Effects of culture conditions on Pectinatus cerevisiiphilus and Pectinatus frisingensis metabolism: a physiological and statistical approach

The genus Pectinatus has been often reported in beer spoilage with off-flavours. The bacteria are strictly anaerobic, Gram-negative rods. Propionate and acetate are the main fermentation products from glucose in the two species belonging to the genus, P. cerevisiiphilus and P. frisingensis. Amino acids routinely present at a high level in beer were not growth substrates for both species, and a significant accumulation of succinate was observed with lactate as growth substrate. Both Pectinatus ssp. showed almost identical fermentation balances on glucose. Growth kinetics of both glucose-grown species were unchanged under a N₂, H₂ or 20% CO₂-containing atmosphere. Combinations of culture medium pH values from pH 3·9 to pH 7·2, of glucose levels between 5 and 55 mmol l^-1, and of lactate concentrations varied from 4 to 40 mmol l^-1 demonstrated that biomass and volatile fatty acids production were proportional to glucose concentration for both Pectinatus species. A significant increase of volatile fatty acid production was measured for both species at the lowest pH values with a lactate or a glucose concentration increase. The maximum biomass production was observed at pH 6·2 for P. cerevisiiphilus , and between pH 4·5 and pH 4·9 for P. frisingensis. Glucose and lactate or pH value were dependent with regard to propionate and acetate production in P. frisingensis. On the other hand, the variations of these three parameters were independent with regard to biomass production for both strains, and to volatile fatty acids production for P. cerevisiiphilus. Addition of ethanol to glucose-grown cultures completely inhibited growth at 1·3 mol l^-1 ethanol for P. cerevisiiphilus , and at 1·8 mol l^-1 for P. frisingensis.     

Variability in survival of Pectinatus cerevisiiphilus, strictly anaerobic bacteria, under different oxygen conditions

Survival of Pectinatus cerevisiiphilus DSM 20466 in pure culture at variable temperatures under different oxygen concentrations was measured. Survival of P. cerevisiiphilus in co-culture with Saccharomyces cerevisiae under both saturated oxygen and brewing conditions was also studied. The survival of strictly anaerobic bacteria to oxygen seems to follow the classical laws of heat resistance. The D(oxy) values of P. cerevisiiphilus , calculated as a function of oxygen level, shows that the oxygen level is important for the survival duration of the bacteria. The temperature greatly influences the oxygen resistance of P. cerevisiiphilus, which increases when the temperature decreases. P. cerevisiiphilus resists better in co-culture than in pure culture under saturated oxygen conditions. Therefore, the oxygenation of the wort does not totally eliminate the risk of beer contamination by this bacterium. Under brewing conditions in co-culture at 8 degrees C, P. cerevisiiphilus grows slowly to reach a final cell concentration up to 10(6) cells/mL in beer, which is undrinkable. Pectinatus is a strictly anaerobic bacterium; however, it is resistant under certain oxygen conditions of incubation. This resistance is considerably higher in the presence of Saccharomyces cerevisiae .     

Carbon and energy balances of glucose fermentation with hydrogenproducing bacterium Citrobacter amalonaticus Y19

For the newly isolated H2-producing chemoheterotrophic bacterium Citrobacter amalonaticus Y19, anaerobic glucose metabolism was studied in batch cultivation at varying initial glucose concentrations (3.5- 9.5 g/l). The carbon-mass and energy balances were determined and utilized to analyze the carbon metabolic-pathways network. The analyses revealed (a) variable production of major metabolites (H2, ethanol, acetate, lactate, CO2, and cell mass) depending on initial glucose levels; (b) influence of NADH regeneration on the production of acetate, lactate, and ethanol; and (c) influence of the molar production of ATP on the production of biomass. The results reported in this paper suggest how the carbon metabolic pathway(s) should be designed for optimal H2 production, especially at high glucose concentrations, such as by blocking the carbon flux via lactate dehydrogenase from the pyruvate node.     

Anaerobic Growth of Corynebacterium glutamicum via Mixed-Acid Fermentation

Corynebacterium glutamicum, a model organism in microbial biotechnology, is known to metabolize glucose under oxygen-deprived conditions to l-lactate, succinate, and acetate without significant growth. This property is exploited for efficient production of lactate and succinate. Our detailed analysis revealed that marginal growth takes place under anaerobic conditions with glucose, fructose, sucrose, or ribose as a carbon and energy source but not with gluconate, pyruvate, lactate, propionate, or acetate. Supplementation of glucose minimal medium with tryptone strongly enhanced growth up to a final optical density at 600 nm (OD₆₀₀) of 12, whereas tryptone alone did not allow growth. Amino acids with a high ATP demand for biosynthesis and amino acids of the glutamate family were particularly important for growth stimulation, indicating ATP limitation and a restricted carbon flux into the oxidative tricarboxylic acid cycle toward 2-oxoglutarate. Anaerobic cultivation in a bioreactor with constant nitrogen flushing disclosed that CO₂ is required to achieve maximal growth and that the pH tolerance is reduced compared to that under aerobic conditions, reflecting a decreased capability for pH homeostasis. Continued growth under anaerobic conditions indicated the absence of an oxygen-requiring reaction that is essential for biomass formation. The results provide an improved understanding of the physiology of C. glutamicum under anaerobic conditions.     

Propionibacterium freudenreichii thrives in microaerobic conditions by complete oxidation of lactate to CO2

In this study we show increased biomass formation for four species of food-grade propionic acid bacteria (Acidipropionibacterium acidipropionici, Acidipropionibacterium jensenii, Acidipropionibacterium thoenii and Propionibacterium freudenreichii) when exposed to oxygen, implicating functional respiratory systems. Using an optimal microaerobic condition, P. freudenreichii DSM 20271 consumed lactate to produce propionate and acetate initially. When lactate was depleted propionate was oxidized to acetate. We propose to name the switch from propionate production to consumption in microaerobic conditions the ‘propionate switch’. When propionate was depleted the ‘acetate switch’ occurred, resulting in complete consumption of acetate. Both growth rate on lactate (0.100 versus 0.078 h⁻¹) and biomass yield (20.5 versus 8.6 g* mol⁻¹ lactate) increased compared to anaerobic conditions. Proteome analysis revealed that the abundance of proteins involved in the aerobic and anaerobic electron transport chains and major metabolic pathways did not significantly differ between anaerobic and microaerobic conditions. This implicates that P. freudenreichii is prepared for utilizing O₂ when it comes available in anaerobic conditions. The ecological niche of propionic acid bacteria can conceivably be extended to environments with oxygen gradients from oxic to anoxic, so-called microoxic environments, as found in the rumen, gut and soils, where they can thrive by utilizing low concentrations of oxygen.     

Effects of Thymol on Ruminal Microorganisms

Thymol (5-methyl-2-isopropylphenol) is a phenolic compound that is used to inhibit oral bacteria. Because little is known regarding the effects of this compound on ruminal microorganisms, the objective of this study was to determine the effects of thymol on growth and lactate production by the ruminal bacteria Streptococcus bovis JB1 and Selenomonas ruminantium HD4. In addition, the effect of thymol on the in vitro fermentation of glucose by mixed ruminal microorganisms was investigated. Neither 45 nor 90 μg/ml of thymol had any significant effect on growth or lactate production by S. bovis JB1, but 180 μg/ml of thymol completely inhibited growth and lactate production. In the case of S. ruminantium HD4, 45 μg/ml of thymol had little effect on growth and lactate production; however, 90 μg/ml of thymol completely inhibited growth of S. ruminantium HD4. Thymol also decreased glucose uptake by whole cells of both bacteria. When mixed ruminal microorganisms were incubated in medium that contained glucose, 400 μg/ml of thymol increased final pH and the acetate to propionate ratio and decreased concentrations of methane, acetate, propionate, and lactate. In conclusion, thymol was a potent inhibitor of glucose fermentation by S. bovis JB1 and S. ruminantium HD4. Even though thymol treatment decreased methane and lactate concentrations and increased final pH in mixed ruminal microorganism fermentations of glucose, concentrations of acetate and propionate were also reduced. Received: 13 May 2000 / Accepted: 14 June 2000     

Evaluation of metabolism using stoichiometry in fermentative biohydrogen

We first constructed full stoichiometry, including cell synthesis, for glucose mixed-acid fermentation at different initial substrate concentrations (0.8-6 g-glucose/L) and pH conditions (final pH 4.0-8.6), based on experimentally determined electron-equivalent balances. The fermentative bioH2 reactions had good electron closure (-9.8 to +12.7% for variations in glucose concentration and -3 to +2% for variations in pH), and C, H, and O errors were below 1%. From the stoichiometry, we computed the ATP yield based on known fermentation pathways. Glucose-variation tests (final pH 4.2-5.1) gave a consistent fermentation pattern of acetate + butyrate + large H2, while pH significantly shifted the catabolic pattern: acetate + butyrate + large H2 at final pH 4.0, acetate + ethanol + modest H2 at final pH 6.8, and acetate + lactate + trivial H2 at final pH 8.6. When lactate or propionate was a dominant soluble end product, the H2 yield was very low, which is in agreement with the theory that reduced ferredoxin (Fd(red)) formation is required for proton reduction to H2. Also consistent with this hypothesis is that high H2 production correlated with a high ratio of butyrate to acetate. Biomass was not a dominant sink for electron equivalents in H2 formation, but became significant (12%) for the lowest glucose concentration (i.e., the most oligotrophic condition). The fermenting bacteria conserved energy similarly at approximately 3 mol ATP/mol glucose (except 0.8 g-glucose/L, which had approximately 3.5 mol ATP/mol glucose) over a wide range of H2 production. The observed biomass yield did not correlate with ATP conservation; low observed biomass yields probably were caused by accelerated rates of decay or production of soluble microbial products.     

Metabolism of glucose and cellobiose by cellulolytic mesophilic Clostridium sp. strain H10.

The metabolism of strain H10, a cellulolytic mesophilic Clostridium sp., was studied on glucose and cellobiose as energy and carbon sources. The main products of fermentation of both sugars were acetate, lactate, and ethanol. At low sugar levels, molar growth yields were better for cellobiose than for glucose. In both cases, an inhibition of growth was observed between 1 and 2 g/liter and a total inhibition after the latter concentration. Inhibition was not the result of low pH due to acid formation; growth under static pH conditions was limited in the same way. On the other hand, acetate and lactate had no inhibitory effect when added at concentrations equal to the final titers. Concomitant with the inhibition of growth was a change in metabolic pathways for sugar concentrations between 1 and 2 g/liter, i.e., the production of lactate was higher. After complete inhibition of growth, an accumulation of carbohydrates which were neither glucose nor cellobiose was observed.     

Aerobic and anaerobic metabolism of Listeria monocytogenes in defined glucose medium.

A defined medium with glucose as the carbon source was used to quantitatively determine the metabolic end products produced by Listeria monocytogenes under aerobic and anaerobic conditions. Of 10 strains tested, all produced acetoin under aerobic conditions but not anaerobic conditions. Percent carbon recoveries of end products, typified by strain F5069, were as follows: lactate, 28%; acetate, 23%; and acetoin, 26% for aerobic growth and lactate, 79%; acetate, 2%; formate, 5.4%; ethanol, 7.8%; and carbon dioxide, 2.3% for anaerobic growth. No attempt to determine carbon dioxide under aerobic growth conditions was made. The possibility of using acetoin production to assay for growth of L. monocytogenes under defined conditions should be considered.     

Anaerobic cells of Bacillus cereus F4430/73 respond to low oxidoreduction potential by metabolic readjustments and activation of enterotoxin expression

In the present study, a food-borne pathogen strain of Bacillus cereus (F4430/73) was anaerobically grown in controlled-batch conditions under low initial oxidoreduction potential (ORP=–148 mV) using hydrogen gas as reducing agent. Its physiological characteristics, including growth, glucose fermentation capacity and enterotoxin production, were compared with anaerobic conditions generated by nitrogen gas (ORP=+ 45 mV). The results showed that low ORP affected growth mainly during the early stages. Maximal specific rates of growth and glucose consumption were reduced, and drastic changes in time profiles of fermentation product concentration were observed. Production of lactate was promoted at the expense of acetate. Nevertheless, low ORP did not affect final biomass yield. Under both ORP conditions, Non-haemolytic enterotoxin (Nhe) was produced early during the exponential growth phase as a first enterotoxin and Haemolysin BL (Hbl) later during the early stationary growth phase as a second enterotoxin. The major effect of low ORP was the strong stimulation of Hbl production and, to a lesser extent, Nhe production. This control was complex, involving different levels of regulation. We discussed the regulation of enterotoxin expression and the involvement of the pleiotropic regulator PlcR.     

Epitope mapping of monoclonal antibodies specific for the directly cross-linked mesodiaminopimelic acid peptidoglycan found in the anaerobic beer spoilage bacterium Pectinatus cerevisiiphilus.

Nineteen monoclonal antibodies (Mabs) were isolated based on reactivity with disrupted Pectinatus cerevisiiphilus cells. All of the Mabs reacted with cells from which the outer membrane had been stripped by incubation with sodium dodecyl sulphate, suggesting the peptidoglycan (PG) layer was involved in binding. Mab reactivity with purified PG confirmed this. Epitope mapping revealed the Mabs in total recognize four binding sites on the PG. Mabs specific for each of the four sites also bound strongly to disrupted Pectinatus frisingensis, Selenomonas lacticifix, Zymophilus paucivorans, and Zymophilus raffinosivorans cells, but weakly to disrupted Megasphaera cerevisiae cells. No antibody reactivity was seen with disrupted cells of 11 other species of Gram-negative bacteria. These results confirm that a common PG structure is used by several species of anaerobic Gram-negative beer spoilage bacteria. These results also indicate that PG-specific Mabs can be used to rapidly detect a range of anaerobic Gram-negative beer spoilage bacteria, provided the bacterial outer membrane is first removed to allow antibody binding.     

A kinetic model describing Shewanella oneidensis MR-1 growth, substrate consumption, and product secretion

Aerobic growth of Shewanella oneidensis MR-1 in minimal lactate medium was studied in batch cultivation. Acetate production was observed in the middle of the exponential growth phase and was enhanced when the dissolved oxygen (DO) concentration was low. Once the lactate was nearly exhausted, S. oneidensis MR-1 used the acetate produced during growth on lactate with a similar biomass yield as lactate. A two-substrate Monod model, with competitive and uncompetitive substrate inhibition, was devised to describe the dependence of biomass growth on lactate, acetate, and oxygen and the acetate growth inhibition across a broad range of concentrations. The parameters estimated for this model indicate interesting growth kinetics: lactate is converted to acetate stoichiometrically regardless of the DO concentration; cells grow well even at low DO levels, presumably due to a very low K(m) for oxygen; cells metabolize acetate (maximum specific growth rate, micro(max,A) of 0.28 h(-1)) as a single carbon source slower than they metabolize lactate (micro(max,L) of 0.47 h(-1)); and growth on acetate is self-inhibiting at a concentration greater than 10 mM. After estimating model parameters to describe growth and metabolism under six different nutrient conditions, the model was able to successfully estimate growth, oxygen and lactate consumption, and acetate production and consumption under entirely different growth conditions.     

Phylogenetic characterization of Centipeda periodontii, Selenomonas sputigena and Selenomonas species by 16S rRNA gene sequence analysis.

The nearly complete 16S rRNA gene sequences for oral Gram-negative anaerobic motile bacteria, Centipeda periodontii, Selenomonas sputigena and Selenomonas species (formerly S. sputigena type strain), were determined in order to unveil their relationship to other oral motile bacteria. To determine the phylogenetic characterization of these bacteria, their 16S rRNA gene sequences were obtained and compared with those from the ribosomal sequence databases previously reported. The 16S rRNA gene sequences of these bacteria were similar to those of Selenomonas ruminantium and Schwartzia succinivorans isolated from rumens, and to Pectinatus cerevisiiphilus isolated from spoiled beer. Among oral bacteria, the nucleotide sequence analysis of these bacteria revealed high nucleotide similarity to Veillonella species, whereas low similarity to oral motile bacteria such as Campylobacter species. Phylogenetic analysis clearly confirmed that C. periodontii and two Selenomonas species were classified as relatives of a group besides Selenomonas, Schwartzia, and Pectinatus species, and not as close relatives to oral motile bacteria, such as Campylobacter species. These results suggest that such oral Gram-negative anaerobic motile bacteria are close relatives of oral bacteria.     

Physiological characterization of brewer’s yeast in high-gravity beer fermentations with glucose or maltose syrups as adjuncts

High-gravity brewing, which can decrease production costs by increasing brewery yields, has become an attractive alternative to traditional brewing methods. However, as higher sugar concentration is required, the yeast is exposed to various stresses during fermentation. We evaluated the influence of high-gravity brewing on the fermentation performance of the brewer’s yeast under model brewing conditions. The lager brewer’s strain Weihenstephan 34/70 strain was characterized at three different gravities by adding either glucose or maltose syrups to the basic wort. We observed that increased gravity resulted in a lower specific growth rate, a longer lag phase before initiation of ethanol production, incomplete sugar utilization, and an increase in the concentrations of ethyl acetate and isoamyl acetate in the final beer. Increasing the gravity by adding maltose syrup as opposed to glucose syrup resulted in more balanced fermentation performance in terms of higher cell numbers, respectively, higher wort fermentability and a more favorable flavor profile of the final beer. Our study underlines the effects of the various stress factors on brewer’s yeast metabolism and the influence of the type of sugar syrups on the fermentation performance and the flavor profile of the final beer.     

Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase.

Mutants of Escherichia coli deficient in the fermentative NAD-linked lactate dehydrogenase (ldh) have been isolated. These mutants showed no growth defects under anaerobic conditions unless present together with a defect in pyruvate formate lyase (pfl). Double mutants (pfl ldh) were unable to grow anaerobically on glucose or other sugars even when supplemented with acetate, whereas pfl mutants can do so. The ldh mutation was found to map at 30.5 min on the E. coli chromosome. The ldh mutant FMJ39 showed no detectable lactate dehydrogenase activity and produced no lactic acid from glucose under anaerobic conditions as estimated by in vivo nuclear magnetic resonance measurements. We also found that in wild-type strains the fermentative lactate dehydrogenase was conjointly induced by anaerobic conditions and an acidic pH. Despite previous findings that phosphate concentrations affect the proportion of lactic acid produced during fermentation, we were unable to find any intrinsic effect of phosphate on lactate dehydrogenase activity, apart from the buffering effect of this ion.     

Effect of mixed carbon substrate on exopolysaccharide production of cyanobacterium Nostoc flagelliforme in mixotrophic cultures

The effects of organic carbon sources on cell growth and exopolysaccharide (EPS) production of dissociated Nostoc flagelliforme cells under mixotrophic batch culture were investigated. After 7?days of cultivation, glycerol, acetate, sucrose, and glucose increased the final cell density and final EPS concentrations, and mixotrophic growth achieved higher biomass concentrations. The increase in cell growth was particularly high when glucose was added as the sole carbon source. On the other hand, EPS production per dry cell weight was significantly enhanced by adding acetate. For more effective EPS production, the effects of the mixture of glucose and acetate were investigated. Increasing the ratio of glucose to acetate resulted in higher growth rate with BG-11 medium and higher EPS productivity with BG-11₀ medium (without NaNO₃). When the medium was supplemented with a mixture of glucose (4.0?g?L^?1) and acetate (2.0?g?L^?1), 1.79?g?L^?1 biomass with BG-11 medium and 879.6?mg?L^?1 of EPS production with BG-11₀ medium were achieved. Adopting this optimal ratio of glucose to acetate established in flask culture, the culture was also conducted in a 20-L photobioreactor with BG-11 medium for 7?days. A maximum biomass of 2.32?g?L^?1 was achieved, and the EPS production was 634.6?mg?L^?1.     

The kinetics of glucose fermentation bySelenomonas ruminantium HD4 grown in continuous culture

Summary The production of organic acids (acetate, lactate, and propionate) by the anaerobic, ruminal bacteriumSelenomonas ruminantium HD4 was investigated in both glucose-limited and glucose-sufficient (phosphate-limited) continuous cultures. The fermentation pattern of products exhibited a shift upon release of glucose limitation from acetate and propionate to lactate at a dilution rate of 0.2 h^–1. Glucose sufficiency brought about two-to fourfold increase in specific glucose utilization rate, lactate productivity, and lactate yield relative to glucose-limited growth conditions. The increased lactate production under glucose-sufficient growth conditions was attributed to the overutilization of excess glucose.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Effect of environmental pH on the fermentation balance of Lactobacillus reuteri

The influence of environmental pH on the regulation of glucose catabolism by Lactobacillus reuteri was examined in anaerobic batch cultures. Under acidic conditions both glucose consumption and end-products formation were low. Maximum biomass was reached at pH 5·0, with a specific growth rate of µ= 0·78 h^-1. The shift in pH values from 4.3 to 6.5 reflected an increase in glucose uptake as well as in the yield ( Y _p/x) of acetate, lactate and ethanol after 12 h of incubation. Ethanol was the major metabolite produced at all pH values assayed.     

Studies of the Acetate Kinase-Phosphotransacetylase and the Butanediol-Forming Systems in Aerobacter aerogenes

Mutants of Aerobacter aerogenes devoid of acetate kinase and phosphotransacetylase activities were isolated by selection for resistance to fluoroacetate on lactate medium. The mutants were used to study the role of the acetate kinase-phosphotransacetylase system in growth on acetate and glucose. Acetate kinase-negative and phosphotransacetylase-negative mutants were unable to grow on acetate minimal medium. Their growth rates on glucose minimal medium were identical with that of the parent strain under aerobic conditions, but lower growth rates were observed in the mutant strains during anaerobic growth on glucose medium. The mutants were unable to incorporate [2-(14)C]-acetate rapidly while growing on glycerol. Variations in acetate kinase and phosphotransacetylase levels during growth on glucose were studied. The specific activities of the enzymes increased approximately fivefold during aerobic growth on glucose in batch culture. The enzyme levels were also studied during anaerobic growth on glucose at constant pH (pH 5.8 and 7.0). Smaller increases in specific activities were found under these conditions. The role of acetate in the induction of the diacetyl (acetoin) reductase was investigated using a mutant deficient in both acetate kinase and phosphotransacetylase. The effect of pH on the induction of this enzyme during growth on glucose under anaerobic conditions was tested. The data support the idea that free acetic acid is the inducer for the enzymes of the butanediol-forming pathway in A. aerogenes.     

Multiple growth inhibition of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> in 1,3-propanediol fermentation

The inhibition of substrate and product on the growth of Klebsiella pneumoniae in anaerobic and aerobic batch fermentation for the production of 1,3-propanediol was studied. The cells under anaerobic conditions had a higher maximum specific growth rate of 0.19 h^–1 and lower tolerance to 110 g glycerol l^–1, compared to the maximum specific growth rate of 0.17 h^–1 and tolerance to 133 g glycerol l^–1 under aerobic conditions. Acetate was the main inhibitory metabolite during the fermentation under anaerobic conditions, with lactate and ethanol the next most inhibitory. The critical concentrations of acetate, lactate and ethanol were assessed to be 15, 19, 26 g l^–1, respectively. However, cells grown under aerobic conditions were more resistant to acetate and lactate but less resistant to ethanol. The critical concentrations of acetate, lactate and ethanol were assessed to be 24, 26, and 17 g l^–1, respectivelyRevisions requested 8 september; Revisions received 2 November 2004