TOYOCERIN - Word Related Documents




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367000.8363Quantification of tetracycline and chloramphenicol resistance in digestive tracts of bulls and piglets fed with Toyocerin®, a feed additive containing Bacillus toyonensis spores. The complete genome sequencing of Bacillus toyonensis, the active ingredient of the feed additive Toyocerin(®), has revealed the presence of tetM and cat genes, a tetracycline and a chloramphenicol resistance gene, respectively. The aim of this study was to determine whether the use of Toyocerin(®) (viable spores of B. toyonensis) as a probiotic in feedstuff increased the abundance of tetracycline and chloramphenicol resistant bacteria in the intestinal tracts of piglets and Holstein bulls. To this end, qPCRs were designed to quantify the abundances of tetM and cat genes and B. toyonensis in the intestinal content of animals treated and non-treated with Toyocerin(®). Additionally, the culturable bacterial populations resistant to tetracycline or chloramphenicol were enumerated by plate counting. No statistical significances were detected between the concentrations of tetracycline or chloramphenicol resistant bacterial populations in treated and non-treated animals. The concentrations of tetM and cat in most of the treated animals were similar to those of B. toyonensis. Furthermore, tetM and cat genes were also detected in some non-treated animals, although in low concentrations. These results suggest that tetM and cat genes are already circulating among the commensal microbiota regardless of the use of Toyocerin(®). The use of Toyocerin(®) as a supplement in feedstuff does not increase the abundances of tetracycline and chloramphenicol resistant bacteria in the intestinal tracts of piglets and Holstein bulls beyond the contribution directly associated to the introduction of B. toyonensis spores through diet.201425085518
601010.8219The role of two families of bacterial enzymes in putrescine synthesis from agmatine via agmatine deiminase. Putrescine, one of the main biogenic amines associated to microbial food spoilage, can be formed by bacteria from arginine via ornithine decarboxylase (ODC), or from agmatine via agmatine deiminase (AgDI). This study aims to correlate putrescine production from agmatine to the pathway involving N-carbamoylputrescine formation via AdDI (the aguA product) and N-carbamoylputrescine amidohydrolase (the aguB product), or putrescine carbamoyltransferase (the ptcA product) in bacteria. PCR methods were developed to detect the two genes involved in putrescine production from agmatine. Putrescine production from agmatine could be linked to the aguA and ptcA genes in Lactobacillus hilgardii X1B, Enterococcus faecalis ATCC 11700, and Bacillus cereus ATCC 14579. By contrast Lactobacillus sakei 23K was unable to produce putrescine, and although a fragment of DNA corresponding to the gene aguA was amplified, no amplification was observed for the ptcA gene. Pseudomonas aeruginosa PAO1 produces putrescine and is reported to harbour aguA and aguB genes, responsible for agmatine deiminase and N-carbamoylputrescine amidohydrolase activities. The enzyme from P. aeruginosa PAO1 that converts N-carbamoylputrescine to putrescine (the aguB product) is different from other microorganisms studied (the ptcA product). Therefore, the aguB gene from P. aeruginosa PAO1 could not be amplified with ptcA-specific primers. The aguB and ptcA genes have frequently been erroneously annotated in the past, as in fact these two enzymes are neither homologous nor analogous. Furthermore, the aguA, aguB and ptcA sequences available from GenBank were subjected to phylogenetic analysis, revealing that gram-positive bacteria harboured ptcA, whereas gram-negative bacteria harbour aguB. This paper also discusses the role of the agmatine deiminase system (AgDS) in acid stress resistance.201021404211
873520.8199The Effect of Ice-Nucleation-Active Bacteria on Metabolic Regulation in Evergestis extimalis (Scopoli) (Lepidoptera: Pyralidae) Overwintering Larvae on the Qinghai-Tibet Plateau. Evergestis extimalis (Scopoli) is a significant pest of spring oilseed rape in the Qinghai-Tibet Plateau. It has developed resistance to many commonly used insecticides. Therefore, biopesticides should be used to replace the chemical pesticides in pest control. In this study, the effects of ice-nucleation-active (INA) microbes (Pseudomonas syringae 1.7277, P. syringae 1.3200, and Erwinia pyrifoliae 1.3333) on E. extimalis were evaluated. The supercooling points (SCP) were markedly increased due to the INA bacteria application when they were compared to those of the untreated samples. Specifically, the SCP of E. extimalis after its exposure to a high concentration of INA bacteria in February were -10.72 °C, -13.73 °C, and -14.04 °C. Our findings have demonstrated that the trehalase (Tre) genes were up-regulated by the application of the INA bacteria, thereby resulting in an increased trehalase activity. Overall, the INA bacteria could act as effective heterogeneous ice nuclei which could lower the hardiness of E. extimalis to the cold and then freeze them to death in an extremely cold winter. Therefore, the control of insect pests with INA bacteria goes without doubt, in theory.202236292857
813030.8187Garden fruit chafer (Pachnoda sinuata L.) accelerates recycling and bioremediation of animal waste. Bioconversion of livestock wastes using insect larvae represents an emerging and effective strategy for waste management. However, knowledge on the role of the garden fruit chafer (Pachnoda sinuataL.) in waste recycling and influence on the diversity ofmicrobial community infrass fertilizeris limited. Here, we determined whether and to what extent the conversion of cattle dung into insect frass fertilizer byP. sinuatainfluences the frass' microbial community and its associated antibiotic resistance genes abundance. Pachnoda sinuata larvae were used to valorise cattle dung into frass fertilizer; samples were collected weekly to determine the composition of bacteria and fungi, and antibiotic resistant genes using molecular tools. Results revealed that bioconversion of cattle dung byP. sinuatalarvae significantly increased the richness of beneficial bacteria in the frass fertilizer by 2.5-folds within 28 days, but fungal richness did not vary during the study. Treatment of cattle dung withP. sinuatalarvae caused 2 - 3-folds decrease in the genes conferring resistance to commonly used antibiotics such as aminoglycoside, diaminopyrimidine, multidrug, sulfonamide and tetracycline within 14 days. Furthermore, the recycling cattle dung using considerably reduced the abundance of mobile genetic elements known to play critical roles in the horizontal transfer of antibiotic resistance genes between organisms. This studyhighlights the efficiency ofsaprohytic insects in recycling animal manure and suppressing manure-borne pathogens in the organic fertilizer products, opening new market opportunities for innovative and safe bio-based products and achieving efficient resource utilization in a circular and green economy.202437989012
811740.8186Composting of oxytetracycline fermentation residue in combination with hydrothermal pretreatment for reducing antibiotic resistance genes enrichment. Hydrothermal pretreatment can efficiently remove the residual antibiotics in oxytetracycline fermentation residue (OFR), but its effect on antibiotic resistance genes (ARGs) during composting remains unclear. This study compared the shifts in bacterial community and evolutions in ARGs and integrons during different composting processes of OFRs with and without hydrothermal pretreatment. The results demonstrated that hydrothermal pretreatment increased the bacterial alpha diversity at the initial phase, and increased the relative abundances of Proteobacteria and Actinobacteria but decreased that of Bacteroidetes at the final phase by inactivating mycelia and removing residual oxytetracycline. Composting process inevitably elevated the abundance and relative abundance of ARGs. However, the increase in ARGs was significantly reduced by hydrothermal pretreatment, because the removal of oxytetracycline decreased their potential host bacteria and inhibited their horizontal gene transfer. The results demonstrated that hydrothermal pretreatment is an efficient strategy to reduce the enrichment of ARGs during the OFR composting.202033099099
798550.8186Differential response of nonadapted ammonia-oxidising archaea and bacteria to drying-rewetting stress. Climate change is expected to increase the frequency of severe drought events followed by heavy rainfall, which will influence growth and activity of soil microorganisms, through osmotic stress and changes in nutrient concentration. There is evidence of rapid recovery of processes and adaptation of communities in soils regularly experiencing drying/rewetting and lower resistance and resilience in nonadapted soils. A microcosm-based study of ammonia-oxidising archaea (AOA) and bacteria (AOB), employing a grassland soil that rarely experiences drought, was used to test this hypothesis and also whether AOB were more resistant and resilient, through greater tolerance of high ammonia concentrations produced during drought and rewetting. Treated soils were dried, incubated for 3 weeks, rewetted, incubated for a further 3 weeks and compared to untreated soils, maintained at a constant moisture content. Nitrate accumulation and AOA and AOB abundance (abundance of respective amoA genes) and community composition (DGGE analysis of AOA amoA and AOB 16S rRNA genes) were poorly adapted to drying-rewetting. AOA abundance and community composition were less resistant than AOB during drought and less resilient after rewetting, at times when ammonium concentration was higher. Data provide evidence for poor adaptation of microbial communities and processes to drying-rewetting in soils with no history of drought and indicate niche differentiation of AOA and AOB associated with high ammonia concentration.201425070168
753560.8184The effects of pig manure application on the spread of tetracycline resistance in bulk and cucumber rhizosphere soils: a greenhouse experiment. It is important to understand the dynamics of tetracycline-resistant bacteria (TRB) and tetracycline resistance genes (TRGs) in bulk and rhizosphere soils for evaluating the spread of TRGs from pig manure to human. In this work, a greenhouse experiment was conducted to investigate the difference in abundance of TRB, tetracycline-resistant Escherichia coli (TRE), tetracycline-resistant Pseudomonas spp. (TRP), and TRGs between bulk and cucumber rhizosphere soils. The application of pig manure resulted in the long-term persistence of TRB, TRE, TRP, and TRGs in bulk soil and rhizosphere of cucumber for at least 65 days. Pig manure application dose was the major driving force in altering the abundances of TRB and TRE, whereas TRP was disturbed mainly by compartment (bulk soil or rhizosphere). Both TRE and the percentage of TRE in bulk and rhizosphere soils increased linearly with an increase in dose of pig manure. The exponential relationships between pig manure dose and TRP along with TRP percentage were also noted. There were significant differences in the relative abundances of TRGs between bulk and cucumber rhizosphere soils, suggesting the use of pig manure exerted a more lasting impact on the spread of TRGs in the rhizosphere than in the bulk soil.201728222270
605270.8180Safety and technological application of autochthonous Streptococcus thermophilus cultures in the buffalo Mozzarella cheese. Thermophilic and mesophilic lactic acid bacteria (LAB), such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus, and Lactococcus lactis, play a crucial role in the technological and sensory quality of Mozzarella cheese. In this study, the safety (genes encoding virulence factors and antibiotic resistance) and acidifying activity of autochthonous S. thermophilus cultures were evaluated in order to choose the most suitable strain for industrial application. The safe and good acidifying culture was tested in two buffalo Mozzarella cheese batches: Mozzarella cheeses produced with autochthonous culture (SJRP107) and commercial culture (STM5). The cultivable LAB was evaluated by culture-dependent method (plate counting) and the quantification of S. thermophilus cultures (commercial and autochthonous) were evaluated by culture-independent method RealT-qPCR (real-time quantitative polymerase chain reaction). The texture, physicochemical and proteolytic properties of the Mozzarella cheeses were similar for both batches. The nonstarter LAB count was higher during manufacture than in the storage, and the RealT-qPCR indicated the presence of S. thermophilus culture until the end of storage. S. thermophilus SJRP107 presented high potential for safety application in the production of Mozzarella cheese. Furthermore, considering the culture characteristics and their relationship with product quality, further studies could be helpful to determine their effect on the sensory characteristics of the cheese.202031948624
794680.8179New Insights into the Microbial Diversity of Cake Layer in Yttria Composite Ceramic Tubular Membrane in an Anaerobic Membrane Bioreactor (AnMBR). Cake layer formation is an inevitable challenge in membrane bioreactor (MBR) operation. The investigations on the cake layer microbial community are essential to control biofouling. This work studied the bacterial and archaeal communities in the cake layer, the anaerobic sludge, and the membrane cleaning solutions of anaerobic membrane bioreactor (AnMBR) with yttria-based ceramic tubular membrane by polymerase chain reaction (PCR) amplification of 16S rRNA genes. The cake layer resistance was 69% of the total membrane resistance. Proteins and soluble microbial by-products (SMPs) were the dominant foulants in the cake layer. The pioneering archaeal and bacteria in the cake layer were mostly similar to those in the anaerobic bulk sludge. The dominant biofouling bacteria were Proteobacteria, Bacteroidetes, Firmicutes, and Chloroflexi and the dominant archaeal were Methanosaetacea and Methanobacteriacea at family level. This finding may help to develop antifouling membranes for AnMBR treating domestic wastewater.202133546268
753890.8177Short-term thermophilic treatment cannot remove tetracycline resistance genes in pig manures but exhibits controlling effects on their accumulation and spread in soil. In this work, a microcosm experiment was conducted to merely mimic thermophilic phase in aerobic composting with pig manures in order to explore: (i) the effect of thermophilic phase in composting on the abundances of tetracycline resistance genes (TRGs); and (ii) the impacts of the treated manures on the abundances of TRGs in soil. It was found that 4days of thermophilic process reduced the abundance of TRGs in pig manures by ∼1 lg unit compared to the samples without treatments, suggesting that other phases in composting may play significant roles in removal of TRGs. Once pig manures with thermophilic treatment were applied to soil, TRGs abundances decreased to the levels in unfertilized soil. With correlation analyses, it was concluded that pig manure derived tetracycline-resistant bacteria (TRB) and nutrients exerted different effects on TRGs abundances in soil. In conclusion, short-term thermophilic treatment cannot remove tetracycline resistance genes in pig manures but exhibits controlling effects on their accumulation and spread in soil. Nutrients enrichment in soil following manuring of treated pig manures, together with a large proportion of gram-positive TRB left in treated pig manures with less risk to TRGs spread, contributed to the controlling effects.201728715744
8120100.8176Insight into the fate of antibiotic resistance genes and bacterial community in co-composting green tea residues with swine manure. Green tea residues (GTRs) are byproducts of tea production and processing, and this type of agricultural waste retains nutritious components. This study investigated the co-composting of GTRs with swine manure, as well as the effects of GTRs on antibiotic resistance genes (ARGs) and the bacterial community during co-composting. The temperature and C/N ratio indicate compost was mature after processing. The addition of GTRs effectively promoted the reduction in the abundances of most targeted ARGs (tet and sul genes), mobile genetic element (MGE; intI1), and metal resistance genes (MRGs; pcoA and tcrB). Redundancy analysis (RDA) showed that GTRs can reduce the abundance of MRGs and ARGs by reducing the bioavailability of heavy metals. Network analysis shows that Firmicutes and Actinobacteria were the main hosts of ARGs and ARGs, MGEs, and MRGs shared the same potential host bacteria. Adding GTRs during composting may reduce ARGs transmission through horizontal gene transfer (HGT). GTRs affected the bacterial community, thereby influencing the variations in the ARG profiles and reducing the potential risk associated with the compost product.202032310121
7884110.8173Underlying the inhibition mechanisms of sulfate and lincomycin on long-term anaerobic digestion: Microbial response and antibiotic resistance genes distribution. This study evaluated the resilience of a long-term anaerobic treatment system exposed to sulfate, lincomycin (LCM) and their combined stress. LCM was found to impede anaerobic propionate degradation, while sulfate for restraining methanogenic acetate utilization. The combined stress, with influent LCM of 200 mg/L and sulfate of 1404 mg/L, revealed severer inhibition on anaerobic digestion than individual inhibition, leading to 73.9 % and 38.5 % decrease in methane production and sulfate removal, respectively. Suppression on propionate-oxidizing bacteria like unclassified_f__Anaerolineae and unclassified_f__Syntrophaceae further demonstrated LCM's inhibitory effect on propionate degradation. Besides, the down-regulation of genes encoding dissimilatory sulfate reduction enzymes caused by LCM triggered great inhibition on sulfate reduction. A notable increase in ARGs was detected under sulfate-stressed condition, owing to its obvious enrichment of tetracycline-resistant genes. Genera including unclassified_f__Syntrophaceae, unclassified_f__Geobacteraceae and unclassified_f__Anaerolineaceae were identified as dominant host of ARGs and enriched by sulfate addition. Overall, these results could provide the theoretical basis for further enhancement on anaerobic digestion of pharmaceutical wastewater containing sulfate and lincomycin.202438185146
8112120.8172Fate of antibiotic resistance bacteria and genes during enhanced anaerobic digestion of sewage sludge by microwave pretreatment. The fate of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) were investigated during the sludge anaerobic digestion (AD) with microwave-acid (MW-H), microwave (MW) and microwave-H2O2-alkaline (MW-H2O2) pretreatments. Results showed that combined MW pretreatment especially for the MW-H pretreatment could efficiently reduce the ARB concentration, and most ARG concentrations tended to attenuate during the pretreatment. The subsequent AD showed evident removal of the ARB, but most ARGs were enriched after AD. Only the concentration of tetX kept continuous declination during the whole sludge treatment. The total ARGs concentration showed significant correlation with 16S rRNA during the pretreatment and AD. Compared with unpretreated sludge, the AD of MW and MW-H2O2 pretreated sludge presented slightly better ARB and ARGs reduction efficiency.201626970692
7241130.8171In situ analysis of antibiotic resistance genes in anaerobically digested dairy manure and its subsequent disposal facilities. The metagenomic and quantitative polymerase chain reaction approaches were combined to evaluate the profiles of ARGs and plasmids in anaerobically digested dairy manure in situ and reveal the persistence and elevation of typical ARGs and plasmids in its subsequent disposal facilities in CAFOs, respectively. Our results indicated that the typical ARGs and plasimd were mainly sul2, mefa, tetm-01, tetm-02, tetw, aph3iiia, and clostridioides difficile strain 12,038 plasmid unnamed in CAFOs, some of which greatly enriched in AD residue after its storage, especially sul1 and sul2. Meantime, the AD slurry recycling introduced the bacteria carrying ARGs into soil, especially Romboutsia genus, which greatly enriched sul2, tetm-01, tetm-02, aphiiia, and mefa. In the present study, ARGs occurrence, persistence and distribution were understood through in situ analysis of their profiles during dairy manure AD treatment and subsequent disposals in CAFOs, which are helpful for controlling the potential environmental risks from dairy manure recycling.202133894444
8118140.8171Effects of biocontrol Bacillus and fermentation bacteria additions on the microbial community, functions and antibiotic resistance genes of prickly ash seed oil meal-biochar compost. This study evaluated the effects of biocontrol Bacillus and fermenting bacteria addition on the microbial community, metabolic functions and antibiotic resistance genes (ARGs) of new prickly ash seed oil meal (PSOM)-biochar composting. The results showed that the addition of Bacillus subtilis and fermentation bacteria significantly increased the NH(4)(+)-N, bacterial abundance and fungal diversity of compost while decreasing the relative abundances (RAs) of carbon metabolism genes in mature compost. NH(4)(+)-N was significantly correlated with microbial abundance and diversity, and its increase was closely related to microbial amino acid metabolism. The addition of biocontrol and fermenting bacteria changed the RAs of ARGs, which was caused by changes in the potential hosts Proteobacteria, Bacteroidota and Firmicutes in the compost. Consequently, adding Bacillus and fermenting bacteria into PSOM to make composting was suggested as an effective method to promote nutrient transformation, regulate microbial activity and decrease RAs of tetracycline and vancomycin ARGs.202134339999
8113150.8170Fate of antibiotic resistance genes in mesophilic and thermophilic anaerobic digestion of chemically enhanced primary treatment (CEPT) sludge. Anaerobic digestion (AD) of chemically enhanced primary treatment (CEPT) sludge and non-CEPT (conventional sedimentation) sludge were comparatively operated under mesophilic and thermophilic conditions. The highest methane yield (692.46±0.46mL CH(4)/g VS(removed) in CEPT sludge) was observed in mesophilic AD of CEPT sludge. Meanwhile, thermophilic conditions were more favorable for the removal of total antibiotic resistance genes (ARGs). In this study, no measurable difference in the fates and removal of ARGs and class 1 integrin-integrase gene (intI1) was observed between treated non-CEPT and CEPT sludge. However, redundancy analysis indicated that shifts in bacterial community were primarily accountable for the variations in ARGs and intI1. Network analysis further revealed potential host bacteria for ARGs and intI1.201728797965
7887160.8168Double-edged sword effects of sulfate reduction process in sulfur autotrophic denitrification system: Accelerating nitrogen removal and promoting antibiotic resistance genes spread. This study proposed the double-edged sword effects of sulfate reduction process on nitrogen removal and antibiotic resistance genes (ARGs) transmission in sulfur autotrophic denitrification system. Excitation-emission matrix-parallel factor analysis identified the protein-like fraction in soluble microbial products as main endogenous organic matter driving the sulfate reduction process. The resultant sulfide tended to serve as bacterial modulators, augmenting electron transfer processes and mitigating oxidative stress, thereby enhancing sulfur oxidizing bacteria (SOB) activity, rather than extra electron donors. The cooperation between SOB and heterotroph (sulfate reducing bacteria (SRB) and heterotrophic denitrification bacteria (HDB)) were responsible for advanced nitrogen removal, facilitated by multiple metabolic pathways including denitrification, sulfur oxidation, and sulfate reduction. However, SRB and HDB were potential ARGs hosts and assimilatory sulfate reduction pathway positively contributed to ARGs spread. Overall, the sulfate reduction process in sulfur autotrophic denitrification system boosted nitrogen removal process, but also increased the risk of ARGs transmission.202439122125
7537170.8168Swine-manure composts induce the enrichment of antibiotic-resistant bacteria but not antibiotic resistance genes in soils. Composting is a common and effective strategy for reducing antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) from animal manure. However, it is unclear whether the advantages of composting for the control of ARGs and ARB can be further increased in land application. This study investigated the fate of ARB and ARGs after land application of swine-manure composts (SMCs) to three different soil types (red soil, loess and black soil). The results showed that although the SMCs caused an increase in the abundance of total ARGs in the soil in the short period, they significantly reduced (p < 0.01) the abundance of total ARGs after 82 days compared to the control. The decay rate of ARGs reflected by the half-life times (t(1/2)) varied by soil type, with red soil being the longest. The SMCs mainly introduced ermF, tetG and tetX into the soils, while these ARGs quickly declined to the control level. Notably, SMCs increased the number of ARB in the soils, especially for cefotaxime-resistant bacteria. Although SMCs only affected the microbiome significantly during the early stage (p < 0.05), it took a much longer time for the microbiome to recover compared to the control. Statistical analysis indicated that changes in the microbial community contributed more to the fate of ARGs during SMCs land application than other factors. Overall, it is proposed that the advantages of ARGs control in the composting process for swine manure can be further increased in land application, but it can still bring some risks in regard to ARB.202337536132
8713180.8165Genomic Analysis of 18th-Century Kazakh Individuals and Their Oral Microbiome. The Asian Central Steppe, consisting of current-day Kazakhstan and Russia, has acted as a highway for major migrations throughout history. Therefore, describing the genetic composition of past populations in Central Asia holds value to understanding human mobility in this pivotal region. In this study, we analyse paleogenomic data generated from five humans from Kuygenzhar, Kazakhstan. These individuals date to the early to mid-18th century, shortly after the Kazakh Khanate was founded, a union of nomadic tribes of Mongol Golden Horde and Turkic origins. Genomic analysis identifies that these individuals are admixed with varying proportions of East Asian ancestry, indicating a recent admixture event from East Asia. The high amounts of DNA from the anaerobic Gram-negative bacteria Tannerella forsythia, a periodontal pathogen, recovered from their teeth suggest they may have suffered from periodontitis disease. Genomic analysis of this bacterium identified recently evolved virulence and glycosylation genes including the presence of antibiotic resistance genes predating the antibiotic era. This study provides an integrated analysis of individuals with a diet mostly based on meat (mainly horse and lamb), milk, and dairy products and their oral microbiome.202134943238
7649190.8164Pathogenic bacteria in biogas plants using cattle, swine, and poultry manure. Fugate, a waste product from biogas production, regularly used in agriculture as a fertiliser, may contain bacterial pathogens that cause zoonoses. Anaerobic digestion (AD) can inactivate viable pathogens, including parasites, viruses, and pathogens containing antibiotic resistance genes. This study aimed to compare the numbers of pathogenic bacteria and diversity of potential bacterial pathogens in the fugate using three different types of slurry: cattle, swine, and poultry manure. The swine fugate showed higher numbers of Clostridium perfringens and Campylobacter sp. than the poultry and cattle fugate. In the cattle fugate, the lowest total number of pathogenic bacteria and a low number of coliforms were detected after the AD. The use of cattle manure in biogas plants presents a lower potential for soil contamination with pathogens. The fugate produced using poultry or swine manure can be used carefully to avoid possibility of contamination of aquifers or surface waters. Also fugate produced from manure of cows suffering from chronic botulism can be used only with carefulness because of the presence of Clostridium botulinum spores in biogas waste of diseased cows.202540735305