Rapid microbial growth in a pH auxostat

Summary Feeding nutrient to meet demand dilutes slow-growing organisms from continuous culture and greatly favors rapid growth. Doubling times of roughly 10 minutes have been verified in a pH auxostat by viable cell counts and by direct counting with a Petroff-Hausser chamber. Effects of wall attachment were negligible because a fresh reactor was substituted frequently before wall growth could become established.     

Characteristics of some fermentative bacteria from a thermophilic methane-producing fermenter

Anaerobic bacteria from a 55C methane-producing beef waste fermenter were enumerated, isolated, and characterized. Direct microscopic bacterial counts were 5.2–6.8×10¹⁰ per g fermenter effluent. Using a nonselective roll-tube medium which contained 40% fermenter effluent, 8.5–14.1% of the microscopic count was culturable. Deletion of fermenter effluent significantly reduced the viable count. Sixty-four randomly picked strains were characterized. All were pleomorphic, gram-negative, anaerobic rods, many of which were difficult to grow in liquid media. The strains were divided into 5 major groups based on glucose fermentation, hydrogen sulfide production, starch hydrolysis, fermentation products, and morphology. Glucose was fermented by 75% of the isolates, 76% utilized starch, 25% produced hydrogen sulfide, 76% produced hydrogen, 37% produced indole, 21% hydrolyzed gelatin, and 13% were sporeformers. Ethanol, lactate, formate, acetate, and hydrogen were common fermentation products. Twenty-four representative strains had 1–12 flagella. Growth was observed between 35 and 73C. These studies indicate that species diversity among the isolated organisms was low.     

Development of a multivariable auxostat

Summary Auxostat operation was extended to two set points in an aerobic yeast fermentation of Saccharomyces cerevisiae. The concentration of NH₄ ⁺ was controlled directly with an on-line NH₃ probe and control of pH indirectly fixed the glucose concentration, thus demonstrating an NH₄ ⁺:pH-auxostat. Multivariable control in the auxostat was superior to a chemostat for studying nutrient interaction and exploring multisubstrate limitations on metabolism.     

Stable carbon isotope fractionation by acetotrophic sulfur-reducing bacteria

Acetate is the most important intermediate in anaerobic degradation of organic matter. The carbon isotope effects associated with the oxidation of acetate (ɛ_ac) were examined for four acetotrophic sulfur reducers, Desulfuromonas acetoxidans, Desulfuromonas thiophila, Desulfurella acetivorans , and Hippea maritima . During the consumption of acetate and sulfur, acetate was enriched in ¹³C by 11.5 and 11.2‰ in Desulfuromonas acetoxidans and Desulfuromonas thiophila , respectively. By contrast, isotope fractionation in D. acetivorans and H. maritima resulted in isotope enrichment factors of ɛ_ac=−6.3‰ and −8.4‰, respectively. These sulfur-reducing bacteria all metabolize acetate via the tricarboxylic acid cycle, but have different mechanisms for the initial activation of acetate. In Desulfuromonas acetoxidans , acetyl-CoA is formed by succinyl-CoA : acetate-CoA-transferase, and in D. acetivorans by acetate kinase and phosphate acetyltransferase. Hence, values of ɛ_ac seem to be characteristic for the type of activation of acetate to acetyl-CoA in acetotrophic sulfur reducers. Summarizing ɛ_ac-values in anaerobic acetotrophic microorganisms, it appears that isotope fractionation depends on the mechanism of acetate activation to acetyl-CoA, on the key enzyme of the acetate dissimilation pathway, and on the bioavailability of acetate, which all have to be considered when using δ¹³C of acetate in environmental samples for diagnosis of the involved microbial populations.     

Commensalism of methane-producing and motile aerobic bacteria in certain freshwater wetlands

It is pointed out that the methane flux measured experimentally for certain ponds and swamps is quantitatively consistent with a commensal dependence of Methanobacteria on O₂-chemotactic motile aerobic bacteria. The Methano species is thereby shielded from oxygen and provided with carbon dioxide for the anaerobic production of methane.     

A study of mixed continuous cultures of sulfate-reducing and methane-producing bacteria

Ecological relationships between sulfate-reducing and methane-producing bacteria in mud of Lake Vechten have been studied by continuous culture studies using the chemostat technique. The maximum specific growth rate (μ _max) and saturation constant (K _s) were, respectively, 0.36 hr⁻¹ and 0.047 mM for lactate-limited growth ofDesulfovibrio desulfuricans and 0,011 hr⁻¹ and 0.17 mM for acetate-limited growth ofMethanobacterium sp. Calculated values for the true molar growth yieldsY _G) and maintenance coefficients (m) were 30.6 g bacterial mass/mole of lactate and 0.53 g substrate/g dry wt hr forD. desulfuricans and 37.8 g bacterial mass/mole of acetate and 0.54 g substrate/g dry wt hr forMethanobacterium. No growth ofMethanobacterium was observed at apS²⁻ value (the hydrogen sulfide potential) of more than 11 and there was no effect on the growth atpS²⁻ values above 13. In mixed continuous culture experiments the concentration of acetate decreased in the secondstage growth vessel, whereas that of methane increased stoichiometrically. If the substrate concentration in the reservoirs (S _r) was increased from 0.1 to 0.5 mg/ml, the population ofDesulfovibrio increased and that ofMethanobacterium was washed out of the culture vessel, since the concentration of hydrogen sulfide reached apS²⁻ value of 10.5. From the mixed continuous culture experiments a commensalism between the two species can be described, i.e., the acetate-fermentingMethanobacterium benefits from the acetate released byDesulfovibrio which is, in turn, not affected in the presence of the former.     

Methane production from organic acid-rich wastewaters by immobilized thermophilic methane-producing bacteria

Thermophilic methane-producing bacteria isolated from a wastewater treatment facility have been immobilized in acetylcellulose filter with agar. The immobilized cells produced methane from wastewaters in rich organic acid (acetic, propionic and butyric acids) at the rate of 1.4 μmol mg protein⁻¹ h⁻¹. The optimum conditions for methane production by immobilized whole cells were 52–55°C and pH 7.0–8.0. The immobilized cells retained 80% of the initial activity after exposure to air. The immobilized thermophilic bacteria produced methane continuously over 10 days at 52°C.     

Interrelations between sulfate-reducing and methane-producing bacteria in bottom deposits of a fresh-water lake. II. Inhibition experiments

A possible substrate interrelationship between methane-producing and sulfate-reducing bacteria has been studied in bottom deposits of Lake Vechten. Inhibition of methanogenesis in mud samples by chlorine-containing analogues of methane resulted in accumulation of acetate. Fluoroacetate reduced the concentration of methane by about 75%. With carbon tetrachloride, accumulation of hydrogen gas was observed. These results indicate that acetate is the main precursor of methanogenesis in mud. After addition of β-fluorolactate, lactate accumulated and H₂S was no longer produced, which indicates that lactate is the main source of energy for sulfate reduction in mud. At the same time the concentration of methane increased possibly due to the lower concentration of H₂S, which has a toxic effect on methanogenesis. Experiments with intact mud cores provide evidence that the described phenomena occur also in situ.     

Interrelations between sulfate-reducing and methane-producing bacteria in bottom deposits of a fresh-water lake. I. Field observations

Observations on the seasonal periodicity in bottom deposits of Lake Vechten indicated an ecological relationship between sulfate-reducing and methane-producing bacteria. Sulfate reducers are most abundant at depths of 0 to 2 cm in the mud at pS^2- values of about 11 and redox potential values of-100 to-150 mV. Maximum number of methane producers are situated at depths of 3 to 6 cm in the mud at pS^2- values of about 14, redox potential values of-250 to-300 mV and maximum values of the methane concentration. During summer stratification the numbers of bacteria increased considerably. However the number of methane producers rose much more than that of the sulfate reducers. Sulfate in the interstitial water of the sediments is reduced by the sulfate reducers and the sulfate concentration limited the latter's abundance. Methane producers are found deeper in the mud at lower concentrations of hydrogen sulphide. Therefore the different localities of the two bacterial groups may be due to sensitivity of methane producers to hydrogen sulphide. Differential counting of the mixed population of methane-producing bacteria showed that acetate-and methyl-alcohol-fermenting types are most abundant at a depth of 5, and formate-and CO₂/H₂-fermenting types at a depth of 3 cm in the mud.     

Isolation and characterization of a thermophilic acetotrophic strain of Methanothrix

An enrichment culture which converted acetate to methane at 60°C was obtained from a thermophilic anaerobic bioreactor. The predominant morphotype in the enrichment was a sheathed gas-vacuolated rod with marked resemblence to the mesophile Methanothrix soehngenii. This organism was isolated using vancomycin treatments and serial dilutions and was named Methanothrix sp. strain CALS-1. Strain CALS-1 grew as filaments typically 2–5 cells long, and cultures showed opalescent turbidity rather than macroscopic clumps. The cells were enclosed in a striated subunit-type sheath and there were distinct cross-walls between the cells, similar to M. soehngenii. The gas vesicles in cells were typically 70 nm in diameter and up to 0.5 m long, and were collapsed by pressures over 3 atm (ca. 300 kPa). Stationary-phase cells tended to have a higher vesicle content than did growing cells, and occasionally bands of cells were seen floating at the top of the liquid in stationary-phase cultures. Acetate was the only substrate of those tested which was used for methanogenesis by strain CALS-1, and acetate was decarboxylated by the aceticlastic reaction. The optimum temperature for growth of strain CALS-1 was near 60°C (doubling time=24–26 h), with no growth occurring at 70°C and 37°C. The optimum pH value for growth was near 6.5 in bicarbonate/CO₂ buffered medium and no growth occurred at pH 5.5 or pH 8.4. No growth was obtained below pH 7 when the medium was buffered with 20 mM phosphate. Strain CALS-1 grew in a chemically defined medium and required biotin. Sulfide concentrations over 1 mM were inhibitory to the culture, and growth was more rapid with 1 mM 2-mercaptoethane sulfonate (coenzyme M) or 1 mM titanium citrate as an accessory reductant than with 1 mM cysteine. It is likely that strain CALS-1 represents a new species in the genus Methanothrix.     

Interrelations between sulfate-reducing and methane-producing bacteria in bottom deposits of a fresh-water lake. III. Experiments with 14C-labeled substrates

An ecological substrate relationship between sulfate-reducing and methane-producing bacteria in mud of Lake Vechten has been studied in experiments using ¹⁴C-labeled acetate and lactate as substrates. Fluoroacetate strongly inhibited the formation of ¹⁴CO₂ from [U-¹⁴C]-acetate and β-fluorolactate gave an inhibition of similar magnitude of the breakdown of [U-¹⁴C]-l-lactate to ¹⁴CO₂ thus confirming earlier results on the specific action of these inhibitors. The turnover-rate constant of l-lactate was 2.37 hr^-1 and the average l-lactate pool size was 12.2 μg per gram of wet mud, giving a turnover rate of 28.9 μg of lactate/gram of mud per hr. The turnover-rate constant of acetate was 0.35 hr^-1 and the average pool size was 5.7 μg per gram of wet mud, giving a rate of disappearance of 1.99 μg of acetate/gram of mud per hr. Estimations of the acetate turnover rate based upon the formation of ¹⁴CO₂ from [U-¹⁴C]-acetate or [1-¹⁴C]-acetate yielded figures of the same magnitude (range 0.45 to 1.74). These and other results suggest that only a portion of the lactate dissimilated is turned over through the acetate pool. The ratio of ¹⁴CO₂/¹⁴CH₄ produced from [U-¹⁴C]-acetate by mud was 1.32; indicating that 0.862 moles of CH₄ and 1.138 moles of CO₂ are formed per mole of acetate. From the rate of disappearance of acetate (0.027 μmoles/gram wet mud per hr) and the rate of methane production (0.034 μmoles/gram wet mud per hr), it may be concluded that acetate is an important precursor of methanogenesis in mud (approximately 70%). A substrate relationship between the two groups of bacteria is likely since ¹⁴CH₄ was formed from [U-¹⁴C]-l-lactate.     

Acceleration of mass transfer in methane-producing loop reactors

Gas bubbles entrapped in methanogenic granules subjected to hydrostatic pressure oscillations during recirculation in loop reactors will induce intraparticle liquid flows, and thereby enhance mass transfer in excess of diffusion. This breathing particle concept was clearly demonstrated in a well defined inorganic model system. The experimental results could be described satisfactory with a structured mathematical model, while a 30% improvement is predicted for methanogenic loop reactors as compared to constant pressure systems. It is concluded that acceleration of mass transfer in gas-producing systems offers challenging perspectives for both heterogeneous catalysis and biological fermentations.     

Growth determinations for unattached bacteria in a contaminated aquifer

Growth rates of unattached bacteria in groundwater contaminated with treated sewage and collected at various distances from the source of contamination were estimated by using frequency of dividing cells and tritiated-thymidine uptake and compared with growth rates obtained with unsupplemented, closed-bottle incubations. Estimates of bacterial generation times [(In 2)/mu] along a 3-km-long transect in oxygen-depleted (0.1 to 0.7 mg of dissolved oxygen liter-1) groundwater ranged from 16 h at 0.26 km downgradient from an on-land, treated-sewage outfall to 139 h at 1.6 km and correlated with bacterial abundance (r2 = 0.88 at P less than 0.001). Partitioning of assimilated thymidine into nucleic acid generally decreased with distance from the contaminant source, and one population in heavily contaminated groundwater assimilated little thymidine during a 20-h incubation. Several assumptions commonly made when frequency of dividing cells and tritiated-thymidine uptake are used were not applicable to the groundwater samples.