increased membrane permeability by 2.2-fold, and downregulated microbial energy metabolism by 25.1%, leading to increased apoptosis of anaerobes by 1.9-fold and release of intracellular substances by 2.9-fold, further constraining methanogenesis. The higher (1.6 vs. 1.1 copies per gyrA) metabolic activity of acetate-dependent methanogenesis implied more efficient methane production in a steady mesophilic, MT-mediated system. Metagenomic binning and network analyses indicated that ST induced dysbiosis in keystone species and greatly enhanced microbial functional redundancy, causing loss of microbial syntrophic interactions and redundant metabolic pathways. In contrast, the greater microbial interconnections (average degrees 44.9 vs. 22.1) in MT at a steady mesophilic state suggested that MT could better maintain necessary system functionality and stability through microbial syntrophy or specialized pathways. Adopting MT to transform thermophilic digesters into mesophilic digesters is feasible and could potentially enhance the further optimization and broader application of practical anaerobic engineering.

The dominant-substrate driven the enhanced performance in co-digestion of Pennisetum hybrid and livestock waste.

Co-digestion has been considered a promising method to improve methane yield. The effect of the proportion of dominant substrate on the performance and microbial community of anaerobic digestion of Pennisetum hybrid (PH) and livestock waste (LW) was investigated. An obvious synergistic effect was obtained with an increase of 15.20%-17.45% in specific methane yield compared to the predicted value. Meanwhile, the dominant substrate influenced the relational model between methane yield enhancement rate and mixture ratio. For the LW-dominant systems, a parabolic model between enhancement rate and mixture ratio was observed with a highest value of 392.16 mL/g VS achieved at a PH:LW ratio of 2:8. While a linear pattern appeared for PH-dominant systems with the highest methane yield of 307.59 mL/g VS. Co-digestion selectively enriched the relative abundance of Clostridium_sensu_stricto_1, Terrisporobacter, Syntrophomonas, Methanosarcina and Methanobacterium, which boosted the performance of hydrolysis, acidogenesis, acetogenesis and methanogenesis processes.

Integrated aerobic-anaerobic digestion of highly solids-loaded corn stover and swine manure under dynamic aeration: Temperature rise, physicochemical characteristics, and methane production.

This research aimed to design an integrated aerobic-anaerobic reactor with dynamic aeration that was automatically regulated based on real-time oxygen concentration and investigate the aerobic pretreatment and subsequent dry co-anaerobic digestion (co-AD) characteristics of highly solids-loaded corn stover and swine manure in terms of temperature rise, physiochemical characteristics, and methane production. The high-temperature feedstocks from the aerobic pretreatment phase rapidly entered the AD phase without transportation and effectively improved the start-up and methane production of the co-AD. Oxygen concentration range, aeration rate, and pretreatment time affected the cumulative aeration time, temperature rise, and organic matter removal interactively during aerobic pretreatment, and a low aeration rate was relatively preferable. Although the lignocellulose removal increased with the increase in pretreatment duration, the maximal lignin elimination efficiency only reached 1.30%. The inoculum injection in the transition phase from aerobic pretreatment to co-AD and the leachate reflux during co-AD were also critical for producing methane steadily apart from aerobic pretreatment. The cold air weakened the temperature rise of aerobic pretreatment, and the low-temperature leachate reduced the methane production in the co-AD process. An oxygen concentration range of 13%-17%, aeration rate of 0.10 m3/(min·m3), pretreatment time of 84 h, inoculum loading of 40%, leachate refluxing thrice per day, and double-layer inoculation were optimum for improving the integrated aerobic-anaerobic digestion system's ability to resist low temperatures and achieving high methane production. The maximal cumulative and volatile solids (VS) methane yields of corn stover and swine manure reached 444.58 L and 266.30 L/kg VS.

Roles of quorum-sensing molecules in methane production from anaerobic digestion aided by biochar.

Biochar has been used to enhance methane generation from anaerobic digestion through establishing direct interspecific electron transfer between microorganisms. However, the microbial communication is still inadequate, thereby limiting further methane production improvement contributed by biochar. This study investigated the roles of quorum-sensing molecules, acylated homoserine lactone (AHL), in anaerobic digestion of waste activated sludge aided by biochar. Results showed that the co-addition of separated biochar and AHL achieved best methane production performance, with the maximal methane yield of 154.7 mL/g volatile suspended solids, which increased by 51.9%, 47.2%, 17.9%, and 39.4% respectively compared to that of control, AHL-loaded biochar, sole AHL, and sole biochar groups. The reason was that the co-addition of separated biochar and AHL promoted the stages of hydrolysis and acidification, promoting the conversion of organic matters and short-chain fatty acids, and optimizing the accumulation of acetate acid. Moreover, the methanogenesis stage also performed best among experimental groups. Correspondingly, the highest activities of electron transfer and coenzyme F420 were obtained, with increase ratios of 33.2% and 27.2% respectively compared to that of control. Furthermore, biochar did more significant effects on the evolution of microbial communities than AHL, and the direct interspecific electron transfer between fermentative bacteria and methanogens were possibly promoted.

S-containing molecular markers of dissolved organic carbon attributing to riverine dissolved methane production across different land uses.

The emission of methane (CH4) from streams and rivers contributes significantly to its global inventory. The production of CH4 is traditionally considered as a strictly anaerobic process. Recent investigations observed a "CH4 paradox" in oxic waters, suggesting the occurrence of oxic methane production (OMP). Human activities promoted dissolved organic carbon (DOC) in streams and rivers, providing significant substrates for CH4 production. However, the underlying DOC molecular markers of CH4 production in river systems are not well known. The identification of these markers will help to reveal the mechanism of methanogenesis. Here, Fourier transform ion cyclotron mass spectrometry and other high-quality DOC characterization, ecosystem metabolism, and in-situ net CH4 production rate were employed to investigate molecular markers attributing to riverine dissolved CH4 production across different land uses. We show that endogenous CH4 production supports CH4 oversaturation and positively correlates with DOC concentrations and gross primary production. Furthermore, sulfur (S)-containing molecules, particularly S-aliphatics and S-peptides, and fatty acid-like compounds (e.g., acetate homologs) are characterized as markers of water-column aerobic and anaerobic CH4 production. Watershed characterization, including riverine discharge, allochthonous DOC input, turnover, as well as autochthonous DOC, affects the CH4 production. Our study helps to understand riverine aerobic or anaerobic CH4 production relating to DOC molecular characteristics across different land uses.

Magnetic porous microspheres altering interfacial thermodynamics of sewage sludge to drive metabolic cooperation for efficient methanogenesis.

Controllable and recyclable magnetic porous microspheres (MPMs) have been proposed as a means for enhancing the anaerobic digestion (AD) of sludge, as they do not require continuous replenishment and can serve as carriers for anaerobes. However, the effects of MPMs on the interfacial thermodynamics of sludge and the biological responses triggered by abiotic effects in AD systems remain to be clarified. Herein, the underlying mechanisms by which MPMs alter the solid-liquid interface of sludge to drive methanogenesis were investigated. A significant increase in the contents of 13C and 2H (D) in methane molecules was observed in the presence of MPMs, suggesting that MPMs might enhance the CO2-reduction methanogenesis and participation of water in methane generation. Experimental results demonstrated that the addition of MPMs did not promote the anaerobic bioconversion of soluble organics for methanogenesis, suggesting that the enhanced methanogenesis and water participation were not achieved through promotion of the bioconversion of original liquid-state organics in sludge. Analyses of the capillary force, surface adhesion force, and interfacial proton-coupled electron transfer (PCET) of MPMs revealed that MPMs can enhance mass transfer, effective contact, and electron-proton transfer with sludge. These outcomes were confirmed by the statistical analyses of variations in the interfacial thermodynamics and PCET of sludge with and without MPMs during AD. It was thus proposed that the MPMs enhanced the PCET of sludge and PCET-driven release of protons from water by promoting the interfacial Lewis acid-base interactions of sludge, thereby resulting in the enrichment of free and attached methanogenic consortia and the high energy-conserving metabolic cooperation. This proposition was further confirmed by identifying the predominant syntrophic partners, suggesting that PCET-based efficient methanogenesis was attributable to the enrichment of genomes harbouring CO2-reducing pathway and genes encoding water-mediated proton transfer. These findings offer new insights into how substrate properties can be altered by exogenous materials to enable highly efficient methanogenesis.

Biochar alleviates inhibition effects of humic acid on anaerobic digestion: Insights to performances and mechanisms.

To recover methane from waste activated sludge through anaerobic digestion (AD) is one promising alternative to achieve carbon neutrality for wastewater treatment plants. However, humic acids (HAs) are one of the major compositions in waste activated sludge, and their accumulation performs inhibition effects on AD. This study investigated the potentials of biochar (BC) in alleviating inhibition effects of HAs on AD. Results showed that although the accumulated HAs reduced methane yield by 9.37% compared to control, the highest methane yield, 132.6 mL CH4/g VSS, was obtained after adding BC, which was 45.9% higher than that in HA group. Mechanism analysis showed that BC promoted the activities of hydrolase such as protease and α-glucosidase, which were 69.7% and 29.7% higher than those in HA group, respectively. The conversion of short-chain fatty acids was accelerated. In addition, the evolutions of electroactive microorganisms like Clostridium_sensu_stricto_13 and Methanosaeta were consistent with the activitiies of electron transfer and the contents of cytochrome c. Furthermore, parts of HAs rather than all of them were adsorbed by BC, and the remaining free HAs and BC formed synergistic effects on methanogenesis, then both CO2 reduction and acetoclastic methanogenesis pathways were improved. The findings may provide some solutions to alleviate inhibition effects of HAs on AD.

Heterogeneous g-C3N4/polyaniline composites enhanced the conversion of organics into methane during anaerobic wastewater treatment.

In this study, g-C3N4/PANI was prepared by in situ oxidative polymerization. Graphite-phase carbon nitride (g-C3N4) with surface defects was deposited onto the surface of conductive polyaniline (PANI) to form a p-n heterojunction. This construction aimed to create an efficient heterogeneous catalyst, increasing the surface defect level and active sites of the composite, and augmenting its capability to capture and transfer extracellular electrons under anaerobic conditions. This addresses the challenge of low efficiency in direct interspecies electron transfer between bacteria and archaea during anaerobic digestion for methane production. The results showed that the prepared g-C3N4/PANI increased the CH4 yield and CH4 production rate by 82% and 96%, respectively. Notably, the conductivity and XPS test results showed that the ratio of g-C3N4 to PANI was 0.15, and the composite exhibited favorable conductivity, with a uniform distribution of pyrrolic nitrogen, pyridinic nitrogen, and graphitic nitrogen, each accounting for approximately 30%. Furthermore, g-C3N4/PANI effectively enhanced the metabolic efficiency of intermediate products such as acetate and butyrate. Analysis of the microbial community structure revealed that g-C3N4/PANI led to a significant increase in the abundance of hydrogenotrophic methanogen Methanolinea (from 48% to 64%) and enriched Clostridium (a rise of 1%) with direct interspecies electron transfer capability. Microbial community function analysis demonstrated that the addition of g-C3N4/PANI boosted the activities of key enzymes involved in anaerobic digestion, including phosphate transacetylase (PTA), phospho-butyryl transferase (PTB), and NAD-independent lactate dehydrogenase (NNLD), by 47%, 135%, and 153%, respectively. This acceleration in enzymatic activity promoted the metabolism of acetyl-CoA, butyryl-CoA, and pyruvate. Additionally, the function of ABC transporters was enhanced, thereby improving the efficiency of material and energy exchange among microorganisms.

Deciphering Fe@C amendment on long-term anaerobic digestion of sulfate and propionate rich wastewater: Driving microbial community succession and propionate metabolism.

This study evaluated the reflection of long-term anaerobic system exposed to sulfate and propionate. Fe@C was found to efficiently mitigate anaerobic sulfate inhibition and enhance propionate degradation. With influent propionate of 12000mgCOD/L and COD/SO42- ratio of 3.0, methane productivity and sulfate removal were only 0.06 ± 0.02L/gCOD and 63 %, respectively. Fe@C helped recover methane productivity to 0.23 ± 0.03L/gCOD, and remove sulfate completely. After alleviating sulfate stress, less organic substrate was utilized to form extracellular polymeric substances for self-protection, which enhanced mass transfer in anaerobic sludge. Microbial community succession, especially for alteration of key sulfate-reducing bacteria and propionate-oxidizing bacteria, was driven by Fe@C, thus enhancing sulfate reduction and propionate degradation. Acetotrophic Methanothrix and hydrogenotrophic unclassified_f_Methanoregulaceae were enriched to promote methanogenesis. Regarding propionate metabolism, inhibited methylmalonyl-CoA degradation was a limiting step under sulfate stress, and was mitigated by Fe@C. Overall, this study provides perspective on Fe@C's future application on sulfate and propionate rich wastewater treatment.

Novel insights into the mechanism of dynamic changes in microstructure and physicochemical properties of corn straw pretreated by ball milling and feasibility analysis of anaerobic digestion.

In this study, the effects of Ball milling (BM) pretreatment (0-240 min) on the microstructure, physicochemical properties and subsequent methanogenesis performance of corn straw (CS) were explored, and the feasibility analysis was carried out. The results showed that BM pretreatment destroyed the dense structure of the CS, and the particle size was significantly reduced (D50: 13.85 µm), transforming it into a cell-scale granular form. The number of mesopores increased, the pore volume (PV) (0.032 cm3/g) and specific surface area (SSA) (4.738 m2/g) considerably increased, and the water-absorbent property was improved. The crystalline order of cellulose was disrupted and the crystallinity (CrI) (8.61 %) and crystal size (CrS) (3.37) were remarkably reduced. The cross-links between lignocelluloses were broken, and the relative content and functional groups did not alter obviously. The bulk density (BD), repose angle (RA) and slip angle (SA) dramatically increased. As a result, CS was more readily accessible, attached and utilized by microorganisms and enzymes, causing the hydrolysis and acidification of AD to be greatly facilitated. Compared with the untreated group, the cumulative methane production (CMP) increased by 35.83 %-101.97 %, and the lag phase time (λ) was shortened by 33.04 %-71.17 %. The results of redundancy analysis, Pearson analysis and Mantel test showed that BM pretreatment affects the process of AD by changing the physicochemical factors of CS. The normalization analysis showed that particle size (D90) and BD can be used as direct indicators to evaluate the performance of AD and predict the threshold of biodegradation of CS. Energy analysis and energy conversion assessment showed that BM is a green and efficient AD pretreatment strategy. This result provides a theoretical basis for the industrial application of BM pretreatment towards more energy-efficient and sustainable development.

Cellulase production by Aspergillus fumigatus A4112 and the potential use of the enzyme in cooperation with surfactant to enhance floating oil recovery and methane production from palm oil mill effluent.

This research performed cellulase production by Aspergillus fumigatus A4112 and evaluated its potential use in palm oil mill effluent (POME) hydrolysis to recover oil simultaneously with the generation of fermentable sugar useful for biofuel production under non-sterilized conditions. Empty fruit bunch (EFB) without pretreatment was used as carbon source. The combination of nitrogen sources facilitated CMCase production. The maximum activity (3.27 U/mL) was obtained by 1.0 g/L peptone and 1.5 g/L (NH4)2SO4 and 20 g/L EFB at 40 °C for 7 days. High level of FPase activity (39.51 U/mL) was also obtained. Interestingly, the enzyme retained its cellulase activities more than 60% at ambient temperature over 15 days. In enzymatic hydrolysis, Triton X-100 was an effective surfactant to increase total oil recovery in the floating form. High yield of reducing sugar (50.13 g/L) and 21% (v/v) of floating oil was recoverable at 65 °C for 48 h. Methane content of the raw POME increased from 41.49 to 64.94% by using de-oiled POME hydrolysate which was higher than using the POME hydrolysate (59.82%). The results demonstrate the feasibility of the constructed process for oil recovery coupled with a subsequent step for methane yield enhancement in biogas production process that benefits the palm oil industry.