High-Throughput RPLC-MS/MS Quantification of Short- and Medium-Chain Fatty Acids.

Short- and medium-chain fatty acids (SMCFA) are monocarboxylic acids with a carbon chain length of 1-12 carbon atoms. They are mainly produced in humans by the gut microbiota, play crucial metabolic roles, are vital for intestinal health, and have multifaceted impact on immune and neurological functions. Accurate detection and quantification of SMCFA in different human biofluids is achieved using 3-nitro phenylhydrazine (3-NPH) derivatization of the free fatty acids followed by reverse phase liquid chromatography (RPLC) separation and detection by tandem mass spectrometry (MS/MS). Here, we describe the simultaneous measurement of 14 SMCFA and lactate in detail. All 3-NPH-SMCFA-hydrazones are separated in less than 5 min with an 8-min total run time (injection-to-injection). Linear dynamic range over 0.1-500 µM is achieved for most SCFAs, while it is 0.05-100 µM for MCFAs. Validation of the procedure depicts good linearity (R2 > 0.98) and repeatability (CV ≤ 20%). The lower limit of detection (LLOD) is 10-30 nM. The lower limit of quantification (LLOQ) is 50-100 nM for most analytes, while it is 0.5 µM for acetate. In conclusion, the method offers several benefits compared to alternative methods regarding throughput, selectivity, sensitivity, and robustness.

Low-pressure continuous dynamic extraction from oak chips combined with passive micro-oxygenation to tune red wine properties.

Static infusion of oak chips in wine is a common practice during wine ageing, aimed at improving sensory properties and stability of wines. The wine/chips contact required to reach the desired effect can last several weeks or months. A low-pressure continuous dynamic (LPCD) extractor in which a closed-circle, low-pressure continuous flow of wine passes through an extraction cell filled with chips, was evaluated as a tool to tune red wine properties in few hours. The aim of this work was to evaluate the effect of the use of a LPCD extractor the effect on color, volatile compounds and sensory properties of a Primitivo wine, as well as to assess the combined effect of LPCD extractor, passive microxygenation through polyethylenetereftalate (PET) containers and exogenous tannins. Their combined effect caused a significant increase of stabilized pigments was observed, without compromising the aroma profile. LPCD extraction, passive micro-oxygenation through plastic materials and enological tannins can be considered as a low-cost, and potentially low-impact, integrated technological platform suitable to tune wine sensory properties and stability, when either traditional approaches (such as barrel aging) or other assisted extraction technologies are not applicable or preferred, even in small wineries.

Carbon chain elongation characterizations of electrode-biofilm microbes in electro-fermentation.

The higher efficiency of electro-fermentation in synthesizing medium-chain fatty acids (MCFAs) compared to traditional fermentation has been acknowledged. However, the functional mechanisms of electrode-biofilm enhancing MCFAs synthesis remain research gaps. To address this, this study proposed a continuous flow electrode-biofilm reactor for chain elongation (CE). After 225 days of operation, stable electrode-biofilms formed and notably improved caproate yield by more than 38 %. The electrode-biofilm was enriched with more CE microorganisms and electroactive bacteria compared to the suspended sludge microorganisms, including Caproicibacterium, Oscillibacter and Pseudoramibacter. Besides, the upregulated CE pathways were evaluated by metagenomic analysis, and the results indicated that the pathways such as acetyl-CoA and malonyl-[acp] formation, reverse beta-oxidation, and fatty acid biosynthesis pathway were all markedly enhanced in cathodic biofilm, more than anodic biofilm and suspended microorganisms. Moreover, microbial community regulated processes like bacterial chemotaxis, flagellar assembly and quorum sensing, crucial for electrode-biofilm formation. Electron transfer, energy metabolism, and microbial interactions were found to be prominently upregulated in the cathodic biofilm, surpassing levels observed in anodic biofilm and suspended sludge microorganisms, which further enhanced CE efficiency. In addition, the statistical analyses further highlighted key microbial functions and interactions within the cathodic biofilm. Oscillospiraceae_bacterium was identified to be the most active microbe, alongside pivotal roles played by Caproiciproducens_sp._NJN-50, Clostridiales_bacterium, Prevotella_sp. and Pseudoclavibacter_caeni. Eventually, the proposed microbial collaboration mechanisms of cathodic biofilm were ascertained. Overall, this study uncovered the biological effects of the electrode-biofilm on MCFAs electrosynthesis, thereby advancing biochemicals production and filling the knowledge gaps in CE electroactive biofilm reactors.

ß-hydroxybutyrate and mitochondria mediate the association between medium-chain fatty acids, DHA and mild cognitive impairment: a nested case-control study.

BACKGROUND: Medium-chain fatty acids (MCFAs) and docosahexaenoic acid (DHA) could affect the occurrence of mild cognitive impairment (MCI). ß-hydroxybutyrate (BHB), mitochondrial DNA copy number (mtDNAcn) and mitochondrial DNA (mtDNA) deletions might be their potential mechanisms. This study aimed to explore the relationship between MCFAs, DHA and MCI, and potential mechanisms. METHODS: This study used data from Tianjin Elderly Nutrition and Cognition (TENC) cohort study, 120 individuals were identified with new onset MCI during follow-up, 120 individuals without MCI were selected by 1:1 matching sex, age, and education levels as the control group from TENC. Conditional logistic regression analysis and mediation effect analysis were used to explore their relationship. RESULTS: Higher serum octanoic acid levels (OR: 0.633, 95% CI: 0.520, 0.769), higher serum DHA levels (OR: 0.962, 95% CI: 0.942, 0.981), and more mtDNAcn (OR: 0.436, 95% CI: 0.240, 0.794) were associated with lower MCI risk, while more mtDNA deletions was associated with higher MCI risk (OR: 8.833, 95% CI: 3.909, 19.960). Mediation analysis suggested that BHB and mtDNAcn, in series, have mediation roles in the association between octanoic acid and MCI risk, and mtDNA deletions have mediation roles in the association between DHA and MCI risk. CONCLUSION: Higher serum octanoic acid and DHA levels were associated with lower MCI risk. Octanoic acid could affect the incidence of MCI through BHB, then mitochondria function, or through mitochondria function, or directly. Serum DHA level could affect the incidence of MCI through mitochondria function, or directly.

Impact of cell voltage on synthesis of caproic acid from carbon dioxide and ethanol in direct current powered microbial electrosynthesis cell.

Production of medium chain fatty acids (MCFAs) from CO2 through microbial electrosynthesis (MES) holds great potential. The present study investigated the effect of cathode voltages of - 0.8 V (MES-1), -1.0 V (MES-2) and -1.2 V vs Ag/AgCl (MES-3), on the production of MCFAs from CO2 and ethanol using an enriched culture. Direct current (DC) power supply was used to maintain constant cathode voltages. The highest amounts of caproic acid were produced in MES-2 at an average concentration of 1.51 ± 0.14 g/L with a maximum selectivity of 68 ± 7 %. Microbial diversity analysis showed abundance of the Clostridiaceae family that allowed chain elongation in all MES reactors. This study shows that potentiostatic control approach for MCFA synthesis, can be replaced by DC power supply in future MES setups. Using selective culture enrichment, MES efficiently produces MCFAs from CO2 and ethanol, with -1.0 V yielding the highest caproic acid.

Influence of Carbohydrate Intake on Caprylic Acid (C8:0)-Induced Ketogenesis-A Systematic Review and Meta-Analysis.

The ketogenic diet is used worldwide to treat various diseases, especially drug-resistant epilepsies. Medium-chain triglycerides or medium-chain fatty acids, primarily the major ketogenic compound caprylic acid (C8; C8:0), can significantly support ketogenesis. This review examines the effects of concurrent carbohydrate intake on C8-induced ketogenesis. A systematic literature search (PubMed and Web of Science) with subsequent data extraction was performed according to PRISMA guidelines and the Cochrane Handbook. Studies investigating the metabolic response to C8-containing MCT interventions with carbohydrate intake were included. The studies did not include a ketogenic diet. Three intervention groups were created. The quality of the studies was assessed using the RoB II tool, and the meta-analysis was performed using the Cochrane RevMan software. A total of 7 trials, including 4 RCTs, met the inclusion criteria. Ketone production was lower when C8 was combined with carbohydrates compared to MCT intake alone. The lower C8 dose group (11 g) did not show a significantly lower ketogenic effect than the higher dose group (19 g). Forest plot analysis showed heterogeneous data. The data suggest a non-linear relationship between C8, carbohydrate intake and ketone production. Further studies are needed to investigate the influence of different carbohydrates on C8-induced ketogenesis. Limitations include heterogeneous intervention conditions, such as different types of dispersions, caffeine intake, limited number of studies and variability in study design.

Unlocking lager's flavour palette by metabolic engineering of Saccharomyces pastorianus for enhanced ethyl ester production.

Despite being present in trace amounts, ethyl esters play a crucial role as flavour compounds in lager beer. In yeast, ethyl hexanoate, ethyl octanoate and ethyl decanoate, responsible for fruity and floral taste tones, are synthesized from the toxic medium chain acyl-CoA intermediates released by the fatty acid synthase complex during the fatty acid biosynthesis, as a protective mechanism. The aim of this study was to enhance the production of ethyl esters in the hybrid lager brewing yeast Saccharomyces pastorianus by improving the medium chain acyl-CoA precursor supply. Through CRISPR-Cas9-based genetic engineering, specific FAS1 and FAS2 genes harbouring mutations in domains of the fatty acid synthesis complex were overexpressed in a single and combinatorial approach. These mutations in the ScFAS genes led to specific overproduction of the respective ethyl esters: overexpression of ScFAS1I306A and ScFAS2G1250S significantly improved ethyl hexanoate production and ScFAS1R1834K boosted the ethyl octanoate production. Combinations of ScFAS1 mutant genes with ScFAS2G1250S greatly enhanced predictably the final ethyl ester concentrations in cultures grown on full malt wort, but also resulted in increased levels of free medium chain fatty acids causing alterations in flavour profiles. Finally, the elevated medium chain fatty acid pool was directed towards the ethyl esters by overexpressing the esterase ScEEB1. The genetically modified S. pastorianus strains were utilized in lager beer production, and the resulting beverage exhibited significantly altered flavour profiles, thereby greatly expanding the possibilities of the flavour palette of lager beers.

Comparison of the Fatty Acid Profiles of Sow and Goat Colostrum.

Currently, the utilization of hyperprolific sows has stimulated the search for supplements aimed at enhancing piglet survival, as these sows yield more offspring than they can adequately feed with their colostrum production. In contrast, intensive goat farming often yields surplus colostrum, thus necessitating its removal, since kids are exclusively fed colostrum through lactation solely within the initial day of birth. The objective of this study was to examine and compare the fatty acid (FA) profiles of colostrum from sows and goats, together with possible influencing factors such as sow parity and the postpartum day of the goat, for possible use as an energy supplement for neonatal piglets. Swine colostrum was collected from sows with a 0-5 parity. In addition, samples of goat colostrum were collected on their first (D1) and second (D2) days of postpartum milking. The FA profiles of the colostrum were analyzed via gas chromatography. The parity value of the sows did not affect (p > 0.05) the FA colostrum composition. High proportions of palmitic, oleic, and linoleic acids were found in both types of colostrum. Levels of palmitic, oleic, and linoleic acids were significantly higher in D1 goat colostrum, whereas saturated FAs of less than 14 carbons (4:0, 6:0, 8:0, 10:0, and 12:0) were found in higher proportions in D2. These FAs play an important role in colostrum as they are a readily available source of energy and have also been attributed strong antibacterial activity. Therefore, goat colostrum, especially D2, could be used as an alternative energy supplement for newborn piglets, in particular for the weakest and smallest of the litter, which are the most in need.

Advancements in medium chain fatty acids production through chain elongation: Key mechanisms and innovative solutions for overcoming rate-limiting steps.

The depletion of fossil fuels has prompted an urgent search for alternative chemicals from renewable sources. Current technology in medium chain fatty acids (MCFAs) production though chain elongation (CE) is becoming increasingly sustainable, hence the motivation for this review, which provides the detailed description, insights and analysis of the metabolic pathways, substrates type, inoculum and fermentation process. The main rate-limiting steps of microbial MCFAs production were comprehensively revealed and the corresponding innovative solutions were also critically evaluated. Innovative strategies such as substrate pretreatment, electrochemical regulation, product separation, fermentation parameter optimization, and electroactive additives have shown significant advantages in overcoming the rate-limiting steps. Furthermore, novel regulatory strategies such as quorum sensing and electronic bifurcation are expected to further increase the MCFAs yield. Finally, the techno-economic analysis was carried out, and the future research focuses were also put forward.

Dietary medium-chain fatty acids reduce hepatic fat accumulation via activation of a CREBH-FGF21 axis.

OBJECTIVE: Dietary medium-chain fatty acids (MCFAs), characterized by chain lengths of 8-12 carbon atoms, have been proposed to have beneficial effects on glucose and lipid metabolism, yet the underlying mechanisms remain elusive. We hypothesized that MCFA intake benefits metabolic health by inducing the release of hormone-like factors. METHODS: The effects of chow diet, high-fat diet rich in long-chain fatty acids (LCFA HFD) fed ad libitum or pair-fed to a high-fat diet rich in MCFA (MCFA HFD) on glycemia, hepatic gene expression, circulating fibroblast growth factor 21 (FGF21), and liver fat content in both wildtype and Fgf21 knockout mice were investigated. The impact of a single oral dose of an MCFA-rich oil on circulating FGF21 and hepatic Fgf21 mRNA expression was assessed. In flag-tagged Crebh knockin mice and liver-specific Crebh knockout mice, fed LCFA HFD or MCFA HFD, active hepatic CREBH and hepatic Fgf21 mRNA abundance were determined, respectively. RESULTS: MCFA HFD improves glucose tolerance, enhances glucose clearance into brown adipose tissue, and prevents high-fat diet-induced hepatic steatosis in wildtype mice. These benefits are associated with increased liver expression of CREBH target genes (Apoa4 and Apoc2), including Fgf21. Both acute and chronic intake of dietary MCFAs elevate circulating FGF21. Augmented hepatic Fgf21 mRNA following MCFA HFD intake is accompanied by higher levels of active hepatic CREBH; and MCFA-induced hepatic Fgf21 expression is blocked in mice lacking Crebh. Notably, while feeding male and female Fgf21 wildtype mice MCFA HFD results in reduced liver triacylglycerol (TG) levels, this liver TG-lowering effect is blunted in Fgf21 knockout mice fed MCFA HFD. The reduction in liver TG levels observed with MCFA HFD was independent of weight loss. CONCLUSIONS: Dietary MCFAs reduce liver fat accumulation via activation of a CREBH-FGF21 signaling axis.

Differential Effects of Three Medium-Chain Fatty Acids on Mitochondrial Quality Control and Skeletal Muscle Maturation.

Nutritional interventions are one focus of sarcopenia treatment. As medium-chain fatty acids (MCFAs) are oxidized in the mitochondria and produce energy through oxidative phosphorylation (OXPHOS), they are key parts of nutritional interventions. We investigated the in vitro effects of three types of MCFA, caprylic acid (C8), capric acid (C10), and lauric acid (C12), in skeletal muscle cells. Compared with C10 and C12, C8 promoted mitophagy through the phosphatase and tensin homolog (PTEN)-induced kinase 1-Parkin pathway and increased the expression of peroxisome proliferator-activated receptor gamma coactivator 1-α and dynamin-related protein 1 to reduce mitochondrial oxidative stress and promote OXPHOS. Furthermore, the expression of myogenic differentiation 1 and myosin heavy chain increased in myotubes, thus promoting muscle differentiation and maturation. These results suggest that C8 improves mitochondrial quality and promotes skeletal muscle maturation; in contrast, C10 and C12 poorly promoted mitochondrial quality control and oxidative stress and suppressed energy production. Future animal experiments are required to establish the usefulness of C8 for nutritional interventions for sarcopenia.

The Undeniable Potential of Thermophiles in Industrial Processes.

Extremophilic microorganisms play a key role in understanding how life on Earth originated and evolved over centuries. Their ability to thrive in harsh environments relies on a plethora of mechanisms developed to survive at extreme temperatures, pressures, salinity, and pH values. From a biotechnological point of view, thermophiles are considered a robust tool for synthetic biology as well as a reliable starting material for the development of sustainable bioprocesses. This review discusses the current progress in the biomanufacturing of high-added bioproducts from thermophilic microorganisms and their industrial applications.

Serum Medium-Chain Fatty Acids and the Risk of Incident Diabetes: Findings from the 4C Study.

CONTEXT: Emerging studies have revealed associations between dietary medium-chain fatty acids (MCFAs) and glucose homeostasis. However, the relationship between serum MCFAs and the incidence of diabetes, and potential interactions with genetic predisposition, remains unclear in prospective cohort studies. OBJECTIVE: To investigate associations and genetic susceptibility between serum MCFAs and diabetes risk. METHODS: We investigated baseline serum MCFAs (n=5) in a nested case-control study comprising incident diabetes cases (n=1,707) and matched normoglycemic control subjects (n=1,707) from the China Cardiometabolic Disease and Cancer Cohort Study. Associations between MCFAs and type 2 diabetes mellitus (T2DM) were examined, both overall and stratified by diabetes genetic susceptibility. Genetic risk scores (GRS) were calculated based on 86 T2DM-associated genetic variants. RESULTS: In the fully adjusted conditional logistic regression model, serum octanoic acid and nonanoic acid exhibited inverse dose-response relationships with diabetes risk, showing odds ratios (95% confidence intervals) of 0.90 (0.82-0.98) and 0.84 (0.74-0.95), respectively. Subgroup analysis demonstrated that inverse associations between MCFAs and incident diabetes were more pronounced among individuals with physical inactivity (Pinteraction = 0.042, 0.034, and 0.037, for octanoic, nonanoic and decanoic acid, respectively). Moreover, inverse associations of octanoic acid with diabetes risk were notably enhanced among individuals with high genetic risk compared to those with low genetic risk. Significant interactions were observed between octanoic acid and GRS on T2DM risk (Pinteraction = 0.003). CONCLUSIONS: These findings provide evidence supporting inverse associations between serum MCFAs and T2DM risk, and reveal potential interplay between genetic susceptibility and circulating octanoic acid in modulating diabetes risk.

A Novel Sustainable and Self-Sufficient Biotechnological Strategy for Directly Transforming Sewage Sludge into High-Value Liquid Biochemicals.

Sewage sludge, as a carbon-rich byproduct of wastewater treatment, holds significant untapped potential as a renewable resource. Upcycling this troublesome waste stream represents great promise in addressing global escalating energy demands through its wide practice of biochemical recovery concurrently. Here, we propose a biotechnological concept to gain value-added liquid bioproducts from sewage sludge in a self-sufficient manner by directly transforming sludge into medium-chain fatty acids (MCFAs). Our findings suggest that yeast, a cheap and readily available commercial powder, would involve ethanol-type fermentation in chain elongation to achieve abundant MCFA production from sewage sludge using electron donors (i.e., ethanol) and acceptors (i.e., short-chain fatty acids) produced in situ. The enhanced abundance and transcriptional activity of genes related to key enzymes, such as butyryl-CoA dehydrogenase and alcohol dehydrogenase, affirm the robust capacity for the self-sustained production of MCFAs. This is indicative of an effective metabolic network established between yeast and anaerobic microorganisms within this innovative sludge fermentation framework. Furthermore, life cycle assessment and techno-economic analysis evidence the sustainability and economic competitiveness of this biotechnological strategy. Overall, this work provides insights into sewage sludge upgrading independent of additional carbon input, which can be applied in existing anaerobic sludge fermentation infrastructure as well as to develop new applications in a diverse range of industries.

Perspectives on carboxylates generation from Ecuadorian agro-wastes.

Carboxylates generation from banana (peel and pulp), coffee, and cacao fermentation agro-waste, upon uncontrolled and controlled pHs of 6.6 (heat-driven methanogens inactivation) and 5.2 (pH inactivation), was studied. Regarding volatile fatty acids (VFAs), acetic was the highest for cocoa (96.2 g kg-1TVS) at pH 4.5. However, butyric was relevant for banana pulp (90.7 g kg-1TVS), at controlled pH 6.6. The highest medium chain fatty acid (MCFAs) level was hexanoic (cocoa, 3.5 g kg-1TVS), while octanoic reached a maximum of 2.8 g kg-1TVS for coffee at pH 6.6. At pH 5.2 MCFAs yield was relatively low. Uncontrolled pH conditions, using banana resulted in superior VFAs production compared to controlled conditions. Thus, pH became a determining variable when deciding the time and kind of carboxylic acid to be recovered. The bacterial community at the end of the chain elongation process was dominated by phyla Firmicutes, and Clostridium as the most common genera.

Octanoic acid mitigates busulfan-induced blood-testis barrier damage by alleviating oxidative stress and autophagy.

BACKGROUND: The management of male infertility continues to encounter an array of challenges and constraints, necessitating an in-depth exploration of novel therapeutic targets to enhance its efficacy. As an eight-carbon medium-chain fatty acid, octanoic acid (OCA) shows promise for improving health, yet its impact on spermatogenesis remains inadequately researched. METHODS: Mass spectrometry was performed to determine the fatty acid content and screen for a pivotal lipid component in the serum of patients with severe spermatogenesis disorders. The sperm quality was examined, and histopathological analysis and biotin tracer tests were performed to assess spermatogenesis function and the integrity of the blood-testis barrier (BTB) in vivo. Cell-based in vitro experiments were carried out to investigate the effects of OCA administration on Sertoli cell dysfunction. This research aimed to elucidate the mechanism by which OCA may influence the function of Sertoli cells. RESULTS: A pronounced reduction in OCA content was observed in the serum of patients with severe spermatogenesis disorders, indicating that OCA deficiency is related to spermatogenic disorders. The protective effect of OCA on reproduction was tested in a mouse model of spermatogenic disorder induced by busulfan at a dose 30 mg/kg body weight (BW). The mice in the study were separated into distinct groups and administered varying amounts of OCA, specifically at doses of 32, 64, 128, and 256 mg/kg BW. After evaluating sperm parameters, the most effective dose was determined to be 32 mg/kg BW. In vivo experiments showed that treatment with OCA significantly improved sperm quality, testicular histopathology and BTB integrity, which were damaged by busulfan. Moreover, OCA intervention reduced busulfan-induced oxidative stress and autophagy in mouse testes. In vitro, OCA pretreatment (100 µM) significantly ameliorated Sertoli cell dysfunction by alleviating busulfan (800 µM)-induced oxidative stress and autophagy. Moreover, rapamycin (5 µM)-induced autophagy led to Sertoli cell barrier dysfunction, while OCA administration exerted a protective effect by alleviating autophagy. CONCLUSIONS: This study demonstrated that OCA administration suppressed oxidative stress and autophagy to alleviate busulfan-induced BTB damage. These findings provide a deeper understanding of the toxicology of busulfan and a promising avenue for the development of novel OCA-based therapies for male infertility.

Simplification of Dietary Treatment in Pharmacoresistant Epilepsy: Impact of C8 and C10 Fatty Acids on Sirtuins of Neuronal Cells In Vitro.

Pharmacotherapy is the therapeutic mainstay in epilepsy; however, in about 30% of patients, epileptic seizures are drug-resistant. A ketogenic diet (KD) is an alternative therapeutic option. The mechanisms underlying the anti-seizure effect of a KD are not fully understood. Epileptic seizures lead to an increased energy demand of neurons. An improvement in energy provisions may have a protective effect. C8 and C10 fatty acids have been previously shown to activate mitochondrial function in vitro. This could involve sirtuins (SIRTs) as regulatory elements of energy metabolism. The aim of the present study was to investigate whether ß-hydroxybutyrate (ßHB), C8 fatty acids, C10 fatty acids, or a combination of C8 and C10 (250/250 µM) fatty acids, which all increase under a KD, could up-regulate SIRT1, -3, -4, and -5 in HT22 hippocampal murine neurons in vitro. Cells were incubated for 1 week in the presence of these metabolites. The sirtuins were measured at the enzyme (fluorometrically), protein (Western blot), and gene expression (PCR) levels. In hippocampal cells, the C8, C10, and C8 and C10 incubations led to increases in the sirtuin levels, which were not inferior to a ßHB incubation as the 'gold standard'. This may indicate that both C8 and C10 fatty acids are important for the antiepileptic effect of a KD. A KD may be replaced by nutritional supplements of C8 and C10 fatty acids, which could facilitate the diet.

Feedback of chain elongation microorganisms on iron-based conductive materials: Enhanced microbial functions and biotoxicity adaptation mechanisms.

Despite the increased research efforts aimed at understanding iron-based conductive materials (CMs) for facilitating chain elongation (CE) to produce medium chain fatty acids (MCFAs), the impact of these materials on microbial community functions and the adaptation mechanisms to their biotoxicity remain unclear. This study found that the supply of zero-valent iron (ZVI) and magnetite enhanced the MCFAs carbon-flow distribution by 26 % and 52 %, respectively. Metagenomic analysis revealed the upregulation of fatty acid metabolism, pyruvate metabolism and ABC transporters with ZVI and magnetite. The predominant functional microorganisms were Massilibacterium and Tidjanibacter with ZVI, and were Petrimonas and Candidatus_Microthrix with magnetite. Furthermore, it was demonstrated that CE microorganisms respond and adapt to the biotoxicity of iron-based CMs by adjusting Two-component system and Quorum sensing for the first time. In summary, this study provided a new deep-insight on the feedback mechanisms of CE microorganisms on iron-based CMs.

Synergistic antifungal effects of the preservative ammonium propionate and medium chain fatty acids against dormant and germinating conidia, germ tubes and hyphae of Aspergillus chevalieri, a feed spoilage fungus.

In feed, propionic acid is the weak organic acid of choice to prevent growth of spoilage fungi. For safe and easy industrial handling this antifungal agent is applied in the presence of neutralizing ammonium, which however has the disadvantage to negatively affect the efficacy of fungus-inhibiting properties of the formulation. In the present study we investigated the impact of medium chain fatty acids (MCFA) on the antifungal efficacy of an ammonium propionate formulation on dormant- and germinating conidia as well as germ tubes and hyphae of Aspergillus chevalieri, a xerophilic fungus predominant on moulded feed. Dormant conidia were not affected by 32 mM of ammonium propionate after a 28 h-treatment in demi water. Similar results were obtained with solely 0.52 mM MCFA. However, the combination of both components nearly eradicated formation of colonies from these conidia and was accompanied by distortion of the cellular structure as was visible with light- and transmission electron microscopy. Germination of conidia, characterised by swelling and germ tube formation, was significantly decreased in the presence of 16 mM ammonium propionate and 0.26 mM MCFA, while the latter component itself did not significantly decrease germination. We conclude that a combination of ammonium propionate and MCFA had a synergistic antifungal effect on dormant and germinating conidia. When the combination of ammonium propionate and MCFA was tested on hyphae for 30 min, we observed that cell death was significantly increased in comparison to components alone. Treatment of the hyphae with 16 mM of ammonium propionate caused aberrant mitochondria, as evidenced by irregularly shaped and enlarged mitochondria that contained electron-dense inclusions as observed by transmission electron microscopy. When the combination of ammonium propionate and MCFA was applied against the hyphae, more severe cell damage was observed, with signs of autophagy. Summarised, our results demonstrate synergistic antifungal effects of ammonium propionate and medium chain fatty acids on fungal survival structures, during their germination and after a short (sudden) treatment of growing cells. This is of potential importance for several areas of feed and food storage and shelf-life.

Deciphering the reduction of antibiotic resistance genes (ARGs) during medium-chain fatty acids production from waste activated sludge: Driven by inhibition of ARGs transmission and shift of microbial community.

Medium-chain fatty acids (MCFAs) production from waste activated sludge (WAS) by chain extension (CE) is a promising technology. However, the effects and mechanisms of CE process on the fate of antibiotic resistance genes (ARGs) remain unclear. In this study, the results showed that the removal efficiency of ARGs was 81.15 % in CE process, suggesting its efficacy in reducing environmental risks. Further, the observed decrease in mobile genetic elements (MGEs) indicated that CE process restricted the horizontal gene transfer (HGT). Complementing this, the increase in soluble organic matters and extracellular 16 S rDNA confirmed that MCFAs production caused bacterial damage. Decreased intracellular ARGs and increased extracellular ARGs further revealed that MCFAs production impaired ARGs hosts, thereby limiting the vertical gene transfer (VGT) of ARGs. Shift of microbial community combined with co-occurrence network analysis demonstrated that functional bacteria without host potential for ARGs were enriched, but potential ARGs and MGEs hosts decreased, showing the role of functional bacterial phylogeny and selection pressure of MCFAs in reducing ARGs. Finally, partial least squares path model was used to systematic verify the mechanism of ARGs removal in CE process, which was attributed to the inhibition of ARGs transmission (HGT and VGT) and shift of microbial community.