Prophage Carriage and Genetic Diversity within Environmental Isolates of Clostridioides difficile.

Clostridioides difficile is an important human pathogen causing antibiotic-associated diarrhoea worldwide. Besides using antibiotics for treatment, the interest in bacteriophages as an alternative therapeutic option has increased. Prophage abundance and genetic diversity are well-documented in clinical strains, but the carriage of prophages in environmental strains of C. difficile has not yet been explored. Thus, the prevalence and genetic diversity of integrated prophages in the genomes of 166 environmental C. difficile isolates were identified. In addition, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems were determined in the genomes of prophage regions. Predicted prophages and CRISPR-Cas systems were identified by using the PHASTER web server and CRISPRCasFinder, respectively. Phylogenetic relationships among predicated prophages were also constructed based on phage-related genes, terminase large (TerL) subunits and LysM. Among 372 intact prophages, the predominant prophages were phiCDHM1, phiCDHM19, phiMMP01, phiCD506, phiCD27, phiCD211, phiMMP03, and phiC2, followed by phiMMP02, phiCDKM9, phiCD6356, phiCDKM15, and phiCD505. Two newly discovered siphoviruses, phiSM101- and phivB_CpeS-CP51-like Clostridium phages, were identified in two C. difficile genomes. Most prophages were found in sequence types (STs) ST11, ST3, ST8, ST109, and ST2, followed by ST6, ST17, ST4, ST5, ST44, and ST58. An obvious correlation was found between prophage types and STs/ribotypes. Most predicated prophages carry CRISPR arrays. Some prophages carry several gene products, such as accessory gene regulator (Agr), putative spore protease, and abortive infection (Abi) systems. This study shows that prophage carriage, along with genetic diversity and their CRISPR arrays, may play a role in the biology, lifestyle, and fitness of their host strains.

Growth Behavior of E. coli, Enterococcus and Staphylococcus Species in the Presence and Absence of Sub-inhibitory Antibiotic Concentrations: Consequences for Interpretation of Culture-Based Data.

Culture-based approaches are used to monitor, e.g., drinking water or bathing water quality and to investigate species diversity and antibiotic resistance levels in environmental samples. For health risk assessment, it is important to know whether the growing cultures display the actual abundance of, e.g., clinically relevant antibiotic resistance phenotypes such as vancomycin-resistant Enterococcus faecium/Enterococcus faecalis (VRE) or methicillin-resistant Staphylococcus aureus. In addition, it is important to know whether sub-inhibitory antibiotic concentrations, which are present in surface waters, favor the growth of antibiotic-resistant strains. Therefore, clinically relevant bacteria were isolated from different water sources and the growth behavior of 58 Escherichia coli, 71 Enterococcus, and 120 Staphylococcus isolates, belonging to different species and revealing different antibiotic resistance patterns, was studied with respect to "environmental" antibiotic concentrations. The finding that VRE could only be detected after specific enrichment can be explained by their slow growth compared to non-resistant strains. Interpreting their absence in standardized culture-based methods as nonexistent might be a fallacy. Sub-inhibitory antibiotic concentrations that were detected in sewage and receiving river water did not specifically promote antibiotic-resistant strains. Generally, those antibiotics that influenced cell metabolism directly led to slightly reduced growth rates and less than maximal optical densities after 48 h of incubation.

Concentration of facultative pathogenic bacteria and antibiotic resistance genes during sewage treatment and in receiving rivers.

Whereas the hygienic condition of drinking and bathing water by law must be monitored by culture-based methods, for quantification of microbes and antibiotic resistance in soil or the aquatic environment, often molecular genetic assays are used. For comparison of both methods, knowledge of their correlation is necessary. Therefore the population of total bacteria, Escherichia coli, enterococci and staphylococci during sewage treatment and in receiving river water was compared by agar plating and quantitative polymerase chain reaction (qPCR) assays. In parallel, all samples were investigated for clinically relevant antibiotic resistance genes. Whereas plating and qPCR data for total bacteria correlated well in sewage after primary treatment, qPCR data of river water indicated higher cell numbers for E. coli. It is unknown if these cells are 'only' not growing under standard conditions or if they are dead. Corresponding to the amount of non-culturable cells, the 'breakpoints' for monitoring water quality should be adapted. The abundances of clinically relevant antibiotic resistance genes in river water were in the same order of magnitude or even higher than in treated sewage. For estimation of the health risk it is important to investigate which species carry respective genes and whether these genes are disseminated via gene transfer.

Staphylococcus argensis sp. nov., a novel staphylococcal species isolated from an aquatic environment.

A staphylocoagulase-negative, novobiocin-susceptible strain (M4S-6T) of a species of the genus Staphylococcus was isolated from the river Argen in Southern Germany. It was assigned to the genus Staphylococcus due to the presence of the fatty acids, ai-C15 : 0, i-C15 : 0, i-C17 : 0, ai-C17 : 0, and of menaquinone (MK-7) in the cytoplasmic membrane, which are typical of coagulase-negative staphylococci. The polar lipid profile consisted of phosphatidylglycerol, diphosphatidylglycerol, an unknown phospholipid and an unknown glycolipid. Although the 16S gene sequence of strain M4S-6T revealed a 98% similarity with its closest relative, Staphylococcus pettenkoferi, it could be distinguished by several phenotypical and physiological markers. In contrast to S. pettenkoferi, M4S-6T was ornithine decarboxylase-positive, urease-negative and could use formiate and l-histidine as carbon-sources; nitrate was not reduced. Whereas S. pettenkoferi could grow with d(-)-mannitol, d-sorbitol, gluconic acid, l-proline, carboxymethylcellulose and lignosulfonate, M4S-6T was not able to grow with these substances. The results of 16S rRNA gene sequence analysis and of phenotypic testing indicated that M4S-6T was a representative of a novel species for which the name Staphylococcus argensis sp. nov., is proposed with the type strain M4S-6T (DSM 29875T = CIP 110904T).

Pilot-scale chitin extraction from shrimp shell waste by deproteination and decalcification with bacterial enrichment cultures.

Extraction of chitin from mechanically pre-purified shrimp shells can be achieved by successive NaOH/HCl treatment, protease/HCl treatment or by environmentally friendly fermentation with proteolytic/lactic acid bacteria (LAB). For the last mentioned alternative, scale-up of shrimp shell chitin purification was investigated in 0.25 L (F1), 10 L (F2), and 300 L (F3) fermenters using an anaerobic, chitinase-deficient, proteolytic enrichment culture from ground meat for deproteination and a mixed culture of LAB from bio-yoghurt for decalcification. Protein removal in F1, F2, and F3 proceeded in parallel within 40 h at an efficiency of 89-91 %. Between 85 and 90 % of the calcit was removed from the shells by LAB in another 40 h in F1, F2, and F3. After deproteination of shrimp shells in F3, spent fermentation liquor was re-used for a next batch of 30-kg shrimp shells in F4 (300 L) which eliminated 85.5 % protein. The purity of the resulting chitin was comparable in F1, F2, F3, and F4. Viscosities of chitosan, obtained after chitin deacetylation and of chitin, prepared biologically or chemically in the laboratory, were much higher than those of commercially available chitin and chitosan.

Sensitivity of antibiotic resistant and antibiotic susceptible Escherichia coli, Enterococcus and Staphylococcus strains against ozone.

Tolerance of antibiotic susceptible and antibiotic resistant Escherichia coli, Enterococcus and Staphylococcus strains from clinical and wastewater samples against ozone was tested to investigate if ozone, a strong oxidant applied for advanced wastewater treatment, will affect the release of antibiotic resistant bacteria into the aquatic environment. For this purpose, the resistance pattern against antibiotics of the mentioned isolates and their survival after exposure to 4 mg/L ozone was determined. Antibiotic resistance (AR) of the isolates was not correlating with higher tolerance against ozone. Except for ampicillin resistant E. coli strains, which showed a trend towards increased resistance, E. coli strains that were also resistant against cotrimoxazol, ciprofloxacin or a combination of the three antibiotics were similarly or less resistant against ozone than antibiotic sensitive strains. Pigment-producing Enterococcus casseliflavus and Staphylococcus aureus seemed to be more resistant against ozone than non-pigmented species of these genera. Furthermore, aggregation or biofilm formation apparently protected bacteria in subsurface layers from inactivation by ozone. The relatively large variance of tolerance against ozone may indicate that resistance to ozone inactivation most probably depends on several factors, where AR, if at all, does not play a major role.

Transformation of the matrix structure of shrimp shells during bacterial deproteination and demineralization.

BACKGROUND: After cellulose and starch, chitin is the third-most abundant biopolymer on earth. Chitin or its deacetylated derivative chitosan is a valuable product with a number of applications. It is one of the main components of shrimp shells, a waste product of the fish industry. To obtain chitin from Penaeus monodon, wet and dried shrimp shells were deproteinated with two specifically enriched proteolytic cultures M1 and M2 and decalcified by in-situ lactic acid forming microorganisms. The viscosity of biologically processed chitin was compared with chemically processed chitin. The former was further investigated for purity, structure and elemental composition by several microscopic techniques and (13)C solid state NMR spectroscopy. RESULTS: About 95% of the protein of wet shrimp shells was removed by proteolytic enrichment culture M2 in 68 h. Subsequent decalcification by lactic acid bacteria (LAB) took 48 h. Deproteination of the same amount of dried shrimps that contained a 3 × higher solid content by the same culture was a little bit faster and was finished after 140 h. The viscosity of chitin was in the order of chemically processed chitin > bioprocessed chitin > commercially available chitin. Results revealed changes in fine structure and chemical composition of the epi-, exo- and endocuticle of chitin from shrimp shells during microbial deproteination and demineralization. From transmission electron microscopy (TEM) overlays and electron energy loss spectroscopy (EELS) analysis, it was found that most protein was present in the exocuticle, whereas most chitin was present in the endocuticle. The calcium content was higher in the endocuticle than in the exocuticle.13C solid state NMR spectra of different chitin confirmed < 3% impurities in the final product. CONCLUSIONS: Bioprocessing of shrimp shell waste resulted in a chitin with high purity. Its viscosity was higher than that of commercially available chitin but lower than that of chemically prepared chitin in our lab. Nevertheless, the biologically processed chitin is a promising alternative for less viscous commercially available chitin. Highly viscous chitin could be generated by our chemical method. Comprehensive structural analyses revealed the distribution of the protein and Ca matrix within the shrimp shell cuticle which might be helpful in developing shrimp waste processing techniques.

Interspecies distances between propionic acid degraders and methanogens in syntrophic consortia for optimal hydrogen transfer.

A mixed culture from an anaerobic biowaste digester was enriched on propionate and used to investigate interspecies hydrogen transfer in dependence of spatial distances between propionate degraders and methanogens. From 20.3 mM propionate, 20.8 mM acetate and 15.5 mM methane were formed. Maximum specific propionate oxidation and methane formation rates were 49 and 23 mmol mg(-1) day(-1), respectively. Propionate oxidation was inhibited by only 20 mM acetate by about 50 %. Intermediate formate formation during inhibited methanogensis was observed. The spatial distribution and the biovolume fraction of propionate degraders and of methanogens in relation to the total population during aggregate formation were determined. Measurements of interbacterial distances were conducted with fluorescence in situ hybridization by application of group-specific 16S rRNA-targeted probes and 3D image analyses. With increasing incubation time, floc formation and growth up to 54 µm were observed. Propionate degraders and methanogens were distributed randomly in the flocs. The methanogenic biovolume fraction was high at the beginning and remained constant over 42 days, whereas the fraction of propionate degraders increased with time during propionate feeding. Interbacterial distances between propionate degraders and methanogens decreased with time from 5.30 to 0.29 µm, causing an increase of the maximum possible hydrogen flux from 1.1 to 10.3 nmol ml(-1) min(-1). The maximum possible hydrogen flux was always higher than the hydrogen formation and consumption rate, indicating that reducing the interspecies distance by aggregation is advantageous in complex ecosystems.

Effect of carbon sources and of sulfate on microbial arsenic mobilization in sediments of West Bengal, India.

Arsenic (As) dissolution from sediments into groundwater in the Bengal Delta/West India was investigated. Two experimental sites were choosen with contrasting As concentrations in shallow groundwater. Apparently patches of high-As and low-As sediments occured in close neigbourhood. A fast As mobilization with lactate or ethanol as carbon sources and sulfate as an electron acceptor and a possible influence of indigenous flora because of higher As amounts and an increasing total cell count was observed over a peroid of 110 days. Sucrose was a less suitable carbon source. Inoculation of an arsenate-reducing Pseudomonas putida WB, that was isolated from the sediments did not improve arsenic mobilization. Maximal arsenic concentrations up to 160µg/l were leached out from sediment columns with lactate or ethanol+sulfate in the water at natural groundwater flow, but the majority of the As remained in the sandy sediments. Some correlation of arsenic with Fe, but not with Mn dissolution seems to exist.

Prevalence, Antimicrobial Resistance and Toxin-Encoding Genes of Clostridioides difficile from Environmental Sources Contaminated by Feces.

Clostridioides difficile (C. difficile) is the most common pathogen causing antibiotic-associated intestinal diseases in humans and some animal species, but it can also be present in various environments outside hospitals. Thus, the objective of this study was to investigate the presence and the characteristics of toxin-encoding genes and antimicrobial resistance of C. difficile isolates from different environmental sources. C. difficile was found in 32 out of 81 samples (39.50%) after selective enrichment of spore-forming bacteria and in 45 samples (55.56%) using a TaqMan-based qPCR assay. A total of 169 C. difficile isolates were recovered from those 32 C. difficile-positive environmental samples. The majority of environmental C. difficile isolates were toxigenic, with many (88.75%) positive for tcdA and tcdB. Seventy-four isolates (43.78%) were positive for binary toxins, cdtA and cdtB, and 19 isolates were non-toxigenic. All the environmental C. difficile isolates were susceptible to vancomycin and metronidazole, and most isolates were resistant to ciprofloxacin (66.86%) and clindamycin (46.15%), followed by moxifloxacin (13.02%) and tetracycline (4.73%). Seventy-five isolates (44.38%) showed resistance to at least two of the tested antimicrobials. C. difficile strains are commonly present in various environmental sources contaminated by feces and could be a potential source of community-associated C. difficile infections.

Clostridioides difficile from Fecally Contaminated Environmental Sources: Resistance and Genetic Relatedness from a Molecular Epidemiological Perspective.

Clostridioides difficile is the most important pathogen causing antimicrobial-associated diarrhea and has recently been recognized as a cause of community-associated C. difficile infection (CA-CDI). This study aimed to characterize virulence factors, antimicrobial resistance (AMR), ribotype (RT) distribution and genetic relationship of C. difficile isolates from diverse fecally contaminated environmental sources. C. difficile isolates were recovered from different environmental samples in Northern Germany. Antimicrobial susceptibility testing was determined by E-test or disk diffusion method. Toxin genes (tcdA and tcdB), genes coding for binary toxins (cdtAB) and ribotyping were determined by PCR. Furthermore, 166 isolates were subjected to whole genome sequencing (WGS) for core genome multi-locus sequence typing (cgMLST) and extraction of AMR and virulence-encoding genes. Eighty-nine percent (148/166) of isolates were toxigenic, and 51% (76/148) were positive for cdtAB. Eighteen isolates (11%) were non-toxigenic. Thirty distinct RTs were identified. The most common RTs were RT127, RT126, RT001, RT078, and RT014. MLST identified 32 different sequence types (ST). The dominant STs were ST11, followed by ST2, ST3, and ST109. All isolates were susceptible to vancomycin and metronidazole and displayed a variable rate of resistance to moxifloxacin (14%), clarithromycin (26%) and rifampicin (2%). AMR genes, such as gyrA/B, blaCDD-1/2, aph(3')-llla-sat-4-ant(6)-la cassette, ermB, tet(M), tet(40), and tetA/B(P), conferring resistance toward fluoroquinolone, beta-lactam, aminoglycoside, macrolide and tetracycline antimicrobials, were found in 166, 137, 29, 32, 21, 72, 17, and 9 isolates, respectively. Eleven "hypervirulent" RT078 strains were detected, and several isolates belonged to RTs (i.e., RT127, RT126, RT023, RT017, RT001, RT014, RT020, and RT106) associated with CA-CDI, indicating possible transmission between humans and environmental sources pointing out to a zoonotic potential.

Mass spectrometric identification of 13C-labeled metabolites during anaerobic propanoic acid oxidation.

Biowaste digestion is a possibility to gain biogas as a renewable fuel source. However, the anaerobic food chain may be disrupted by, e.g., substrate overload or by inhibitors, leading to the accumulation of volatile fatty acids (VFAs), predominantly of propanoic acid (PA). VFA Accumulation may cause a rapid pH decrease, less biogas production, or even a total inhibition. To maintain high biogas productivity or to prevent a collapse of methanogenesis, metabolic properties of the degrading microorganisms must be elucidated, e.g., by investigation of the established pathways for degradation of VFAs. A Dani 3950 headspace system (HS), a Varian 431 gas chromatograph (GC), and a Varian 210 mass spectrometer (MS) have been combined to quantify and specifically identify metabolites of PA oxidation. The use of [1-(13)C]-labeled PA as a carbon source for microorganisms allows differentiation between the methyl-malonyl-CoA or the C(6)-dismutation pathway, both resulting in AcOH production. Appearance of the (13)C-moiety either in the COO or Me group of AcO can easily be detected by MS. The methyl-malonyl-CoA pathway was successfully identified as the only pathway of PA degradation by organisms in a lab-scale anaerobic digester. A similar approach can be applied to any degradation pathway involving VFAs.

Long-term effects of antibiotics on the elimination of chemical oxygen demand, nitrification, and viable bacteria in laboratory-scale wastewater treatment plants.

Antibiotics and other pharmaceuticals are contaminants of the environment because of their widespread use and incomplete removal by microorganisms during wastewater treatment. The influence of a mixture of ciprofloxacin (CIP), gentamicin (GM), sulfamethoxazole (SMZ)/trimethoprim (TMP), and vancomycin (VA), up to a final concentration of 40 mg/L, on the elimination of chemical oxygen demand (COD), nitrification, and survival of bacteria, as well as the elimination of the antibiotics, was assessed in a long-term study in laboratory treatment plants (LTPs). In the presence of 30 mg/L antibiotics, nitrification of artificial sewage by activated sludge ended at nitrite. Nitrate formation was almost completely inhibited. No nitrification at all was possible in the presence of 40 mg/L antibiotics. The nitrifiers were more sensitive to antibiotics than heterotrophic bacteria. COD elimination in antibiotic-stressed LTPs was not influenced by ≤20 mg/L antibiotics. Addition of 30 mg/L antibiotic mixture decreased COD removal efficiency for a period, but the LTPs recovered. Similar results were obtained with 40 mg/L antibiotic mixture. The total viable count of bacteria was not affected negatively by the antibiotics. It ranged from 2.2 × 10(6) to 8.2 × 10(6) colony-forming units per milliliter (CFU/mL) compared with the control at 1.4 × 10(6)-6.3 × 10(6) CFU/mL. Elimination of the four antibiotics during phases of 2.4-30 mg/L from the liquid was high for GM (70-90 %), much lower for VA, TMP, and CIP (0-50 %), and highly fluctuating for SMZ (0-95 %). The antibiotics were mainly adsorbed to the sludge and not biodegraded.

Effect of magnetite addition on transcriptional profiles of syntrophic Bacteria and Archaea during anaerobic digestion of propionate in wastewater sludge.

Anaerobic digestion (AD) is an important technology for the effective conversion of waste and wastewater to methane. Here, syntrophic bacteria transfer molecular hydrogen (H2 ), formate, or directly supply electrons (direct interspecies electron transfer, DIET) to the methanogens. Evidence is accumulating that the methanation of short-chain fatty acids can be enhanced by the addition of conductive material to the anaerobic digester, which has often been attributed to the stimulation of DIET. Since little is known about the transcriptional response of a complex AD microbial community to the addition of conductive material, we added magnetite to propionate-fed laboratory-scale reactors that were inoculated with wastewater sludge. Compared to the control reactors, the magnetite-amended reactors showed improved methanation of propionate. A genome-centric metatranscriptomics approach identified the active SCFA-oxidizing bacteria that affiliated with Firmicutes, Desulfobacterota and Cloacimonadota. The transcriptional profiles revealed that the syntrophic bacteria transferred acetate, H2 and formate to acetoclastic and hydrogenotrophic methanogens, whereas transcription of potential determinants for DIET such as conductive pili and outer-membrane cytochromes did not significantly change with magnetite addition. Overall, changes in the transcriptional profiles of syntrophic Bacteria and Archaea in propionate-fed lab-scale reactors amended with magnetite refute a major role of DIET in the studied system.

Microbiome Analysis via OTU and ASV-Based Pipelines-A Comparative Interpretation of Ecological Data in WWTP Systems.

Linking community composition and ecosystem function via the cultivation-independent analysis of marker genes, e.g., the 16S rRNA gene, is a staple of microbial ecology and dependent disciplines. The certainty of results, independent of the bioinformatic handling, is imperative for any advances made within the field. In this work, thermophilic anaerobic co-digestion experimental data, together with primary and waste-activated sludge prokaryotic community data, were analyzed with two pipelines that apply different principles when dealing with technical, sequencing, and PCR biases. One pipeline (VSEARCH) employs clustering methods, generating individual operational taxonomic units (OTUs), while the other (DADA2) is based on sequencing error correction algorithms and generates exact amplicon sequence variants (ASVs). The outcomes of both pipelines were compared within the framework of ecological-driven data analysis. Both pipelines provided comparable results that would generally allow for the same interpretations. Yet, the two approaches also delivered community compositions that differed between 6.75% and 10.81% between pipelines. Inconsistencies were also observed linked to biologically driven variability in the samples, which affected the two pipelines differently. These pipeline-dependent differences in taxonomic assignment could lead to different conclusions and interfere with any downstream analysis made for such mis- or not-identified species, e.g., network analysis or predictions of their respective ecosystem service.

Mechanisms Driving Microbial Community Composition in Anaerobic Co-Digestion of Waste-Activated Sewage Sludge.

Anaerobic co-digestion (Co-AD) is used to increase the effectiveness of anaerobic digestion (AD) using local "wastes", adding economic and environmental benefits. Since system stability is of existential importance for the operation of wastewater treatment plants, thorough testing of potential co-substrates and their effects on the respective community and system performance is crucial for understanding and utilizing Co-AD to its best capacity. Food waste (FW) and canola lecithin (CL) were tested in mesophilic, lab-scale, semi-continuous reactors over a duration of 120 days with stepwise increased substrate addition. Key performance indicators (biogas, total/volatile solids, fatty acids) were monitored and combined with 16S-rRNA amplicon sequencing to assess the impact of co-substrate addition on reactor performance and microbial community composition (MCC). Additionally, the latter was then compared with natural shifts occurring in the wastewater treatment plant (WWTP, source) at the same time. An almost linear increase in biogas production with both co-substrates at an approximate 1:1 ratio with the organic loading rate (OLR) was observed. The MCCs in both experiments were mostly stable, but also prone to drift over time. The FW experiment MCC more closely resembled the original WWTP community and the observed shifts indicated high levels of functional redundancy. Exclusive to the CL co-substrate, a clear selection for a few operational taxonomic units (OTUs) was observed. There was little evidence for a persistent invasion and establishment of microorganisms from typical primary substrates into the stable resident community of the reactors, which is in line with earlier findings that suggested that the inoculum and history mostly define the MCC. However, external factors may still tip the scales in favor of a few r-strategists (e.g., Prolixibacter) in an environment that otherwise favors K-strategists, which may in fact also be recruited from the primary substrate (Trichococcus). In our study, specialization and diversity loss were also observed in response to the addition of the highly specialized CL, which in turn, may have adverse effects on the system's stability and reduced resilience and recovery.

Hydrogen formation by an arsenate-reducing Pseudomonas putida, isolated from arsenic-contaminated groundwater in West Bengal, India.

Anaerobic growth of a newly isolated Pseudomonas putida strain WB from an arsenic-contaminated soil in West Bengal, India on glucose, L: -lactate, and acetate required the presence of arsenate, which was reduced to arsenite. During aerobic growth in the presence of arsenite arsenate was formed. Anaerobic growth of P. putida WB on glucose was made possible presumably by the non-energy-conserving arsenate reductase ArsC with energy derived only from substrate level phosphorylation. Two moles of acetate were generated intermediarily and the reducing equivalents of glycolysis and pyruvate decarboxylation served for arsenate reduction or were released as H(2). Anaerobic growth on acetate and lactate was apparently made possible by arsenate reductase ArrA coupled to respiratory electron chain energy conservation. In the presence of arsenate, both substrates were totally oxidized to CO(2) and H(2) with part of the H(2) serving for respiratory arsenate reduction to deliver energy for growth. The growth yield for anaerobic glucose degradation to acetate was Y (Glucose) = 20 g/mol, leading to an energy coefficient of Y (ATP) = 10 g/mol adenosine-5'-triphosphate (ATP), if the Emden-Meyerhof-Parnas pathway with generation of 2 mol ATP/mol glucose was used. During growth on lactate and acetate no substrate chain phosphorylation was possible. The energy gain by reduction of arsenate was Y (Arsenate) = 6.9 g/mol, which would be little less than one ATP/mol of arsenate.

Nitrification in fixed-bed reactors treating saline wastewater.

Halophilic nitrifiers belonging to the genus Nitrosomonas and Nitrospira were enriched from seawater and marine sediment samples of the North Sea. The maximal ammonia oxidation rate (AOR) in batch enrichments with seawater was 15.1 mg N L(-1) day(-1). An intermediate nitrite accumulation was observed. Two fixed-bed reactors for continuous nitrification with either polyethylene/claysinter lamellas (FBR A) or porous ceramic rings (FBR B)were run at two different ammonia concentrations, three different ammonia loading rates (ALRs), + or - pH adjustment,and at an increased upflow velocity. A better overall nitrification without nitrite accumulation was observed in FBR B. However, FBR A revealed a higher AOR and nitrite oxidation rate of 6 and 7 mg N L(-1) h(-1), compared to FBR B with 5 and 5.9 mg N L(-1) h(-1), respectively. AORs in the FBRs were at least ten times higher than in suspended enrichment cultures. Whereas a shift within the ammoniaoxidizing population in the genus Nitrosomonas at the subspecies level occurred in FBR B with synthetic seawater at an increasing ALR and a decreasing pH, the nitrite oxidizing Nitrospira population apparently did not change.

Syntrophic acetate oxidation replaces acetoclastic methanogenesis during thermophilic digestion of biowaste.

BACKGROUND: Anaerobic digestion (AD) is a globally important technology for effective waste and wastewater management. In AD, microorganisms interact in a complex food web for the production of biogas. Here, acetoclastic methanogens and syntrophic acetate-oxidizing bacteria (SAOB) compete for acetate, a major intermediate in the mineralization of organic matter. Although evidence is emerging that syntrophic acetate oxidation is an important pathway for methane production, knowledge about the SAOB is still very limited. RESULTS: A metabolic reconstruction of metagenome-assembled genomes (MAGs) from a thermophilic solid state biowaste digester covered the basic functions of the biogas microbial community. Firmicutes was the most abundant phylum in the metagenome (53%) harboring species that take place in various functions ranging from the hydrolysis of polymers to syntrophic acetate oxidation. The Wood-Ljungdahl pathway for syntrophic acetate oxidation and corresponding genes for energy conservation were identified in a Dethiobacteraceae MAG that is phylogenetically related to known SAOB. 16S rRNA gene amplicon sequencing and enrichment cultivation consistently identified the uncultured Dethiobacteraceae together with Syntrophaceticus, Tepidanaerobacter, and unclassified Clostridia as members of a potential acetate-oxidizing core community in nine full-scare digesters, whereas acetoclastic methanogens were barely detected. CONCLUSIONS: Results presented here provide new insights into a remarkable anaerobic digestion ecosystem where acetate catabolism is mainly realized by Bacteria. Metagenomics and enrichment cultivation revealed a core community of diverse and novel uncultured acetate-oxidizing bacteria and point to a particular niche for them in dry fermentation of biowaste. Their genomic repertoire suggests metabolic plasticity besides the potential for syntrophic acetate oxidation. Video Abstract.

Treatment of phenolic wastewater in an anaerobic fixed bed reactor (AFBR) - recovery after shock loading.

An anaerobic fixed bed reactor (AFBR) was run for 550 days with a mixed microbial flora to stabilize synthetic wastewater that contained glucose and phenol as main carbon sources. The influent phenol concentration was gradually increased from 2 to 40 mmol/l within 221 days. The microbial flora was able to adapt to this high phenol concentration with an average of 94% phenol removal. Microbial adaptation at such a high phenol concentration is not reported elsewhere. The maximum phenol removal observed before the phenol shock load was 39.47 mmol/l or 3.7 g phenol/l at a hydraulic retention time (HRT) of 2.5 days and an organic loading rate (OLR) of 5.3 g/l.d which amounts to a phenol removal rate of ca. 15.8 mmol phenol/l.d. The chemical oxygen demand (COD) removal before exposing the reactor to a shock load corresponded with phenol removal. A shock load was induced in the reactor by increasing the phenol concentration from 40 to 50 mmol/l in the influent. The maximum phenol removal rate observed after shock load was 18 mmol/l.d at 5.7 g COD/l.d. But this was not a stable rate and a consistent drop in COD and phenol removal was observed for 1 week, followed by a sharp decline and production of fatty acids. Recovery of the reactor was possible only when no feed was provided to the reactor for 1 month and the phenol concentration was increased gradually. When glucose was omitted from the influent, unknown intermediates of anaerobic phenol metabolism were observed for some time.