Anaerobic Digestion Performance Research Articles

Impacts of river snails rice noodle ingredients addition on the kitchen waste anaerobic digestion performances, microbial communities and metabolic pathways

With the rapid development of the river snails rice noodle industry, the treatment of the wastewater generated in the production of river snails rice noodle has attracted people’s attention. This study investigated the performance of the anaerobic digestion of kitchen waste with river snails rice noodle ingredients, including key enzyme activities, microbial community distribution and metabolic pathways in the anaerobic reactor. The average chemical oxygen demand removal efficiencies were 92.5% and 88.7% when 130 g and 260 g river snails rice noodle ingredients were added in the anaerobic reactor, respectively. Acetate kinase activity of the sludge decreased during anaerobic digestion, but increased at 35 °C on days 55. The river snails rice noodle ingredients inhibited the growth of Proteobacteria and the expression of methane metabolism. An increase in temperature promoted the presence of Bacteroidetes and Methanothrix (increased by 3.95% and 2.64% respectively). The later stage of experiment demonstrated an increase in the glycolysis rate-limiting enzymes ATP-dependent phosphofructokinase and pyruvate kinase. It was illustrated that the addition of river snails rice noodle ingredients inhibited the processing capacity of the anaerobic digestion reactor, and the increase of operating temperature of anaerobic reactor alleviated the inhibition of higher content river snails rice noodle ingredients on anaerobic digestion process. It was provided valuable basic information for the future study of treatment of river snails rice noodle wastewater.

Evaluation of activated pyrochar for boosting anaerobic digestion: Performances and microbial community

In this study, the effects of CO2-activated/non-activated pyrochars (PCs) from cornstalk, cotton straw, and rice straw on anaerobic digestion (AD) performances and microbial characteristics were investigated. The maximum biogas production rate (2.2 L/L/d) with a methane content of 73% was obtained from the AD with CO2-activated cotton straw PC. The activated PC mainly played a strengthening role in the early and middle stages of AD. Specifically, the cornstalk PC could greatly relieve acid inhibition, and cotton straw PC had a significantly positive effect on the regulation of ammonia nitrogen concentration. The rare genera like Verrucomicrobia had obvious differences among groups of AD with PCs. Regarding differential metabolites, cornstalk PC-N2 displayed a positive correlation with isoleucyl-alanine, while exhibiting a negative correlation with deoxyinosine, and the corresponding relative expression levels were + 3.0 and −2.4, respectively. Overall, gas-activated PCs could promote methane production and affect the composition of microbial community.

Impact of Thermo-Mechanical Pretreatment of Sargassum muticum on Anaerobic Co-Digestion with Wheat Straw

Sargassum muticum (SM) is an invasive macroalgal species seasonally occurring in large quantities. While generally suitable for anaerobic digestion, recent studies resulted in low specific methane yields (SMYs), presumably due to salt, polyphenol, and high fiber contents of this marine biomass. In this study, the specific biogas yield (SBY) and SMY of SM alone as well as in co-digestion with wheat straw (WS) were investigated in batch tests at process temperatures of 44 ± 1.4 °C with a retention time of approx. 40 d. The pretreatment variants of SM were examined with regard to desalination and disintegration to potentially improve digestibility and to enhance the overall performance in anaerobic digestion. A sole mechanical treatment (pressing) and a thermo-mechanical treatment (heating and pressing) were tested. Batch assays showed that pressing increased the SMY by 15.1% whereas heating and pressing decreased the SMY by 15.7% compared to the untreated variant (87.64 ± 8.72 mL/gVS). Both anaerobic digestion experiments generally showed that co-digestion with WS can be recommended for SM, but the observed SBY and SMY were still similar to those of other studies in which SM was not pretreated. The mechanical pretreatment of SM, however, offers the potential to enhance the SMY in the anaerobic digestion of SM with WS, but further research is necessary to identify the optimum upgrading approaches since the overall SMY of SM is relatively low compared to other substrates that are commonly used in anaerobic digestion. In addition to anaerobic digestion, SM as an already available biomass could also be of interest for further utilization approaches such as fiber production.

Influence of pH on the performance of anaerobic piggery wastewater treatment coupled with membrane-based NH3 extraction

The influence of the pH of piggery wastewater (PWW) on both ammonia recovery and anaerobic digestion performance during PWW treatment was evaluated in a continuous stirred tank reactor coupled with a membrane-based extraction module. The anaerobic digester was operated at a hydraulic retention time of 20 days at 37 °C, while the flat sheet PTFE membrane module operated continuously at liquid recirculation rates of 250 mL min−1. The membrane module was able to gradually decrease the total ammoniacal concentration from 1.27 ± 0.01 to 0.62 ± 0.01 g L−1 after 360 days of operation. Membrane-based NH3 extraction induced a CH4 yield increase of 1.3-fold. Moreover, COD and VS removal efficiencies increased up to 1.2-fold and 1.5-fold, respectively, along with the increase in PWW pH from 7.5 to 12. Total VFAs removal efficiencies were higher at a PWW pH of 9.

Evaluating effects of biochar on anaerobic digestion of dewatered waste activated sludge: Digester performance, microbial co-metabolism and underlying mechanism

Biochar has been proven to be capable of improving the performance of anaerobic digestion (AD). However, the effect of biochar on microbial communities remains ambiguous. In this study, the influence of pH was excluded in a semi-continuous anaerobic digestor for the treatment of dewatered waste activated sludge (WAS) to determine the effect of biochar on microbes. Compared with the control group, the average methane production increased by 24.5% and 23.2% at the organic loading rates (OLRs) of 1.56 and 3.00 gTS/L/d, respectively, in the presence of biochar. This study innovatively found biochar accelerated the enrichment of Methanofastidiosaceae, which competed with Methanobacteriaceae for H2, and its abundance increased from 0.99% at the OLR of 1.56 g TS/L/d to 16.57% and 38.11% at the OLR of 3.00 and 5.60 gTS/L/d, respectively. The efficient metabolic network of f__norank_o__Aminicenantales, syntrophic bacteria, Methanofastidiosaceae and Methanosaetaceae promoted the conversion of WAS to CH4 in the biochar group. In addition, metagenome analysis revealed that biochar optimized the metabolites related to energy conservation and electron transfer, particularly for hydrogenase (frhABG, mbhLHK and hndA-D), confirming that biochar changed the way H2 was involved in methanogenesis. These findings provide novel insights into the direct effect of biochar on microbial evolution and facilitate the reduction of WAS to achieve higher economic benefits in biogas production.

Evaluation of anaerobic digestion performance and techno-economic analysis of different components of sugarcane leaf

Sugarcane leaf (SL) is an attractive lignocellulosic feedstock for methane production by anaerobic digestion (AD), but the resistant structure and irregular components of SL hinder its effective use. To provide a comprehensive understanding of the different SL components, the physicochemical properties, methane production performance, and a techno-economic analysis (TEA) of the mesophyll (SL-MPL) and midrib (SL-MRB) parts of SL were investigated in this study. The results showed that the chemical components, physical properties, and structures of SL-MPL and SL-MRB were markedly different. After AD, SL-MPL showed methane production of 124.2 ± 8.5 mL/g volatile solid (VS), which was 121.4% higher than that of SL-MRB. Besides, the VS removal and hydrolysis rates for SL-MPL were 1.4 and 2.9 times higher than those for SL-MRB, respectively. The AD of SL-MRB was predominantly limited by its high lignin content, low nutrient element contents, smooth surface, and dense spatial structure. The TEA revealed that the revenue from SL-MPL was 74.9% of total revenue in the AD scenario, which was three times that of SL-MRB. Therefore, SL-MPL was more techno-economically efficient than SL-MRB under the AD scenario. This study provides a theoretical basis and data support for the effective utilization of SL.

Dosage effect of micron zero-valent iron during thermophilic anaerobic digestion of waste activated sludge: Performance and functional community

This work examined the performance and microbial traits in a thermophilic anaerobic digestion (TAD) of waste activated sludge that was impacted by micron zero valent iron (mZVI). Results showed that methane production was promoted by 0.8, 11.9, and 12.0 times, respectively, when mZVI was at dosages of 25, 100, and 250 mg/g total solid (TS). Also, the consumption of volatile fatty acids was increased by mZVI at higher dosages (100 and 250 mg/g TS). Furthermore, 16S rRNA sequencing demonstrated that microbial community stabilized after day 18 regardless of the dosage of mZVI, and that different dosages of mZVI induced different shifts in the functional community of the archaea rather than the bacteria involved in TAD. As a result, mZVI at 100 mg/g TS could increase the relative abundance of archaeal genera Methanothermobacter the most, increasing by 22.8% at the end of TAD compared to CK. Besides, redundancy analysis revealed that the physicochemical properties explained 79.65% and 89.10% of the variations of bacterial and archaeal abundance, respectively. Also, the findings of the correlation analysis revealed that total dissolved iron, ferrous iron, pH, and ammonium nitrogen, may be the key divers of altering functional communities, particularly archaea. Moreover, mZVI at 100 and 250 mg/g TS boosted the metabolic pathways of environmental information processing (ABC transporters) in bacteria and carbon metabolism and methane metabolism for archaea, as well as relative abundances of enzymes and their activities involved in various methanogenic pathways. This study provides new perspectives on the application of mZVI in solid wastes treatments.

Insight into the effect of rice-straw ash on enhancing the anaerobic digestion performance of high salinity organic wastewater

Anaerobic digestion is an economic method for treating high salinity organic wastewater (HSOW), but performance enhancement is needed because of the inhibitory effect of high salinity. In this study, rice-straw ash (RSA) was applied to alleviate the inhibitory effect during HSOW anaerobic digestion. The results showed that, when the NaCl content increased from 0% to 3.0%, the methane production decreased by 87.35%, and the TOC removal rate decreased to 34.12%. As a K+ and alkalinity source, RSA addition enhanced the anaerobic digestion performance, and the optimal dosage was 0.88 g/L. Under this dosage, the methane production increased by 221.60%, and TOC removal rate reached 66.42% at 3.0% salinity. The addition of RSA increased the proportion of living cells in the high salinity environment, and enhanced the activity of key enzymes and electron transfer efficiency in the anaerobic digestion process. The addition of RSA with a dosage of 0.88 g/L promoted the accumulation of acetoclastic methanogen Methanothrix. The abundance of substrate transporters, ion transporters and electron transfer related functional genes were enriched, which might be key for promoting HSOW anaerobic digestion performance. The results also showed that RSA addition played an important role in maintaining the stability of the anaerobic digestion system, and it could be a potential strategy for enhancing the anaerobic digestion performance under high salinity conditions.

Hydrothermal pretreatment of Platanus orientalis Linn fallen leaves: effects of process conditions on anaerobic digestion

ABSTRACT Platanus orientalis Linn is widely distributed as a famous street tree, and its fallen leaves are a rich biomass resource. The fallen leaves of Platanus orientalis Linn (FLPOL) can be resourcefully treated by generating biogas through anaerobic digestion. This study aimed to assess the anaerobic digestion (AD) performance of FLPOL under different hydrothermal pretreatment (HTP) conditions (solid–liquid ratio 2–10%, residence times 0.5–2.5 h, and temperatures 120–200°C). The solid–liquid ratio, residence time, and temperature determined by energy balance calculation were 6%, 2.0 h, and 180°C, respectively, and the energy balance was 1.823 kJ/g biomass. Under these conditions, the methane yield of the process water was 56.85 mL/g·VS. The methanogenic potential, hydrolysis rate, maximum methanogenic rate, and the lag period obtained by the Fitzhugh first-order kinetic model and the modified Gompertz model were 101.803 mL/g·VS, 0.130 d−1, 14.207 mL/(g·VS·d) and 1.332 d, respectively. The research results suggest that HTP has an important role in promoting the large-scale AD application of FLPOL.

Sustainable resource recovery and process improvement in anaerobic digesters using hydrochar: A circular bio‐economic perspective

AbstractHydrothermal carbonization (HTC) is a promising technology for waste valorisation and nutrient recovery to achieve sustainability. HTC converts organic waste into hydrochar, a carbon‐rich solid with numerous surface functionalities that can be used for energy and wastewater treatment. In this review, we highlight the potential of hydrochar‐based technology for improving the performance of anaerobic digestion (AD) systems and downstream applications of nutrient‐laden hydrochar. We identify knowledge gaps in hydrochar production, performance in AD systems and nutrient recovery, including the need for larger‐scale production facilities, multielement adsorption studies, and computational modelling. Techno‐economic analysis and life cycle assessment of hydrochar applications are critical to evaluating the commercial viability of this technology. Overall, hydrochar‐based technology offers a sustainable solution for waste management and resource recovery, with potential socioeconomic benefits for developing economies. The deployment of hydrochar‐based technology will directly address key issues highlighted in the United Nations' Sustainable Development Goals such as Clean water and sanitation (SDG 6); Zero hunger (SDG 2); and Climate action (SDG 13) thereby contributing to a more sustainable future.

Reconceptualization of the mechanism of thermal hydrolysis pretreatment to enhance the anaerobic conversion of sludge organic nitrogen: Decisive role of organic nitrogen occurrence

The occurrence state of organic nitrogen (ON) is the key to affect anaerobic biotransformation of sludge. ON in sludge was chemically classified as PA (easily accessible part), PB (moderately accessible) and PC (hardly accessible) according to the modified CNCPS method. The components of them were analyzed by PY-GCMS, and it was identified that PA was extracellular amino acids, peptides and proteins; PB was genetic material, cell wall peptidoglycans and intracellular proteins; PC was ON that cross-linked with complex macromolecules. The conversion characteristics of PA, PB and PC in sludge and their relationship with anaerobic digestion (AD) performance were investigated after thermal hydrolysis pretreatment (THP) at different temperatures (100–180 °C). With the increase of THP temperature, the hydrolysis of PA and the conversion of PB to PA were promoted. At 180-THP, part of PA was converted to PC due to thermochemical reactions. In the fast degradation stage of AD of ON (ON-fast), PA is the main component of degradation; while in the slow degradation stage (ON-slow), the degradation of ON is mainly dominated by PB. Therefore, THP can significantly increase the proportion of ON-fast and reduce the ON fraction in the digestate (ON-hard). Moreover, PA and PB, rather than PC, were identified as dominant in ON-hard with or without THP for the first time, overturning the traditional view (remaining ON after AD was that cross-linked with complex macromolecules). This is due to that PA and PB are the main ON that make up microbial cells. The findings upgraded our perspective on conversion of ON of sludge during AD and inspire the shifted focus from “degrading PC” to “PC accumulation” for later use, through targeted enhanced PA degradation.

Intensifying anaerobic digestion of 5-hydroxymethylfurfural via granular activated carbon supplementation

5-hydroxymethylfurfural (5-HMF) is typically found in hydrolysate produced by thermal pretreatment and wastewater streams, and its presence can significantly inhibit anaerobic digestion (AD) performance. This study comprehensively investigates the impact of adding 1–5 g/L granular activated carbon (GAC) on the AD of 0.5–4 g/L 5-HMF. The innovation of this study is to clarify the mechanism of direct interspecies electron transfer (DIET) in the AD system with 5-HMF as the sole source. Compared to the control group, adding 5 g/L GAC shows the most significant promotion effect, which increases the electron transfer activity of the sludge by 27.0%–75.0% and enhances the methane production peak rate by 27.5%–68.1%. GAC supplementation can rapidly reduce the concentration of 5-HMF by adsorption and alleviate its inhibition of microorganisms. SEM and microbial community analyses show that GAC enriches electroactive microbes and can promote AD by establishing the DIET pathway.

Effects of cold isostatic press pretreatment of rice straw on microstructure and efficiency of anaerobic digestion for methane production

In this study, rice straw was pretreated using by cold isostatic pressure to disrupt its microstructure and improve the performance of anaerobic digestion, and the optimal process parameters were optimized using the response surface methodology. The results showed that cold isostatic pressure pretreatment under optimal conditions (pressure of 400 MPa and holding time of 9 min) was effective in disrupting the structure of rice straw and improving its biodegradability. The cumulative methane production of the cold isostatic pressure pretreatment group increased by 76% compared to the untreated group. In addition, microbial community analysis showed that the relative abundance of Firmicutes, Halobacterota, DMER64 and Methanosaeta was higher in groups pretreated with cold isostatic pressure than in untreated groups. This study demonstrated the potential of pretreatment of rice straw with cold isostatic pressure to increase methane production during anaerobic digestion.

Green and chemical-free pretreatment of corn straw using cold isostatic pressure for methane production

In this study, the effect of cold isostatic pressure (CIP) pretreatment on the physicochemical properties and subsequent anaerobic digestion (AD) performance of corn straw (CS) was explored. The CS was subjected to CIP pretreatment by pressures of 200, 400 and 600 MPa, respectively, while AD was carried out at medium temperature (35 ± 2 °C). The results showed that CIP pretreatment disrupted the dense structure of the CS and altered the crystallinity index and surface hydrophobicity of the CS, thereby affecting the AD process. The presence of CIP pretreatment increased the initial reducing sugar concentration by 0.11–0.27 g/L and increased the maximum volatile fatty acids content by 112.82–436.64 mg/L, which facilitated the process of acidification and hydrolysis of the AD. It was also observed that the CIP pretreatment maintained the pH in the range of 6.37–7.30, maintaining the stability of the overall system. Moreover, the cumulative methane production in the CIP pretreatment group increased by 27.17 %–64.90 % compared to the control group. Analysis of the microbial results showed that CIP pretreatment increased the abundance of cellulose degrading bacteria Ruminofilibacter from 21.50 % to 27.53 % and acetoclastic methanogen Methanosaeta from 45.48 % to 56.92 %, thus facilitating the hydrolysis and methanogenic stages. The energy conversion analysis showed that CIP is a green and non-polluting pretreatment strategy for the efficient AD of CS to methane.

Up-flow anaerobic sludge blanket treatment of swine wastewater: Effect of heterologous and homologous inocula on anaerobic digestion performance and the microbial community

The effects of heterogenous (anaerobic sludge from treating distillery sewage, ASDS) and homologous (anaerobic sludge from treating swine wastewater, ASSW) inocula on anaerobic digestion and the microbial community in an up-flow anaerobic sludge blanket treating swine wastewater were compared. The highest chemical oxygen demand removal efficiencies with ASDS (84.8%) and ASSW (83.1%) were obtained with an organic loading rate of 15 kg COD/m3/d. For ASSW compared with ASDS, methane production efficiency was 15.3% higher and excess sludge production was 73.0% lower. The abundance of the cellulose hydrolyzing bacterium Clostridium sensu stricto_1 with ASDS (36.1%) was 1.5 times that with ASSW, while that of Methanosarcina with ASSW (22.9%) was > 100 times that with ASDS. ASDS reduced the content of pathogenic bacteria by 88.0%, while ASSW maintained a low level of pathogenic bacteria. ASSW greatly improved the methane production efficiency of wastewater and is more suitable for treating swine wastewater.

Enhancement of anaerobic digestion of ciprofloxacin wastewater by nano zero-valent iron immobilized onto biochar

The commonly used antibiotic ciprofloxacin (CIP) can significantly inhibit and interfere with the anaerobic digestion (AD) performance. This work was developed to explore the effectiveness and feasibility of nano iron-carbon composites to simultaneously enhance methane production and CIP removal during AD under CIP stress. The results demonstrated that when the nano-zero-valent iron (nZVI) content immobilized on biochar (BC) was 33% (nZVI/BC-33), the CIP degradation efficiency reached 87% and the methanogenesis reached 143 mL/g COD, both higher than Control. Reactive oxygen species analysis demonstrated that nZVI/BC-33 could effectively mitigate microorganisms subjected to the dual redox pressure from CIP and nZVI, and reduce a series of oxidative stress reactions. The microbial community depicted that nZVI/BC-33 enriched functional microorganisms related to CIP degradation and methane production and facilitated direct electron transfer processes. Nano iron-carbon composites can effectively alleviate the stress of CIP on AD and enhance methanogenesis.

Electro-polarization of the sludge with dynamic magnetic field enhanced the interspecies electron transfer in ZVI-added anaerobic digesters

Zero-valent iron (ZVI) has been widely reported to strengthen anaerobic digestion; however, ZVI passivation prevents its continuous function. In this study, a dynamic magnetic field (DMF) was imposed on a ZVI-added anaerobic digester treating food waste to promote microbial interspecies electron transfer to enhance methane production. Results showed that methane production with DMF was 15.3% higher than that without DMF. Meanwhile, the Fe(II) concentration from ZVI increased by 39.46% with DMF, but the electron equivalent was far less than the increased methane production even if all electrons from the ZVI were used for hydrogenotrophic methanogenesis. In agreement, the relative abundances of hydrogenotrophic methanogens were less changed after DMF. Notably, an induced electric current ranging from −0.017–0.021 μA was detected with DMF. With the DMF-induced current as electrical stimulation, the sludge conductivity and capacitance increased by 242% and 18%, respectively. In-situ electrochemical tests on the functional groups of the sludge showed that the protein-like substances were polarized by the induced electromotive force to enhance the proton-coupled electron transfer in the sludge. Together with the enriched electrotrophic methanogens, the enhanced microbial interspecies electron exchange was the main reason for the improved performance of anaerobic digestion with DMF.

Synthesis and evaluation of metal-impregnated carbon cloth supplementation to improve anaerobic digestion performance from municipal sludge

This study investigated the effect of metal-impregnated carbon cloth (CC) supplementation on thermophilic anaerobic digestion (AD) improvement by utilizing municipal sludge as a biomass input. Four types of metal-impregnated CC nanocomposites (Fe/CC, Ni/CC, Ni/Fe/CC, NiPcS/Fe/CC) were synthesized from activated CC (A-CC) using hydrothermal synthesis and compared with untreated CC (U-CC) to evaluate their functional significance for enhanced AD. The main factors inspected were electrical conductivity (EC) and Brunauer–Emmett–Teller (BET) surface area. A-CC had the highest surface area of 386.15 m2/g, followed by Fe/CC, NiPcS/Fe/CC, Ni/CC and Ni/Fe/CC with 140.96, 52.27, 43.52 and 36.07 m2/g, respectively. The A-CC and Fe/CC reactors produced 3.45 and 2.70 times more methane than Control (without CC) in the first 9 days. In contrast, the methane production for U-CC, Ni/CC, Ni/Fe/CC and NiPcS/Fe/CC was only 0.87, 1.26, 1.01 and 1.29 times, respectively, compared to Control. At a higher BET surface (>350 m2/g), the surface area becomes a more dominant factor, and the increase of EC value (>2.9*104 1/Ω.m) does not have an impact on methane enhancement. Microbial community analysis revealed that A-CC had the highest abundances of Syntrophomonas, Coprothermobacter and methanogenic populations, including Methanothermobacter and Methanosarcina, within 3 days compared to Control and metal-impregnated CC. There were minimal changes in microbial community composition in metal-impregnated CC compared to the U-CC. The result implies that the efficacy of CC supplementation for AD upgrading largely depends on the higher surface area compared to metal impregnation or conductivity.

Process Performance and Functional Microbial Community in the Anaerobic Digestion of Chicken Manure: A Review

Anaerobic digestion is one of the most widely used treatment methods for animal manure. Chicken manure has high methane production potential and is thus a suitable substrate for biogas plants. However, high nitrogen content inhibits the metabolism of anaerobic microorganisms and thus hinders methane production from chicken manure. Enhancing the performance of anaerobic digestion for chicken manure is indeed a long-standing challenge. This review presents new insights into maintaining methanogens’ activities, the decomposition of acetate, and the dynamics of methanogenic pathways under high ammonia stress. This review also analyzed the possible strategies for alleviating ammonia inhibition effects, including supplementing trace elements, co-digestion with nitrogen-less materials, in-situ ammonia removal, and long adaptation of anaerobic consortia to ammonia stress. The insights obtained in this paper may provide helpful information for a better understanding of anaerobic digestion technology for chicken manure and other nitrogen-rich waste and wastewater.

The effect and mechanism of polyethylene terephthalate microplastics on anaerobic co-digestion of sewage sludge and food waste

This research investigated the impact of polyethylene terephthalate (PET) microplastics on the anaerobic co-digestion of sewage sludge and food waste. The performance of anaerobic digester was investigated with different particle sizes (30 µm and 250 µm) PET at concentrations of 0, 0.45, 1.35, and 2.70 mg/g total solid (TS). Results suggested that the CH4 production at 2.70 mg/g TS level decreased by 21.63% (30 µm) and 15.87% (250 µm) compared with the control group, respectively. PET microplastics exerted negative influence through leaching toxic diisobutyl phthalate (DIBP) and dibutyl phthalate (DBP). Microbial community analysis and redundancy analysis showed that DIBP and DBP reduced the abundances of key hydrolysis bacteria (Bacteroides vadin HA17) and acidification bacteria (Clostridium and Sphaerochaeta). Structural equation model analysis showed that DBP and DIBP were the key factors that reduced the methane production, and they had stronger impact on daily methane production in the 30 µm group compared with the 250 µm group.