Chemical profiling and probiotic viability assessment in Gueuze-style beer: Fermentation dynamics, metabolite and sensory characterization, and in vitro digestion resistance.

Probiotic viability, metabolite concentrations, physicochemical parameters, and volatile compounds were characterized in Gueuze beers formulated with probiotic lactic acid bacteria (LAB) and yeast. Additionally, the sensory profile of the beers and the resistance of the probiotics to digestion were determined. The use of 2 International Bitterness Units resulted in high concentrations of probiotic LAB but a decline in probiotic yeast as pH decreased. Secondary fermentation led to the consumption of maltose, citric acid, and malic acid, and the production of lactic and propionic acids. Carbonation and storage at 4 °C had minimal impact on probiotic viability. The addition of probiotic LAB resulted in a distinct aroma profile with improved sensory characteristics. Our results demonstrate that sour beers produced with probiotic LAB and a probiotic yeast, and fermented using a two-step fermentation process, exhibited optimal physicochemical parameters, discriminant volatile compound profiles, promising sensory characteristics, and high probiotic concentrations after digestion.

Use of waste frying oil and coconut pulp for the production, isolation, and characterization of a new lipase from Moesziomyces aphidis.

Lipases comprise the third most commercialized group of enzymes worldwide and those of microbial origin are sought for their multiple advantages. Agro-industrial waste can be an alternative culture medium for producing lipases, reducing production costs and the improper disposal of waste frying oil (WFO). This study aimed to produce yeast lipases through submerged fermentation (SF) using domestic edible oil waste as inducer and alternative culture medium. The optimal culture conditions, most effective inducer, and purification method for a new lipase from Moesziomyces aphidis BRT57 were identified. Yeast was cultured in medium containing green coconut pulp and WFO waste for 72 h. The maximum production of lipases in SF occurred in a culture medium containing WFO and yeast extract at 48 and 72 h of incubation, with enzyme activities of 8.88 and 11.39 U mL-1, respectively. The lipase was isolated through ultrafiltration followed by size exclusion chromatography, achieving a 50.46 % recovery rate. To the best of our knowledge, this is the first study to report the production and purification of lipases from M. aphidis, demonstrating the value of frying oil as inducer and alternative medium for SF, contributing to the production of fatty acids for biodiesel from food waste.

Metagenomics reveals the resistance patterns of electrochemically treated erythromycin fermentation residue.

Erythromycin fermentation residue (EFR) represents a typical hazardous waste produced by the microbial pharmaceutical industry. Although electrolysis is promising for EFR disposal, its microbial threats remain unclear. Herein, metagenomics was coupled with the random forest technique to decipher the antibiotic resistance patterns of electrochemically treated EFR. Results showed that 95.75% of erythromycin could be removed in 2 hr. Electrolysis temporarily influenced EFR microbiota, where the relative abundances of Proteobacteria and Actinobacteria increased, while those of Fusobacteria, Firmicutes, and Bacteroidetes decreased. A total of 505 antibiotic resistance gene (ARG) subtypes encoding resistance to 21 antibiotic types and 150 mobile genetic elements (MGEs), mainly including plasmid (72) and transposase (52) were assembled in EFR. Significant linear regression models were identified among microbial richness, ARG subtypes, and MGE numbers (r2=0.50-0.81, p< 0.001). Physicochemical factors of EFR (Total nitrogen, total organic carbon, protein, and humus) regulated ARG and MGE assembly (%IncMSE value = 5.14-14.85). The core ARG, MGE, and microbe sets (93.08%-99.85%) successfully explained 89.71%-92.92% of total ARG and MGE abundances. Specifically, gene aph(3')-I, transposase tnpA, and Mycolicibacterium were the primary drivers of the resistance dissemination system. This study also proposes efficient resistance mitigation measures, and provides recommendations for future management of antibiotic fermentation residue.

The mechanisms of pH regulation on promoting volatile fatty acids production from kitchen waste.

The anaerobic acid production experiments were conducted with the pretreated kitchen waste under pH adjustment. The results showed that pH 8 was considered to be the most suitable condition for acid production, especially for the formation of acetic acid and propionic acid. The average value of total volatile fatty acid at pH 8 was 8814 mg COD/L, 1.5 times of that under blank condition. The average yield of acetic acid and propionic acid was 3302 mg COD/L and 2891 mg COD/L, respectively. The activities of key functional enzymes such as phosphotransacetylase, acetokinase, oxaloacetate transcarboxylase and succinyl-coA transferase were all enhanced. To further explore the regulatory mechanisms within the system, the distribution of microorganisms at different levels in the fermentation system was obtained by microbial sequencing, results indicating that the relative abundances of Clostridiales, Bacteroidales, Chloroflexi, Clostridium, Bacteroidetes and Propionibacteriales, which were great contributors for the hydrolysis and acidification, increased rapidly at pH 8 compared with the blank group. Besides, the proportion of genes encoding key enzymes was generally increased, which further verified the mechanism of hydrolytic acidification and acetic acid production of organic matter under pH regulation.

Potential of oleaginous microbes for lipid accumulation and renewable energy generation.

Biocomponents (such as lipids) accumulate in oleaginous microorganisms and could be used for renewable energy production. Oleaginous microbes are characterized by their ability to accumulate high levels of lipids, which can be converted into biodiesel. The oleaginous microbes (including microalgae, bacteria, yeast, and fungi) can utilize diverse substrates. Thus, in this study, commercially viable oleaginous microorganisms are comparatively summarized for their growth conditions, substrate utilization, and applications in biotechnological processes. Lipid content is species-dependent, as are culture conditions (such as temperature, pH, nutrients, and culture time) and substrates. Lipid production can be increased by selecting suitable microorganisms and substrates, optimizing environmental conditions, and using genetic engineering techniques. In addition, the emphasis on downstream processes (including harvesting, cell disruption, lipid extraction, and transesterification) highlights their critical role in enhancing cost-effectiveness. Oleaginous microorganisms are potential candidates for lipid biosynthesis and could play a key role in meeting the energy needs of the world in the future.

Investigation of γ-polyglutamic acid production via asynchronous saccharification and fermentation of raw corn starch.

Starch, a crucial raw material, has been extensively investigated for biotechnological applications. However, its application in γ-polyglutamic acid (γ-PGA) production remains unexplored. Based on γ-PGA output of Bacillus subtilis SCP010-1, a novel asynchronous saccharification and fermentation process for γ-PGA synthesis was implemented. The results revealed that a starch concentration of 20%, α-amylase dosage of 75 U/g, liquefaction temperature of 72℃, and γ-PGA yield of 36.31 g/L was achieved. At a glucoamylase dosage of 100 U/g, saccharification 38 h at 60℃, the yield of γ-PGA increased to 48.88 g/L. The contents of total sugar, glucose, maltose and oligosaccharide in saccharified liquid were determined. Through batch fermentation of saccharified liquid in fermentor, the γ-PGA output was elevated to 116.08 g/L. This study can offer a potential cost reduction of 40%, which can be a promising advancement in industrial γ-PGA production. Moreover, our approach can be applied in other starch-based fermentation industries.

Garlic Bioconverted by Bacillus subtilis Stimulates the Intestinal Immune System and Modulates Gut Microbiota Composition.

SCOPE: This study evaluates the potential of bioconverted garlic ferments (BGFs) to stimulate the intestinal immune system and modulate cecal microbiota composition. METHODS AND RESULTS: In vitro, BGF significantly enhances Peyer's patch (PP)-mediated bone marrow cell proliferation and increases the production of interferon-gamma (IFN-γ), granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-6, and immunoglobulin A (IgA) but not IL-4, IL-5, and immunoglobulin E (IgE). Oral administration of BGF to C3H/HeN mice for 4 weeks significantly increases the GM-CSF (42.1-45.8 pg mL-1) and IFN-γ (6.5-12.1 pg mL-1) levels in PP cells. BGF also significantly elevates the levels of tumor necrosis factor-alpha (TNF-α, 165.0-236.3 pg mg-1), GM-CSF (2.4-3.0 ng mg-1), and IFN-γ (1.5-3.2 ng mg-1) in the small intestinal fluid, and TNF-α (2.2-3.1 pg mL-1) and IFN-γ (10.3-0.21.5 pg mL-1) in the mouse serum. Cecal microbial analysis reveals that BGF increases Bacteroidota and Verrucomicrobiota and decreases Actinobacteria and Bacillota at the phylum level in mice. At the genus level, BGF significantly increases the abundance of Fusimonas (250 mg kg-1 BW-1 day-1), Bacteroides (125 and 250 mg kg-1 BW-1 day-1), and Akkermansia (125 mg kg-1 BW-1 day-1) and decreases that of Bifidobacterium (62.5 and 250 mg kg-1 BW-1 day-1) and Limosilactobacillus (125 and 250 mg kg-1 BW-1 day-1). CONCLUSION: This study provides the first evidence of BGF's ability to modulate the intestinal immune system and gut microbiota, supporting its potential as a novel functional material to enhance gut immunity.

Enhancing short-chain fatty acids production from waste activated sludge anaerobic fermentation with addition of red mud: Performance and mechanisms.

Red mud (RM) as hazardous waste produced from aluminum refining industry has threatened the environment and human health. In this study, RM was added into the fermenter to promote short chain fatty acids (SCFAs) production from waste activated sludge (WAS) anaerobic fermentation. Results showed that the addition of RM could effectively improve the SCFAs production, especially, acetic acid. In particular, the production of total SCFAs and acetic acid in 20 g/L RM added fermenter were 1108.1 mg COD/L and 415.5 mg COD/L, which were 116.0% and 1308.0% higher than that in control fermenter. Batch experiment revealed that RM could enhance the hydrolysis and acidification process. Further study indicated that the activity of enzyme related to hydrolysis-acidification, abundance of fermentative bacteria for SCFAs production and functional metabolism genome were all improved with the addition of RM. The potential mechanism maybe that the RM promoted the hydrolysis-acidification process with the contained varies Fe(Ⅲ) oxides as electron acceptor, and the produced Fe2+ could serve as necessary trace elements to synthesize enzyme and then stimulate the expression of enzyme genes.

Yeast culture enhances long-term fermentation of corn straw by ruminal microbes for volatile fatty acid production: Performance and mechanism.

Ruminal microbes can efficiently ferment biomass waste to produce volatile fatty acids (VFAs). However, keeping long-term efficient VFA production efficiency has become a bottleneck. In this study, yeast culture (YC) was used to enhance the VFA production in long-term fermentation. Results showed that YC group improved the volatile solid removal and VFA concentration to 47.8% and 7.82 g/L, respectively, 18.6% and 16.1% higher than the control, mainly enhancing the acetic, propionic, and butyric acid production. YC addition reduced the bacterial diversity, changed the bacterial composition, and improved interactions among bacteria. The regulation mechanism of YC was to increase the abundance and activity of hydrolytic and acidogenic bacteria such as Prevotella and Treponema, improve bacterial interactions, and further promote expression of functional genes. Ultimately, a long-term efficient ruminal fermentation of corn straw into VFAs was achieved.

From agricultural biomass to D form lactic acid in ton scale via strain engineering of Lactiplantibacillus pentosus.

Worsening environmental conditions make lactic acid a sustainable alternative to petroleum-based plastics. This study created a genetically-engineered strain Lactiplantibacillus pentosus PeL containing a disrupted L-lactate dehydrogenase gene to produce high yield and optically pure D-lactic acid. Cellobiose was identified as the optimal sugar in the single carbon source test, yielding the highest lactic acid. In 5-L fermentation tests, pretreated wood chips hydrolysate was the best lignocellulosic substrate for PeL, resulting in a D-lactic acid yield of 900.7 ±â€¯141.4 mg/g of consumed sugars with an optical purity of 99.8 ±â€¯0.0 %. Gradually scaled-up fermentations using this substrate were achieved in 100-, and 9,000-L fermenters; PeL produced remarkably high D-lactic acid yields of 836.3 ±â€¯11.9 and 915.9 ±â€¯4.4 mg/g of consumed sugars, with optical purities of 95.0 ±â€¯0.0 % and 93.8 ±â€¯0.2 %, respectively. This study is the pioneer in demonstrating economical and sustainable ton-scale production of D-lactic acid.

Effects of various levels of coated cysteamine hydrochloride in the diet on feed intake, digestibility, ruminal fermentation, and blood metabolites in growing Charolais crossbred cattle.

This study investigated the impact of different levels of coated cysteamine hydrochloride (CSH) supplementation on ruminal fermentation, nutrient digestibility, and blood metabolites in Charolais cross bulls. Twelve bulls were allotted to three feeding treatments in a replicated 3 × 3 Latin square design: 0% CSH (control), 0.5% CSH, and 1.0% CSH in concentrate. Animals were fed concentrate at 1.5% of body weight. Dry matter intake (DMI) and DMI as a percentage of body weight showed no significant differences among treatments (p > 0.10). Nutrient digestibility was consistent across treatments, except for a slight decrease in NDF digestibility with 1% CSH (p = 0.07). Ruminal pH, ammonia nitrogen, volatile fatty acid (VFA) proportions, and total VFA concentration were similar among treatments (p > 0.05). Total bacteria, fungal zoospores, and protozoa populations in the rumen did not vary significantly (p > 0.05). Blood glucose and triglyceride concentrations remained stable (p > 0.05), while blood urea nitrogen (BUN) levels were higher in CSH-supplemented groups (p < 0.05). In conclusion, incorporating CSH levels ranging from 0.5% to 1.0% into the diet did not adversely affect feed intake, ruminal fermentation, or microbial populations. Additionally, 1.0% CSH improved BUN concentration in growing Charolais cross bulls.

Effects of different temperature and density on quality and microbial population of wilted alfalfa silage.

In this experiment, alfalfa silage with different packing densities (500 kg/m3、600 kg/m3 and 700 kg/m3) was prepared under the conditions of outdoor high temperature and indoor room temperature, respectively. At the same time, the same lactobacillus additive was used for fermentation in each density treatment group. The chemical composition, fermentation quality and microbial community of alfalfa silage were analyzed. The results showed that the contents of dry matter (DM) and water-soluble carbohydrate (WSC) decreased with the increase of density during fermentation at high temperature. At the same time, when the density is 600 kg/m³, CP (crude protein) content is the highest, ADF (acid detergent fiber) content is the lowest. The contents and pH values of neutral detergent fiber (NDF), lactic acid (LA) and lactic acid bacteria (LAB) were significantly affected by temperature (p < 0.05). Density had significant effects on DM, NDF, WSC and LA contents (p < 0.05). The interaction between temperature and density had significant effects on the content of ADF and LAB (p < 0.05). At the same time, the abundance of Lactiplantibacillus plantarum in high temperature fermented silage was lower than that in normal temperature fermented feed. The number of Lactiplantibacillus plantarum in room temperature treatment group decreased with the increase of density. In summary, this study clarified the effects of different temperature and density on alfalfa fermentation quality and microbial community, and clarified that the density should be reasonably controlled within 600 kg/m³ during alfalfa silage, providing theoretical support for production practice.

The effects of applying cellulase and laccase on fermentation quality and microbial community in mixed silage containing corn stover and wet brewer's grains.

Objective: The purpose of this experiment was to explore the effect of adding cellulase and laccase on fermentation quality and microbial community in mixed silage of corn stover and wet brewer's grains. Try to a new approach for the proper preservation and utilization of the agro-industrial by-products (corn stover and wet brewer's grains). Methods: The experiment was divided into four groups: CK (control), C (cellulase, 120 U/g fresh matter [FM]), L (laccase, 50 U/g FM), CL (cellulase 120 U/g FW and laccase 50 U/g FM), and the chemical composition, fermentation quality, microbial population and microbial community in mixed silage of corn stover and wet brewer's grains after 30 day's fermentation were determined. Results: Compared to control, the addition of cellulase significantly increased crude protein (CP), water-soluble carbohydrate (WSC), lactic acid bacteria (LAB) counts, while significantly decreased the neutral detergent fiber (NDF), acid detergent fiber (ADF) content (P < 0.05). Adding laccase significantly decreased the acid detergent lignin (ADL) content (P < 0.05). Combined application of cellulase and laccase significantly increased the CP, WSC content and LAB counts, while significantly decreased pH value, NDF, ADF and ADL content (P < 0.05), thereby improving fermentation quality. In addition, the application of cellulase and laccase increased the abundance of Firmicutes and LAB genera, and decreased microbial diversity level of the sample. Conclusion: The combined application of cellulase and laccase further improved fermentation quality and microbial community in mixed silage of corn stover and wet brewer's grains.

In vivo absorption, in vitro digestion, and fecal fermentation properties of non-starch polysaccharides from Chinese chestnut kernels and their effects on human gut microbiota.

Non-starch polysaccharides are major bioactive components in chestnuts, and can serve as water-soluble polysaccharides with potential prebiotic properties. This study aims to establish an in vitro digestion and fermentation model to reveal the digestive and fermentative characteristics of Non-starch polysaccharides from chestnut kernels (NSPCK). The results indicated that under simulated digestion, NSPCK was partially digested in gastric juice but remained significantly undigested in saliva and intestinal juice, demonstrating considerable resilience against hydrolysis. After digestion, NSPCK still exhibited stable rough, lamellar, and porous structure and maintained strong antioxidant capacity. Animal experiments revealed positive effects of NSPCK on blood lipid level, and colon tissue of mice. Moreover, NSPCK enhanced the accumulation of short-chain fatty acids during fermentation, particularly acetic acid, propionic acid, and butyric acid. Furthermore, NSPCK intervention increased the abundance of beneficial bacteria such as Lactobacillus and Bifidobacterium, and at the same time reduced that of harmful bacteria such as Enterococcus.

Bioethanol production from pineapple fruit waste juice using bakery yeast.

The declining oil production stemming from decreasing raw material reserves has clashes with rising demands and has created a supply-demand gap in the overall energy sector. Excessive consumption of fossil fuel oil exacerbates environmental issues, potentially leading to global climate change and increased natural disasters. Consequently, there are efforts in looking for alternate renewable fuel sources. The study included physical pre-treatment, natural hydrolysis, natural fermentation, fermentation of pineapple waste juice using bakery yeast, and subsequent distillation. The pineapple wastes produced juice with 12.67 °Brix and pH range of 3.16-3.18. The present study reports bioethanol production from pineapple waste mixed with bakery yeast (Saccharomyces cerevisiae ) and pineapple wastes juice without bakery yeast, revealing that the yeast-amended mixture yielded bioethanol with alcohol content of 45 % compared to 36 % from pineapple juice alone. Re-distillation enhanced bioethanol content from 25 % - 45 %-85 % which aligns well with E85 fuel specifications, indicating bioethanol's suitability as fuel. Thus, bioethanol derived from pineapple fruit wastes presents a promising renewable energy solution. This study investigates the production of bioethanol from pineapple waste juice by comparing two methods: one using bakery yeast and the other without yeast. Both methods are conducted at room temperature to evaluate their efficiency and effectiveness in converting pineapple waste juice into bioethanol.

Aquaculture faecal waste generates different products during anaerobic digestion depending on nutrient composition.

The inclusion of carbohydrate-rich ingredients in aquafeeds has resulted in an increased fraction of undigested material, culminating in faecal waste enriched in unutilized nutrients containing carbon, nitrogen and phosphorus. This study explored the impact of faecal composition, as influenced by diet, on the products of anaerobic digestion with a focus on organic acids (OAs). The aim was to in vitro assess the potential of faeces as internal carbon for denitrification, promoting circularity in recirculating aquaculture systems. In this regard, settleable faeces originating from six diets (DDGS, Dried distillers' grains with solubles; HFM, Hydrolyzed feather meal; IM, Insect meal; SCP, Single-cell protein; SSM, Shrimp shell meal; SWP, Seaweed protein) fed to European seabass, were incubated for a 14-day period in anoxic batch reactors. Nutrient solubilization (chemical oxygen demand, total Kjeldhal nitrogen, total phosphorus) was measured over time, and the final yield of OA was studied in relation to prokaryotic community composition. Results showed that digestion of faecal waste with a high crude-protein-to-carbohydrate ratio leads to an increased amount of OA which is greatly dominated by acetate. Among them, SSM faeces exhibited the highest final OA yield, resulting from a continuous increase over time. Despite variations in OA quantity and profile, prokaryotic composition did not substantially differ among treatments at the end of the trial, with only the relative abundance of three genera varying significantly (Anaerostignum, Bythopirellula, Mycobacterium). Yet, lactate concentration positively correlated with several taxa (Trichococcus, Oleispira, Defluviitaleaceae, Anaerocolumna, and Carboxyliverga) and butyrate with Anaerostignum. Alongside, ammonia release was minimal for all treatments, while phosphorus dissolution did not correlate to the faecal phosphorus content but was rather a result of acidification due to OA production. Overall, considering that an optimal carbon source for denitrification should produce a high amount of end OAs (i.e. acetate) along with low dissolved nitrogen and phosphorus, this research suggests that faecal waste originating from certain carbohydrate-rich aquafeed ingredients can be suitable as internal carbon source for denitrification.

Current upstream and downstream process strategies for sustainable yeast lipid production.

An increasing global population demands more lipids for food and chemicals, but the unsustainable growth of plant-derived lipid production and an unreliable supply of certain lipids due to environmental changes, require new solutions. One promising solution is the use of lipids derived from microbial biomass, particularly oleaginous yeasts. This critical review begins with a description of the most promising yeast lipid replacement targets: palm oil substitute, cocoa butter equivalent, polyunsaturated fatty acid source, and animal fat analogue, emphasizing sustainability aspects. Subsequently, the review focuses on the most recent advances in upstream methodologies, particularly fermentation strategies that promote circularity, such as waste valorisation, co-cultivation and co-product biosynthesis. Downstream processing methods for minimising energy consumption and waste generation, including bioflocculation energy-efficient and environmentally friendly cell lysis and extraction, and integrated co-product recovery methods, are discussed. Finally, the current challenges are outlined. Integrating these strategies advances sustainable yeast lipid production for high-value applications.

Screening and stability verification of reference genes in Botrytis cinerea ZX2 fermentation.

Botrytis cinerea, an airborne plant pathogen, holds the potential to synthesize sesquiterpenes, which have been used for the industrial production of abscisic acid. Previously, through our genetic technology, we obtained strain ZX2, whose main product 1´,4´-trans-ABA-diol is physiologically active in plants. In this study, 50 L of fed fermentation was carried out with ZX2 strain to study the stability of expression of TUA, TUB, ATC, EF-1, GAPDH, UCE and GTP genes. Four kinds of software (GeNorm, NormFinder, BestKeeper and Delta Ct) were used to analyze the expression stability of candidate genes, and finally the best reference gene was screened by RefFinder. Based on the results, the ACT was the most stable gene. It was used to normalize the expression levels of two genes related to 1´,4´-trans-ABA-diol production (hmgr and bcaba3) when fed-batch fermentation. Guide the selection of appropriate internal reference genes during the fermentation process to accurately quantify the relative transcription levels of target genes in B.cinerea ZX2.

Online monitoring of methane transfer rates unveils nitrogen fixation dynamics in Methylococcus capsulatus.

This study explores methane utilization by the methanotrophic microorganism Methylococcus capsulatus (Bath) for biomass production, presenting a promising approach to mitigate methane emissions and foster the development sustainable biomaterials. Traditional screening methods for gas cultivations involve either serum flasks without online monitoring or costly, low-throughput fermenters. To address these limitations, the Respiration Activity MOnitoring System was augmented with methane sensors for real-time methane transfer rate (MTR) monitoring in shake flasks. Utilizing online monitoring of the MTR in shake flasks results in enhanced throughput and cost-effectiveness for cultivating M. capsulatus. Simultaneous monitoring of transfer rates for oxygen, methane, and carbon dioxide was conducted in up to eight shake flasks, ensuring the success of the cultivation process. Alterations in methane-to-oxygen transfer rate ratios and carbon fixation rates reveal the impact of transfer limitations on microbial growth. Detection of gas transfer limitations, exploration of process parameter influences, and investigations of medium components were enabled by the introduced method. Optimal nitrogen concentrations could be determined to ensure optimal growth. This streamlined approach accelerates the screening process, offering efficient investigations into metabolic effects, limitations, and parameter influences in gas fermentations without the need for elaborate offline sampling, significantly reducing costs and enhanced reproducibility.

Fate of antibiotic resistance genes during sludge anaerobic fermentation: Roles of different sludge pretreatment.

Excess sludge, the primary by-product of wastewater treatment plants, is the source and sink of antibiotic resistance genes (ARGs). Sludge pretreatments are an indispensable pathway to improve the resource recovery and harmfulness for anaerobic digestion sludge. However, fewer studies have compared the effects of different pretreatment technologies on the distribution of ARGs during anaerobic sludge digestion. Here, this study established seven anaerobic digesters, and four typical ARGs and one integrase gene of class 1 integron (intI1) regarded as the representative mobile genetic elements (MGEs) were examined during the whole anaerobic digestion process. It was found anaerobic digestion could effectively remove ARGs with about 70.86% removal rate of total ARGs. Among these pretreatments, the reduce efficiency of ARGs was the highest in 50 °C pretreatment, followed by oxidant, and the last was acid-alkaline. The microbial community analysis demonstrated the microbial community structure, including ARGs hosts and antibiotic resistant bacteria, was significantly changed and influenced by high temperature pretreatment. In addition, high temperature and K2S2O8 observably decrease the level of ROS production. Macro transcriptome analysis indicated that sludge pretreatment, except for 50 °C pretreatment, up-regulated the genes relevant to lyases and transferase, but down-regulated the genes responsible for peroxidase, antioxidant enzymes and T4SS gene. This study emphasized and compared the different sludge pretreatments on the fate of ARGs in anaerobic sludge, and highlighted concerns regarding the environmental and health risks to our society.