Mathematical modeling for mixed culture growth of two bacterial populations with opposite substrate preferences

An unstructured mathematical model is proposed for mixed culture growth of two different bacterial species that exhibit "opposite" substrate preferences in response to the "same" environmental conditions. The model incorporates enzymatic control mechanisms such as induction, repression, and inhibition in the microorganisms as manifested in their preferential utilization of substrates and microbial interactions such as amensalism and competition. The model predicts cell mass, substrate concentrations, dissolved oxygen tension, as well as key enzyme levels. The predictions of the model are compared with experimental data for pure culture growth and for mixed culture growth on two substrates, glucose and citrate, in a batch reactor.     

Competition of marine psychrophilic bacteria at low temperatures

The occurrence of obligately and facultatively psychrophilic bacteria in the marine environment suggests that environmental conditions exist which can favour each of these groups in competitive processes. Differences were found in the way in which temperature affected the growth rates of obligate and facultative psychrophiles. Maximum specific growth rates of a number of obligately and facultatively psychrophilic bacteria were determined in batch culture and competition experiments were carried out in a chemostat at growth-limiting substrate concentrations. From the results the relation between the specific growth rate and the concentration of the growth-limiting substrate for both types of organisms at different temperatures was deduced. Both at low and high substrate concentrations obligate psychrophiles grew faster than facultative psychrophiles at the lower temperature extreme (? 4 C). These results suggest that obligately psychrophilic chemoorganotrophs are responsible for mineralization processes in cold natural environments such as ocean waters and the arctic and antarctic regions. In these environments they can successfully compete with facultative psychrophiles because they can grow faster.     

Reactivity versus flexibility in thiobacilli

The results of ecophysiological studies on obligately and facultatively chemolithotrophic thiobacilli performed over the past years clearly show that the two types of organisms occupy different ecological niches. Chemostat experiments with cultures of the obligate chemolithotroph Thiobacillus neapolitanus and the facultative chemolithotroph Thiobacillus A2 have been carried out to explain the competitiveness of T. neapolitanus under conditions of strongly fluctuating substrate supply. Thiobacillus neapolitanus appeared to be very resistant to starvation periods whereafter it could oxidize sulfide (or thiosulfate) almost instantaneously at the original rate. Under alternate supply of 4 h sulfide and 4 h sulfate (or acetate which does not support growth of the organism either) to a chemostat culture of T. neapolitanus (D=0.05 h^–1) the sulfide concentration in the growth vessel never reached levels higher than 4m. This strategy is aimed at maximal reactivity. In contrast to T. neapolitanus the facultative chemolithotroph T.A2 appeared to be very flexible with respect to its energy generation. Under alternate supply of 4 h sulfide and 4 h acetate (D=0.05 h^–1) T.A2 was able to grow continuously since it directed its metabolism to either heterotrophy or autotrophy by rapid induction-repression mechanisms. This flexible strategy seems to be incompatible with a reactive strategy within one organism, since the oxidation capacity for sulfide decreased during the acetate period resulting in accumulation of sulfide during the sulfide period. It is concluded that T.A2 needs a continuous supply of an inorganic and an organic substrate to thrive whereas T. neapolitanus needs only a continuous supply of a reduced inorganic sulfur source but also will persist in environments with interrupted addition of sulfide provided that the starvation period does not last too long.     

Energy requirement for maintenance of the transmembrane potassium gradient in Klebsiella aerogenes NCTC 418: A continuous culture study

Molar growth yield studies on chemostat cultures of Thiobacillus neapolitanus grown in thiosulfate-minerals medium have confirmed earlier observations that the dry weight increased linearly with the dilution rate. The observed increase can be explained neither by a change in cell composition nor by the observed excretion of organic compounds. The increase of the molar growth yield over the full range of growth rates, that is also observed in other obligate chemolithotrophs, was not found in the facultatively chemolithotrophic Thiobacillus A2, grown on thiosulfate or formate. The interpretation of the results in terms of maintenance energy requirement is discussed. It is concluded that these results do not allow a mathematical treatment according to the empirical formula of Pirt.Abbreviation APS Adenosine-5-phosphosulfate     

Isolation and characterization of Thiobacillus hydrothermalis sp. nov., a mesophilic obligately chemolithotrophic bacterium isolated from a deep-sea hydrothermal vent in Fiji Basin

A novel obligately chemolithotrophic Thiobacillus species isolated from a deep-sea hydrothermal vent is described. This organism grows lithoautotrophically on thiosulphate, tetrathionate, sulphide and sulphur which are oxidized to sulphate. The isolate is slightly halophilic and markedly halotolerant, showing optimum growth at pH 7.5 and at 35°C. The G+C content of the DNA is 67.1 mol%. The 16S rRNA sequence is distinct from any other Thiobacilli sequences. Phylogenetic analysis shows the organism to be a representative of the -group of proteobacteria and a specific relative of Thiobacillus neapolitanus. The ubiquinone is ubiquinone-8. These characters distinguish the isolate from any other Thiobacillus or Thiomicrospira species previously reported and is a new species described as Thiobacillus hydrothermalis. The type strain is isolate R3, DSM7121.     

Predicting stability of mixed microbial cultures from single species experiments: 2. Physiological model

In this paper, we study the equilibria of a physiological model describing the continuous culture in which two microbial populations compete for two substitutable resources. This work is an extension of the stability analysis of the phenomenological model of mixed microbial growth [M.M. Ballyk, G.S.K. Wolkowicz, Exploitative competition in the chemostat for two perfectly substitutable resources, Math. Biosci. 118 (1993) 127-180; S.S. Pilyugin, G.T. Reeves, A. Narang, Predicting stability of mixed microbial cultures from single species experiments: 2. Phenomenological model]. Here, we investigate the influence of the peripheral enzymes that catabolize the substrate uptake on the stability of the mixed culture. We show that, under steady state conditions, an increase in the concentration of one substrate inhibits the uptake of the other substrate(s). We present the criteria for existence, uniqueness, and stability of various types of equilibria. We formulate these criteria in terms of growth isoclines and consumption curves for each of the competing species. Since both types of curves can be obtained from a single species experiment, our approach provides a direct connection between theory and experiment and allows one to infer the dynamics of mixed cultures from the dynamics of single species cultures. By expressing the stability criteria in terms of intracellular properties, the model establishes a link between ecology and molecular biology.     

A model for continuous fermentations with amylolytic yeasts

A two-stage, associative fermentation process is more effective for continuous yeast biomass production from starch than a single-stage mixed culture fermentation process. By operating two stages, competition for the same growth limiting substrate is reduced leading to efficient starch utilization. In this article, a mathematical model has been proposed for continuous, two-stage fermentation with a pure culture, amylolytic yeast in the first stage and a mixed culture second stage with a faster growing, nonamylolytic yeast. The model parameters were determined for Saccharomycopsis fibuligera and Candida utilis in continuous, single-stage, pure cultures. In the two-stage model, the effects of changes in dilution rate on biomass, amylase, reducing sugar, and starch concentration, and ratio of stage volumes on microbial composition are discussed and compared with experimental data.     

Predicting stability of mixed microbial cultures from single species experiments: 1. Phenomenological model

The growth of mixed microbial cultures on mixtures of substrates is a fundamental problem of both theoretical and practical interest. On the one hand, the literature is abundant with experimental studies of mixed-substrate phenomena [T. Egli, The ecological and physiological significance of the growth of heterotrophic microorganisms with mixtures of substrates, Adv. Microbiol. Ecol. 14 (1995) 305-386]. On the other hand, a number of mathematical models of mixed-substrate growth have been analyzed in the last three decades. These models typically assume specific kinetic expressions for substrate uptake and biomass growth rates and their predictions are formulated in terms of parameters of the model. In this work, we formulate and analyze a general mathematical model of mixed microbial growth on mixtures of substitutable substrates. Using this model, we study the effect of mutual inhibition of substrate uptake rates on the stability of the equilibria of the model. Specifically, we address the following question: How much of the dynamics exhibited by two competing species can be inferred from single species data? We provide geometric criteria for stability of various types of equilibria corresponding to non-competitive exclusion, competitive exclusion, and coexistence of two competing species in terms of growth isoclines and consumption curves. A growth isocline is a curve in the plane of substrate concentrations corresponding to the zero net growth of a given species. In [G.T. Reeves, A. Narang, S.S. Pilyugin, Growth of mixed cultures on mixtures of substitutable substrates: The operating diagram for a structured model, J. Theor. Biol. 226 (2004) 143-157], we introduced consumption curves as sets of all possible combinations of substrate concentrations corresponding to balanced growth of a single microbial species. Both types of curves can be obtained in single species experiments.     

Chemolithotrophic haloalkaliphiles from soda lakes

This paper summarizes recent data on the occurrence and properties of lithotrophic prokaryotes found in extremely alkaline, saline (soda) lakes. Among the chemolithotrophs found in these lakes the obligately autotrophic sulfur-oxidizing bacteria were the dominant, most diverse group, best adapted to haloalkaline conditions. The culturable forms are represented by three new genera, Thioalkalimicrobium, Thioalkalivibrio and Thioalkalispira in the Gammaproteobacteria. Among them, the genus Thioalkalivibrio was most metabolically diverse, including denitrifying, thiocyanate-oxidizing and facultatively alkaliphilic species. Culturable methane-oxidizing populations in the soda lakes belong to the type I methanotroph group in the Gammaproteobacteria, mostly in the genus Methylomicrobium. The nitrifying bacteria in hyposaline soda lakes were represented by a new species Nitrobacter alkalicus (Alphaproteobacteria), and by an alkaliphilic subspecies of Nitrosomonas halophila (Betaproteobacteria). Both belonged to the low salt-tolerant alkaliphiles. The facultatively autotrophic haloalkaliphilic isolates able to grow with hydrogen as electron donor were identified as representatives of the alpha-3 subclass of the Proteobacteria (aerobic) and of the Natronolimnicola - Alkalispirillum group in the gammaproteobacteria (nitrate-reducing). While all chemolithotrophic isolates from soda lakes belong to the alkaliphiles with a pH optimum for growth around 10, only the sulfur-oxidizing group included species able to grow under hypersaline conditions. This indicates that carbon and nitrogen cycles in the hypersaline alkaline lakes might not be closed.     

Breakdown of dimethyl sulphide by mixed cultures and by Thiobacillus thioparus

Abstract Dimethyl sulphide (DMS) was degraded by acclimatized activated sludge and by a mixed culture of Thiobacillus thioparus TK-1 and Pseudomonas sp. AK-2. While both these organisms persisted in stable co-culture on DMS, it was found that T. thioparus TK-1 and the derived strain TK-m grew in pure culture on DMS, and oxidized DMS with an apparent K _m of 4.5 × 10⁻⁵ M. During growth, all the DMS-sulphur was oxidized stoichiometrically to sulphate but no methanol was detected in pure cultures of TK-m. DMS-carbon was probably converted to CO₂, since the fixation of ¹⁴CO₂ was progressively diluted during growth of a culture on ¹⁴CO₂ and DMS. Growth yields were consistent with autotrophic growth, dependent on the oxidation of the methyl residues to CO₂ (probably with formaldehyde as a first intermediate) and the sulphide to sulphate. The organism thus appears to exhibit a mixture, from the one substrate, of chemolithotrophic and methylotrophic energy generation supporting autotrophic growth with CO₂ fixation.     

Isolation and identification of rumen bacteria capable of anaerobic phloroglucinol degradation.

Eight strains of rumen bacteria capable of degrading phloroglucinol (1,3,5-trihydroxybenzene) under anaerobic conditions were isolated from enrichment cultures of the bovine rumen microflora established in a prereduced medium containing 0.02 M phloroglucinol. Five of the strains were facultatively anaerobic Gram-positive streptococci which were identified as Streptococcus bovis. Three strains of obligately anaerobic Gram-positive cocci were assigned to the genus Coprococcus. Anaerobic cultures of the Streptococcus bovis strains in a 40% rumen fluid medium initially containing 0.02 M phloroglucinol degraded 50-80% of the substrate within 2 days, whereas cultures of the Coprococcus strains degraded more than 80% of the substrate under the same conditions. The Streptococcus bovis strains were incapable of degrading phloroglucinol in brain heart infusion or in the medium of de Man, Rogosa, and Sharpe (MRS broth) incubated aerobically.     

Isolation and characterisation of Thiobacillus halophilus sp. nov., a sulphur-oxidising autotrophic eubacterium from a Western Australian hypersaline lake

The isolation of a novel obligately chemolithotrophic, halophilic and extremely halotolerant Thiobacillus from a hypersaline lake is described. Attempts to demonstrate sulphur- and ferrous iron-oxidizing chemolithotrophs in neighbouring hypersaline lakes were unsuccessful. The organism isolated differs from any other Thiobacillus species previously described and is formally named as Thiobacillus halophilus. It possesses ribulose bisphosphate carboxylase and grows chemolithoautotrophically on thiosulphate, tetrathionate and sulphur, oxidising them to sulphate. Kinetic constants for oxidation of sulphide, thiosulphate, trithionate and tetrathionate are presented. The organism is obligately halophilic, growing best with 0.8–1.0 M NaCl, and tolerating up to 4 M NaCl. Optimum growth was obtained at about 30° C and pH 7.0–7.3. It contains ubiquinone Q-8 and its DNA contains 45 mol % G+C. Organisms of this type might contribute significantly to the autotrophic fixation of carbon dioxide in some hypersaline extreme environments of the kind described.     

Heterotrophic growth of Thiobacillus acidophilus in batch and chemostat cultures

Heterotrophic growth of the facultatively chemolithoautotrophic acidophile Thiobacillus acidophilus was studied in batch cultures and in carbon-limited chemostat cultures. The spectrum of carbon sources supporting heterotrophic growth in batch cultures was limited to a number of sugars and some other simple organic compounds. In addition to ammonium salts and urea, a number of amino acids could be used as nitrogen sources. Pyruvate served as a sole source of carbon and energy in chemostat cultures, but not in batch cultures. Apparently the low residual concentrations in the steady-state chemostat cultures prevented substrate inhibition that already was observed at 150 M pyruvate. Molar growth yields of T. acidophilus in heterotrophic chemostat cultures were low. The Y _max and maintenance coefficient of T. acidophilus grown under glucose limitation were 69 g biomass · mol^–1 and 0.10 mmol · g^–1 · h^–1, respectively. Neither the Y _max nor the maintenance coefficient of glucose-limited chemostat cultures changed when the culture pH was increased from 3.0 to 4.3. This indicates that in T. acidophilus the maintenance of a large pH gradient is not a major energy-requiring process. Significant activities of ribulose-1,5-bisphosphate carboxylase were retained during heterotrophic growth on a variety of carbon sources, even under conditions of substrate excess. Also thiosulphate- and tetrathionate-oxidising activities were expressed under heterotrophic growth conditions.     

Oxidative metabolism of inorganic sulfur compounds by bacteria

The history of the elucidation of the microbiology and biochemistry of the oxidation of inorganic sulfur compounds in chemolithotrophic bacteria is briefly reviewed, and the contribution of Martinus Beijerinck to the study of sulfur-oxidizing bacteria highlighted. Recent developments in the biochemistry, enzymology and molecular biology of sulfur oxidation in obligately and facultatively lithotrophic bacteria are summarized, and the existence of at least two major pathways of thiosulfate (sulfur and sulfide) oxidation confirmed. These are identified as the Paracoccus sulfur oxidation (or PSO) pathway and the S4intermediate (or S4I) pathway respectively. The former occurs in organisms such as Paracoccus (Thiobacillus) versutus and P. denitrificans, and possibly in Thiobacillus novellus and Xanthobacter spp. The latter pathway is characteristic of the obligate chemolithotrophs (e.g. Thiobacillus tepidarius, T. neapolitanus, T. ferrooxidans, T. thiooxidans) and facultative species such as T. acidophilus and T. aquaesulis, all of which can produce or oxidize tetrathionate when grown on thiosulfate. The central problem, as yet incompletely resolved in all cases, is the enzymology of the conversion of sulfane-sulfur (as in the outer [S-] atom of thiosulfate [-S-SO3-]), or sulfur itself, to sulfate, and whether sulfite is involved as a free intermediate in this process in all, or only some, cases. The study of inorganic sulfur compound oxidation for energetic purposes in bacteria (i.e. chemolithotrophy and sulfur photolithotrophy) poses challenges for comparative biochemistry. It also provides evidence of convergent evolution among diverse bacterial groups to achieve the end of energy-yielding sulfur compound oxidation (to drive autotrophic growth on carbon dioxide) but using a variety of enzymological systems, which share some common features. Some new data are presented on the oxidation of 35S-thiosulfate, and on the effect of other anions (selenate, molybdate, tu ngstate, chromate, vanadate) on sulfur compound oxidation, including observations which relate to the roles of polythionates and elemental sulfur as intermediates.     

Competition for inorganic substrates among chemoorganotrophic and chemolithotrophic bacteria

In aerobic enrichment experiments with a chemostat, using phosphate-limited lactate medium, aSpirillum sp. predominated at the lower range of dilution rates. At the higher dilution rates an (chemoorganotrophic) unidentified rod-shaped bacterium came to the fore. The same result was obtained in competition experiments with pure cultures of the two bacteria. Growth parameters were: Rod, _max=0.48 hr^–1,k _s(PO₄ ^3–)=6.6×10^–N M;Spirillum, _max=0.24 hr^–1· k_s(PO₄ ^3–) =2.7×10^–8 M. TheSpirillum grew faster than the rod at low dilution rates, not only under phosphate-limitation but also in K⁺-,Mg²⁺-, NH₄ ⁺-, aspartate-, succinate-, and lactate-limited cultures. Both organisms showed little substrate specificity and could utilize a similar range of carbon and energy sources. The results support the view that part of the diversity among bacteria in the natural environment is based on selection toward substrate concentration. Another set of competition experiments was carried out with pure cultures of two marine obligately chemolithotrophic colorless sulfur bacteria,Thiobacillus thioparus andThiomicrospira pelophila. Tms. pelophila outgrewT. thioparus at low dilution rates under iron limitation, while the reverse was true at high dilution rates. It is concluded that the relatively fast growth ofTms. pelophila at low iron concentration may explain its higher sulfide tolerance. Organisms showing a selection advantage at very low concentrations of limiting substrates appear to have a relatively high surface to volume ratio.     

The potential of mining slag as a substrate for microbial growth and the microbiological analysis of slag and slag seepage

The potential of a Cu/Ni mining slag to act as a substrate for the growth of the bacteria Thiobacillus ferrooxidans, Thiobacillus thiooxidants, and Thiobacillus thioparus was examined. As well, slag and seepage samples were screened for the presence of the Thiobacillus species. For the 28 samples employed in the environmental recovery studies, T. ferrooxidans was recovered in 25 samples, T. thiooxidans in 19 samples, and T. thioparus in 27 samples. For T. ferrooxidans, the development of a colour change in the medium corresponded with the presence of motile bacilli as detected microscopically. For T. thiooxidans and T. thioparus, a decrease in culture pH of greater than 0.2 units usually corresponded with the presence of motile bacilli. The potential for growth on slag was determined by adding slag samples to media (devoid of an electron donor) appropriate for the growth of the three Thiobacillus species. All pulverized slag samples supported the growth of the three species.     

Thiomicrospira pelophila,gen. n., sp. n., a new obligately chemolithotrophic colourless sulfur bacterium

From marine mud flats a very thin, comma- or spiral-shaped bacterium was isolated. The new organism was an obligately chemolithotrophic sulfur bacterium. Its physiology was found to be essentially similar to that ofThiobacillus thioparus. Because of the spirillum-like appearance it was proposed to classify this bacterium into a new genusThiomicrospira, with the species nameTms. pelophila. Tms. pelophila and a marineT. thioparus, which was isolated from the same mud, occupy different niches in this habitat.Tms. pelophila has a remarkable sulfide-tolerance as compared withT. thioparus. This property could be used for the specific enrichment ofTms. pelophila. The organism was also readily isolated in pure culture by filtering mud suspensions through a 0.22 Μm membrane filter.     

A mathematical description of the behaviour of mixed chemostat cultures of an autotrophic nitrifier and a heterotrophic nitrifier/aerobic denitrifier; a comparison with experimental data

Abstract A general, unstructed mathematical model has been used to describe the behaviour of nutrient-limited growth of two bacteria in a continuous co-culture. The experimental system consisted of a two-membered mixed culture of the heterotrophic nitrifier/aerobic denitrifier, Thiosphaera pantotropha , and the autotrophic nitrifier, Nitrosomonas europaea , competing for ammonia in chemostat culture. The outcome of competition was only dependent on the Monod constants and the growth yields of the two bacteria. The model shows that both bacteria will oxidize equal amounts of ammonia when the cell ratio of T. pantotropha/N. europaea is 260.     

Interspecific interactions in a methane-utilizing mixed culture

A two-member methane-utilizing mixed culture of bacteria, formed by combining two pure cultures isolated from a naturally occurring methane-utilizing mixed culture, was studied in continuous culture. From the nutritional requirements and substrate ranges of the pure cultures, a mechanism for the interspecific interactions occurring in the mixed culture was proposed. Product formation kinetics were determined in continuous culture for each product involved in the proposed mechanism. From this proposed mechanism a mathematical model was derived based on simple material balance equations around a single-stage chemostat. The steady-state predictions of this model were compared to experimental results obtained from continuous-culture experiments with the two-member methane-utilizing mixed culture. Interspecific interactions occurring in two-member methanol-utilizing and three-member methane-utilizing mixed cultures have also been discussed.