Expanding the repertoire of counterselection markers for markerless gene deletion in the human gut bacterium Phocaeicola vulgatus.

OBJECTIVE: Phocaeicolavulgatus (formerly Bacteroides vulgatus) is a highly abundant and ubiquitous member of the human gut microbiota, associated with human health and disease, and therefore represents an important target for further investigations. In this study a novel gene deletion method was developed for P. vulgatus, expanding the tools available for genetic manipulation of members of the microbial order Bacteroidales. MATERIAL AND METHODS: The study used a combination of bioinformatics and growth experiments in interaction with molecular cloning to validate the applicability of SacB as a counterselection marker in P. vulgatus. RESULTS: In this study, the levansucrase gene sacB from Bacillussubtilis was verified as a functional counterselection marker for P. vulgatus, conferring a lethal sensitivity towards sucrose. Markerless gene deletion based on SacB was applied to delete a gene encoding a putative endofructosidase (BVU1663). The P. vulgatus Δbvu1663 deletion mutant displayed no biomass formation when grown on levan, inulin or their corresponding fructooligosaccharides. This system was also applied for the deletion of the two genes bvu0984 and bvu3649, which are involved in the pyrimidine metabolism. The resulting P. vulgatus Δ0984 Δ3649 deletion mutant no longer showed sensitivity for the toxic pyrimidine analogon 5-fluorouracil, allowing a counterselection with this compound in the double knockout strain. CONCLUSION: The genetic toolbox for P. vulgatus was expanded by a markerless gene deletion system based on SacB as an efficient counterselection marker. The system was employed to successfully delete three genes in P. vulgatus which all resulted in expected phenotypes as confirmed by subsequent growth experiments.

High-yield production and purification of prebiotic inulin-type fructooligosaccharides.

Due to the health-promoting effects and functional properties of inulin-type fructooligosaccharides (I-FOS), the global market for I-FOS is constantly growing. Hence, there is a continuing demand for new, efficient biotechnological approaches for I-FOS production. In this work, crude inulosucrase InuGB-V3 from Lactobacillus gasseri DSM 20604 was used to synthesize I-FOS from sucrose. Supplementation with 1 mM CaCl2, a pH of 3.5-5.5, and an incubation temperature of 40 °C were found to be optimal production parameters at which crude inulosucrase showed high conversion rates, low sucrose hydrolysis, and excellent stability over 4 days. The optimal process conditions were employed in cell-free bioconversion reactions. By elevating the substrate concentration from 570 to 800 g L-1, the I-FOS concentration and the synthesis of products with a low degree of polymerization (DP) could be increased, while sucrose hydrolysis was decreased. Bioconversion of 800 g L-1 sucrose for 20 h resulted in an I-FOS-rich syrup with an I-FOS concentration of 401 ± 7 g L-1 and an I-FOS purity of 53 ± 1% [w/w]. I-FOS with a DP of 3-11 were synthesized, with 1,1-kestotetraose (DP4) being the predominant transfructosylation product. The high-calorie sugars glucose, sucrose, and fructose were removed from the generated I-FOS-rich syrup using activated charcoal. Thus, 81 ± 5% of the initially applied I-FOS were recovered with a purity of 89 ± 1%.

5-Keto-D-Fructose, a Natural Diketone and Potential Sugar Substitute, Significantly Reduces the Viability of Prokaryotic and Eukaryotic Cells.

5-Keto-D-fructose (5-KF) is a natural diketone occurring in micromolar concentrations in honey, white wine, and vinegar. The oxidation of D-fructose to 5-KF is catalyzed by the membrane-bound fructose dehydrogenase complex found in several acetic acid bacteria. Since 5-KF has a sweetening power comparable to fructose and is presumably calorie-free, there is great interest in making the diketone commercially available as a new sugar substitute. Based on a genetically modified variant of the acetic acid bacterium Gluconobacter oxydans 621H, an efficient process for the microbial production of 5-KF was recently developed. However, data on the toxicology of the compound are completely lacking to date. Therefore, this study aimed to investigate the effect of 5-KF on the viability of prokaryotic and eukaryotic cells. It was found that the compound significantly inhibited the growth of the gram-positive and gram-negative model organisms Bacillus subtilis and Escherichia coli in a concentration-dependent manner. Furthermore, cell viability assays confirmed severe cytotoxicity of 5-KF toward the colon cancer cell line HT-29. Since these effects already occurred at concentrations of 5 mM, the use of 5-KF in the food sector should be avoided. The studies performed revealed that in the presence of amines, 5-KF promoted a strong Maillard reaction. The inherent reactivity of 5-KF as well as the Maillard products formed could be the trigger for the observed inhibition of prokaryotic and eukaryotic cells.

Genetic tools for the redirection of the central carbon flow towards the production of lactate in the human gut bacterium Phocaeicola (Bacteroides) vulgatus.

Species of the genera Bacteroides and Phocaeicola play an important role in the human colon. The organisms contribute to the degradation of complex heteropolysaccharides to small chain fatty acids, which are in part utilized by the human body. Furthermore, these organisms are involved in the synthesis of vitamins and other bioactive compounds. Of special interest is Phocaeicola vulgatus, originally classified as a Bacteroides species, due to its abundance in the human intestinal tract and its ability to degrade many plant-derived heteropolysaccharides. We analyzed different tools for the genetic modification of this microorganism, with respect to homologous gene expression of the ldh gene encoding a D-lactate dehydrogenase (LDH). Therefore, the ldh gene was cloned into the integration vector pMM656 and the shuttle vector pG106 for homologous gene expression in P. vulgatus. We determined the ldh copy number, transcript abundance, and the enzyme activity of the wild type and the mutants. The strain containing the shuttle vector showed an approx. 1500-fold increase in the ldh transcript concentration and an enhanced LDH activity that was about 200-fold higher compared to the parental strain. Overall, the proportion of lactate in the general catabolic carbon flow increased from 2.9% (wild type) to 28.5% in the LDH-overproducing mutant. This approach is a proof of concept, verifying the genetic accessibility of P. vulgatus and could form the basis for targeted genetic optimization. KEY POINTS: • A lactate dehydrogenase was overexpressed in Phocaeicola (Bacteroides) vulgatus. • The ldh transcript abundance and the LDH activity increased sharply in the mutant. • The proportion of lactate in the catabolic carbon flow increased to about 30%.

Process of energy conservation in the extremely haloalkaliphilic methyl-reducing methanogen Methanonatronarchaeum thermophilum.

The recently isolated methanogen Methanonatronarchaeum thermophilum is an extremely haloalkaliphilic and moderately thermophilic archaeon and belongs to the novel class Methanonatronarchaeia in the phylum Halobacteriota. The knowledge about the physiology and biochemistry of members of the class Methanonatronarchaeia is still limited. It is known that M. thermophilum performs hydrogen or formate-dependent methyl-reducing methanogenesis. Here, we show that the organism was able to grow on all tested C1 -methylated substrates (methanol, trimethylamine, dimethylamine, monomethylamine) in combination with formate or molecular hydrogen. A temporary accumulation of intermediates (dimethylamine or/and monomethylamine) in the medium occurred during the consumption of trimethylamine or dimethylamine. The energy conservation of M. thermophilum was dependent on a respiratory chain consisting of a hydrogenase (VhoGAC), a formate dehydrogenase (FdhGHI), and a heterodisulfide reductase (HdrDE) that were well adapted to the harsh physicochemical conditions in the natural habitat. The experiments revealed the presence of two variants of energy-conserving oxidoreductase systems in the membrane. These included the H2 : heterodisulfide oxidoreductase system, which has already been described in Methanosarcina species, as well as the novel formate: heterodisulfide oxidoreductase system. The latter electron transport chain, which was experimentally proven for the first time, distinguishes the organism from all other known methanogenic archaea and represents a unique feature of the class Methanonatronarchaeia. Experiments with 2-hydroxyphenazine and the inhibitor diphenyleneiodonium chloride indicated that a methanophenazine-like cofactor might function as an electron carrier between the hydrogenase/ formate dehydrogenase and the heterodisulfide reductase.

High-resolution method for isocratic HPLC analysis of inulin-type fructooligosaccharides.

In recent decades, strategies to improve human health by modulating the gut microbiota have developed rapidly. One of the most prominent is the use of prebiotics, which can lead to a higher abundance of health-promoting microorganisms in the gut. Currently, oligosaccharides dominate the prebiotic sector due to their ability to promote the growth and activity of probiotic bacteria selectively. Extensive efforts are made to develop effective production strategies for the synthesis of prebiotic oligosaccharides, including the use of microbial enzymes. Within the genus Lactobacillus, several inulosucrases have been identified, which are suitable for the synthesis of prebiotic inulin-type fructooligosaccharides (inulin-FOS). In this study, a truncated version of the inulosucrase from Lactobacillus gasseri DSM 20604 was used for the efficient synthesis of inulin-FOS. Product titers of 146.2 ±â€¯7.4 g inulin-FOSL-1 were achieved by the catalytic activity of the purified recombinant protein InuGB-V3. A time and resource-saving HPLC method for rapid analysis of inulin-FOS in isocratic mode was developed and optimized, allowing baseline separated analysis of inulin-FOS up to a degree of polymerization (DP) of five in less than six minutes. Long-chain inulin-FOS with a DP of 17 can be analyzed in under 45 min. The developed method offers the advantages of isocratic HPLC analysis, such as low flow rates, high sensitivity, and the use of a simple, inexpensive chromatographic setup. Furthermore, it provides high-resolution separation of long-chain inulin-FOS, which can usually only be achieved with gradient systems.

Degradation of the low-calorie sugar substitute 5-ketofructose by different bacteria.

There is an increasing public awareness about the danger of dietary sugars with respect to their caloric contribution to the diet and the rise of overweight throughout the world. Therefore, low-calorie sugar substitutes are of high interest to replace sugar in foods and beverages. A promising alternative to natural sugars and artificial sweeteners is the fructose derivative 5-keto-D-fructose (5-KF), which is produced by several Gluconobacter species. A prerequisite before 5-KF can be used as a sweetener is to test whether the compound is degradable by microorganisms and whether it is metabolized by the human microbiota. We identified different environmental bacteria (Tatumella morbirosei, Gluconobacter japonicus LMG 26773, Gluconobacter japonicus LMG 1281, and Clostridium pasteurianum) that were able to grow with 5-KF as a substrate. Furthermore, Gluconobacter oxydans 621H could use 5-KF as a carbon and energy source in the stationary growth phase. The enzymes involved in the utilization of 5-KF were heterologously overproduced in Escherichia coli, purified and characterized. The enzymes were referred to as 5-KF reductases and belong to three unrelated enzymatic classes with highly different amino acid sequences, activities, and structural properties. Furthermore, we could show that 15 members of the most common and abundant intestinal bacteria cannot degrade 5-KF, indicating that this sugar derivative is not a suitable growth substrate for prokaryotes in the human intestine. KEY POINTS: • Some environmental bacteria are able to use 5-KF as an energy and carbon source. • Four 5-KF reductases were identified, belonging to three different protein families. • Many gut bacteria cannot degrade 5-KF.

Characterization of a novel endo-levanase from Azotobacter chroococcum DSM 2286 and its application for the production of prebiotic fructooligosaccharides.

Prebiotics are known for their ability to modulate the composition of the human microbiome and mediate health-promoting benefits. Endo-levanases, which hydrolyze levan into short-chain FOS, could be used for the production of levan-based prebiotics. The novel endo-levanase (LevB2286) from Azotobacter chroococcum DSM 2286, combines an exceptionally high specific activity with advantageous hydrolytic properties. Starting from levan isolated from Timothy grass, LevB2286 produced FOS ranging from DP 2 - 8. In contrast to endo-levanases described in the literature, LevB2286 formed minor amounts of fructose and levanbiose, even with greatly extended incubation. The combined activity of LevB2286 and the levansucrase LevS1417 from Gluconobacter japonicus LMG 1417 led to a one-step synthesis of levan-type FOS from sucrose. 387.4 ± 17.3 g L-1 FOS were produced within 48 h by the production strategy based on crude cell extract of recombinant Escherichia coli expressing levS1417 and levB2286 simultaneously.

An alternative pentose phosphate pathway in human gut bacteria for the degradation of C5 sugars in dietary fibers.

The microbial degradation of pentoses in the human gut is a crucial factor for the utilization of plant-based dietary fibers. A vast majority of gut microbes are able to use these C5-sugars as a carbon and energy source. However, the underlying metabolic pathways are not fully understood. Bioinformatic analysis showed that a large number of abundant gut bacteria lack genes encoding a transaldolase as a key enzyme of the pentose phosphate pathway. Among them was the important human gut microbe Prevotella copri, which was able to grow in minimal media containing xylose or hemicelluloses as the sole carbon source. Therefore, we looked for an alternative pathway for pentose conversion in P. copri using bioinformatics, enzyme activity assays, and the detection of intermediates of pentose metabolism. It became evident that the organism converted C5-sugars via the sedoheptulose-1,7-bisphosphate pathway (SBPP) to connect pentose metabolism with glycolysis. To circumvent the transaldolase reaction, P. copri uses the combined catalysis of a pyrophosphate-dependent phosphofructokinase and a fructose-bisphosphate aldolase. Furthermore, we present strong evidence that the SBPP is widely distributed in important gut bacteria, including members of the phyla Bacteroides, Firmicutes, Proteobacteria, Verrucomicrobia, and Lentisphaerae.

High yield production of levan-type fructans by Gluconobacter japonicus LMG 1417.

Levan, a ß-2,6-glycosidic linked fructan, is a promising alternative for the inulin dominated fructan market. Although levan is already used in some cosmetic products, the commercial availability of the fructan is still limited. Here we show that Gluconobacter japonicus LMG 1417 is a potent levan-forming organism and a promising platform for the industrial production of levan. The levansucrase LevS1417, which is produced by G. japonicus LMG 1417 and secreted by a signal-peptide-independent pathway, exhibited extraordinary high activity (4726 ±â€¯821 U mg-1 at 50 °C). A cell-free levan production based on the supernatant of the investigated strain led to a final levan yield of 157.9 ±â€¯7.6 g L-1. The amount of secreted levansucrase was more than doubled by plasmid-mediated homologous overproduction of LevS1417 in G. japonicus LMG 1417. Accordingly, the space-time yield of cell-free levan production was doubled using the plasmid-bearing mutant.

Characterization of three novel ß-galactosidases from Akkermansia muciniphila involved in mucin degradation.

The gut microbe Akkermansia (A.) muciniphila becomes increasingly important as its prevalence is inversely correlated with different human metabolic disorders and diseases. This organism is a highly potent degrader of intestinal mucins and the hydrolyzed glycan compounds can then serve as carbon sources for the organism itself or other members of the gut microbiota via cross-feeding. Despite its importance for the hosts' health and microbiota composition, exact mucin degrading mechanisms are still mostly unclear. In this study, we identified and characterized three extracellular ß-galactosidases (Amuc_0771, Amuc_0824, and Amuc_1666) from A. muciniphila ATCC BAA-835. The substrate spectrum of all three enzymes was analyzed and the results indicated a preference for different galactosidic linkages for each hydrolase. All preferred target structures are prevalent within mucins of the colonic habitat of A. muciniphila. To check a potential function of the enzymes for the degradation of mucosal glycan structures, porcine stomach mucin was applied as a model substrate. In summary, we could confirm the involvement of all three ß-galactosidases from A. muciniphila in the complex mucin degradation machinery of this important gut microbe. These findings could contribute to the understanding of the molecular interactions between A. muciniphila and its host on a molecular level.

Synthesis of the alternative sweetener 5-ketofructose from sucrose by fructose dehydrogenase and invertase producing Gluconobacter strains.

A promising alternative to high-calorie sugars and artificial sweeteners is the microbially produced fructose derivative 5-ketofructose (5-KF). The key enzyme for biotransformation, fructose dehydrogenase (Fdh), was overproduced in Gluconobacter (G.) oxydans and G. japonicus LMG 26773. Furthermore, the fdh genes were integrated into the chromosome of G. oxydans (G. oxydans Δmgdh::fdh). All mutants showed high fructose oxidation rates forming 5-KF. G. japonicus LMG 26773 fdh was selected for 5-KF production from the cost-efficient and renewable feedstock sucrose because the organism possessed both, a highly active Fdh and an enzyme able to cleave sucrose. However, 5-KF yield was low because the strain formed levan and consumed 5-KF in the second growth phase. Several Gluconobacter strains were screened for sucrose-hydrolyzing enzymes. One of these proteins (Inv1417) was characterized and it was found that the enzyme showed the highest specific activity compared to all mesophilic invertases described so far (Vmax = 2295 ± 243 U mg protein-1). The corresponding gene was expressed in G. oxydans Δmgdh::fdh. The results clearly indicated that both heterologously produced enzymes Fdh and Inv1417 were active in this single-strain system for 5-KF synthesis. Overall 84 ± 2% of the available fructose units of sucrose were converted to 5-KF.

Energy conservation in the gut microbe Methanomassiliicoccus luminyensis is based on membrane-bound ferredoxin oxidation coupled to heterodisulfide reduction.

Methanomassiliicoccus luminyensis was originally isolated from human feces and belongs to the seventh order of methanogens, the Methanomassiliicoccales, which are only distantly related to other methanogenic archaea. The organism forms methane from the reduction of methylamines or methanol using molecular hydrogen as reductant. The energy-conserving system in M. luminyensis is unique and the enzymes involved in this process are not found in this combination in members of the other methanogenic orders. In this context our central question was how the organism is able to generate ATP. Energy transduction was dependent on a membrane-bound ferredoxin: heterodisulfide oxidoreductase composed of reduced ferredoxin as an electron donor, at least one protein in the membrane fraction and the heterodisulfide reductase HdrD, which reduced the electron acceptor CoM-S-S-CoB. Electron transfer of this respiratory chain proceeded with a rate of 145 nmol reduced heterodisulfide min-1 ·mg-1 membrane protein. Methanomassiliicoccus luminyensis is the first example of a methanogenic archaeon that does not require Na+ ions for energy conservation. Only protons were used as coupling ions for the generation of the electrochemical ion gradient. The membrane-bound F420 H2 :phenazine oxidoreductase complex (without the electron input module FpoF) probably catalyzed the oxidation of reduced ferredoxin and potentially acted as primary proton pump in this electron transport system. In summary, the energy-conserving system of M. luminyensis possesses features found in the pathways of hydrogenotrophic and methylotrophic/aceticlastic methanogenesis. Consequently, the composition of the enzymes involved in ion translocation across the cytoplasmic membrane is different from all other methanogenic archaea.

Characterization of a phospholipid-regulated ß-galactosidase from Akkermansia muciniphila involved in mucin degradation.

The gut microbe Akkermansia muciniphila is important for the human health as the occurrence of the organism is inversely correlated with different metabolic disorders. The metabolism of the organism includes the degradation of intestinal mucins. Thus, the gut health-promoting properties are not immediately obvious and mechanisms of bacteria-host interactions are mostly unclear. In this study, we characterized a novel extracellular ß-galactosidase (Amuc_1686) with a preference for linkages from the type Galß1-3GalNAc. Additionally, Amuc_1686 possesses a discoidin-like domain, which enables the interaction with anionic phospholipids. We detected a strong inhibition by phosphatidylserine, phosphatidylglycerol, phosphatidic acid, and lysophosphatidic acid while phosphatidylcholine and phosphatidylethanolamine had no influence. Amuc_1686 is the first example of a prokaryotic hydrolase that is strongly inhibited by certain phospholipids. These inhibiting phospholipids have important signal functions in immune response and cell clearance processes. Hence, Amuc_1686 might be regulated based on the health status of the large intestine and could therefore contribute to the mutualistic relationship between the microbe and the host on a molecular level. In this sense, Amuc_1686 could act as an altruistic enzyme that does not attack the mucin layer of apoptotic epithelial cells to ensure tissue regeneration, for example, in areas with inflammatory damages.

Long-term investigation of microbial community composition and transcription patterns in a biogas plant undergoing ammonia crisis.

Ammonia caused disturbance of biogas production is one of the most frequent incidents in regular operation of biogas reactors. This study provides a detailed insight into the microbial community of a mesophilic, full-scale biogas reactor (477 kWh h-1 ) fed with maize silage, dried poultry manure and cow manure undergoing initial process disturbance by increased ammonia concentration. Over a time period of 587 days, the microbial community of the reactor was regularly monitored on a monthly basis by high-throughput amplicon sequencing of the archaeal and bacterial 16S rRNA genes. During this sampling period, the total ammonia concentrations varied between 2.7 and 5.8 g l-1 [NH4 + -N]. To gain further inside into the active metabolic pathways, for selected time points metatranscriptomic shotgun analysis was performed allowing the quantification of marker genes for methanogenesis, hydrolysis and syntrophic interactions. The results obtained demonstrated a microbial community typical for a mesophilic biogas plant. However in response to the observed changing process conditions (e.g. increasing NH4 + levels, changing feedstock composition), the microbial community reacted highly flexible by changing and adapting the community composition. The Methanosarcina-dominated archaeal community was shifted to a Methanomicrobiales-dominated archaeal community in the presence of increased ammonia conditions. A similar trend as in the phylogenetic composition was observed in the transcription activity of genes coding for enzymes involved in acetoclastic methanogenesis and syntrophic acetate oxidations (Codh/Acs and Fthfs). In accordance, Clostridia simultaneously increased under elevated ammonia concentrations in abundance and were identified as the primary syntrophic interaction partner with the now Methanomicrobiales-dominated archaeal community. In conclusion, overall stable process performance was maintained during increased ammonia concentration in the studied reactor based on the microbial communities' ability to flexibly respond by reorganizing the community composition while remaining functionally stable.

Physiology and central carbon metabolism of the gut bacterium Prevotella copri.

The human gut microbiota is a crucial factor for the host's physiology with respect to health and disease. Metagenomic shotgun sequencing of microbial gut communities revealed that Prevotella copri is one of the most important players in the gastrointestinal tract of many individuals. Because of the importance of this bacterium we analyzed the growth behavior and the central metabolic pathways of P. copri. Bioinformatic data, transcriptome profiling and enzyme activity measurements indicated that the major pathways are based on glycolysis and succinate production from fumarate. In addition, pyruvate can be degraded to acetate and formate. Electron transport phosphorylation depends on fumarate respiration with NADH and reduced ferredoxin as electron donors. In contrast to Bacteroides vulgatus, P. copri showed a more pronounced dependency on the addition of CO2 or bicarbonate for biomass formation, which is a remarkable difference between P. copri and Bacteroides spp. with important implication in the context of gut microbial competition. The analysis of substrate consumption and product concentrations from many P. copri cultures with different optical densities allowed a prediction of the carbon and electron flow in the central metabolism and a detailed calculation of growth yields as well as carbon and redox balances.

Production of the potential sweetener 5-ketofructose from fructose in fed-batch cultivation with Gluconobacter oxydans.

Sweeteners improve the dietary properties of many foods. A candidate for a new natural sweetener is 5-ketofructose. In this study a fed-batch process for the production of 5-ketofructose was developed. A Gluconobacter oxydans strain overexpressing a fructose dehydrogenase from G. japonicus was used and the sensory properties of 5-ketofructose were analyzed. The compound showed an identical sweet taste quality as fructose and a similar intrinsic sweet threshold concentration of 16.4 mmol/L. The production of 5-ketofructose was characterized online by monitoring of the respiration activity in shake flasks. Pulsed and continuous fructose feeding was realized in 2 L stirred tank reactors and maximum fructose consumption rates were determined. 5-Ketofructose concentrations of up to 489 g/L, product yields up to 0.98 g5-KF/gfructose and space time yields up to 8.2 g/L/h were reached highlighting the potential of the presented process.

Production of 5-ketofructose from fructose or sucrose using genetically modified Gluconobacter oxydans strains.

The growing consumer demand for low-calorie, sugar-free foodstuff motivated us to search for alternative non-nutritive sweeteners. A promising sweet-tasting compound is 5-keto-D-fructose (5-KF), which is formed by membrane-bound fructose dehydrogenases (Fdh) in some Gluconobacter strains. The plasmid-based expression of the fdh genes in Gluconobacter (G.) oxydans resulted in a much higher Fdh activity in comparison to the native host G. japonicus. Growth experiments with G. oxydans fdh in fructose-containing media indicated that 5-KF was rapidly formed with a conversion efficiency of 90%. 5-KF production from fructose was also observed using resting cells with a yield of about 100%. In addition, a new approach was tested for the production of the sweetener 5-KF by using sucrose as a substrate. To this end, a two-strain system composed of the fdh-expressing strain and a G. oxydans strain that produced the sucrose hydrolyzing SacC was developed. The strains were co-cultured in sucrose medium and converted 92.5% of the available fructose units into 5-KF. The glucose moiety of sucrose was converted to 2-ketogluconate and acetate. With regard to the development of a sustainable and resource-saving process for the production of 5-KF, sugar beet extract was used as substrate for the two-strain system. Fructose as product from sucrose cleavage was mainly oxidized to 5-KF which was detected in a concentration of over 200 mM at the end of the fermentation process. In summary, the two-strain system was able to convert fructose units of sugar beet extract to 5-KF with an efficiency of 82 ± 5%.

Growth Characteristics of Methanomassiliicoccus luminyensis and Expression of Methyltransferase Encoding Genes.

DNA sequence analysis of the human gut revealed the presence a seventh order of methanogens referred to as Methanomassiliicoccales. Methanomassiliicoccus luminyensis is the only member of this order that grows in pure culture. Here, we show that the organism has a doubling time of 1.8 d with methanol + H2 and a growth yield of 2.4 g dry weight/mol CH4. M. luminyensis also uses methylamines + H2 (monomethylamine, dimethylamine, and trimethylamine) with doubling times of 2.1-2.3 d. Similar cell yields were obtained with equimolar concentrations of methanol and methylamines with respect to their methyl group contents. The transcript levels of genes encoding proteins involved in substrate utilization indicated increased amounts of mRNA from the mtaBC2 gene cluster in methanol-grown cells. When methylamines were used as substrates, mRNA of the mtb/mtt operon and of the mtmBC1 cluster were found in high abundance. The transcript level of mtaC2 was almost identical in methanol- and methylamine-grown cells, indicating that genes for methanol utilization were constitutively expressed in high amounts. The same observation was made with resting cells where methanol always yielded the highest CH4 production rate independently from the growth substrate. Hence, M. luminyensis is adapted to habitats that provide methanol + H2 as substrates.

BEAP profiles as rapid test system for status analysis and early detection of process incidents in biogas plants.

A method was developed to quantify the performance of microorganisms involved in different digestion levels in biogas plants. The test system was based on the addition of butyrate (BCON), ethanol (ECON), acetate (ACON) or propionate (PCON) to biogas sludge samples and the subsequent analysis of CH4 formation in comparison to control samples. The combination of the four values was referred to as BEAP profile. Determination of BEAP profiles enabled rapid testing of a biogas plant's metabolic state within 24 h and an accurate mapping of all degradation levels in a lab-scale experimental setup. Furthermore, it was possible to distinguish between specific BEAP profiles for standard biogas plants and for biogas reactors with process incidents (beginning of NH4+-N inhibition, start of acidification, insufficient hydrolysis and potential mycotoxin effects). Finally, BEAP profiles also functioned as a warning system for the early prediction of critical NH4+-N concentrations leading to a drop of CH4 formation.