Effect of the oxygen supply on pattern of growth and corrinoid and organic acid production of Propionibacterium shermanii

Propionibacterium shermanii CDB 10014 is able to grow even at high oxygen transfer rates (24.0 mmol O₂ l⁻¹ h⁻¹), in contrast to reports in the specialised literature, where all Propionibacteria are considered oxygen-sensitive microorganisms. Propionic acid is the main product in anaerobiosis. The presence of oxygen in the system leads to an inhibition of propionic acid production while acetic acid formation is enhanced. At high oxygen supply rates no propionic acid is produced and acetic acid is the main product. Lactic acid is also produced in reasonable quantities (2.7 g l⁻¹). The growth rate (μ_max) is higher in anaerobiosis (0.19 h⁻¹) than in aerobiosis (0.12–0.15 h⁻¹). The cell yield is higher in aerobiosis (0.18–0.22 g g⁻¹) than in anaerobiosis (0.14 g g⁻¹) suggesting the oxidative metabolism of glucose by Propionibacterium shermanii CDB 10014. No corrinoid production was detected at oxygen transfer rates of more than 13.6 mmol l⁻¹ h⁻¹. Received: 10 September 1997 / Received revision: 6 January 1998 / Accepted: 9 January 1998     

Pyranose 2-oxidase from Phanerochaete chrysosporium– further biochemical characterisation

Pyranose 2-oxidase (P2O) was purified 43-fold to apparent homogeneity from the basidiomycete Phanerochaete chrysosporium using liquid chromatography on phenyl Sepharose, Mono Q (twice) and phenyl Superose. The native enzyme has a molecular mass of about 250 kDa (based on native PAGE) and is composed of four identical subunits of 65 kDa. It contains three isoforms of isoelectric point (pI) 5.0, 5.05 and 5.15 and does not appear to be a glycoprotein. P2O is optimally stable at pH 8.0 and up to 60 °C. It is active over a broad pH range (5.0–9.0) with maximum activity at pH 8.0–8.5 and at 55 °C, and a broad substrate specificity. d-Glucose is the preferred substrate, but 1-β-aurothioglucose, 6-deoxy-d-glucose, l-sorbose, d-xylose, 5-thioglucose, d-glucono-1,5-lactone, maltose and 2-deoxy-d-glucose are also oxidised at relatively high rates. A Ping Pong Bi Bi mechanism was demonstrated for the P2O reaction at pH 8.0, with a catalytic constant (k _cat) of 111.0 s⁻¹ and an affinity constant (K _m) of 1.43 mM for d-glucose and 83.2 μM for oxygen. Whereas the steady-state kinetics for glucose oxidation were unaffected by the medium at pH ≥ 7.0, at low pH both pH and buffer composition affected the P2O kinetics with the k _cat/K _m value decreasing with decreasing pH. The greatest effect was observed in acetate buffer (0.1 M, pH 4.5), where the k _cat decreased to 60.9 s⁻¹ and the K _m increased to 240 mM. The activity of P2O was completely inhibited by 10 mM HgCl₂, AgNO₃ and ZnCl₂, and 50% by lead acetate, CuCl₂ and MnCl₂. Received: 28 August 1996 / Received revision: 25 November 1996 / Accepted: 29 November 1996     

Succession of bacterivorous protists on laboratory-made marine snow

Colonization and succession over time by bacterivorous protistson laboratory-made marine snow were analysed in five assaysduring 1994. Marine snow was made hom natural seawater usingrolling tanks. In all experiments, the macroaggregates werestable in size and consistency after the fourth day, and thecolonization and succession processes were similar. Newly formedmacre aggregates became colonized by heterotrophic nanoflagellateson the fourth day, mmt of them kinete plastids (Bodo designisand Rhynchomonns nasuta) and bicosoecids (Pseudobodo tremulansand Bicosoeca sp.). Sarcodines and ciliates appeared 1 day later.Among the former, the most abundant genus was Vannella sp.,while scuticociliates (Uronema marinum) and hypotrichs (Euplotesvannus and Aspidisca steini) were the most abundant ciliates.Most of the species observed in the study were more common tobenthic habitats than to pelagic ones. The planktonic existenceof the genera Bodo, Rhynchomonas, Bicosoeca, Euplotes and Aspidiscadepends on the presence of surfaces because they are poor swimmersor immotile, and Pseudobodo and Vannella need attachment forfeeding. The only pelagic protist observed was Uronema, probablybecause its opportunistic behaviour leads it to exploit enrichedenvironments such as marine snow. Flagellate and ciliate abundancesin laboratory-made macroaggregates were much higher than insurrounding water, which indicates that marine snow representsan enhanced habitat for protist growth.     

Recurrent seasonal changes in bacterial growth efficiency,metabolism and community composition in coastal waters

The microbial response to environmental changes in coastal waters of the eastern Cantabrian Sea was explored for four years by analysing a broad set of environmental variables along with bacterial community metabolism and composition. A recurrent seasonal cycle emerged, consisting of two stable periods, characterized by low bacterial metabolic activity (winter) from October to March, and high bacterial metabolic activity (summer) from May to August. These two contrasting periods were linked by short transition periods in April (T_A) and September (T_S). The phylogenetic groups Alphaproteobacteria and Bacteroidetes were dominant during winter and summer respectively, and their recurrent alternation was mainly driven by the bloom of eukaryotic phytoplankton before T_A and the bloom of prokaryotic phytoplankton before T_S. Bacterial growth efficiency remained high and stable during the winter and summer periods but dropped during the two short transition periods. Our results suggest that bacterial growth efficiency should be considered a very resilient property that reflects different stages in the adaptation of the bacterial community composition to the environmental changes occurring throughout the seasonal cycle in this coastal ecosystem.     

Imbalanced nutrient recycling in a warmer ocean driven by differential response of extracellular enzymatic activities

Ocean oligotrophication concurrent with warming weakens the capacity of marine primary producers to support marine food webs and act as a CO₂ sink, and is believed to result from reduced nutrient inputs associated to the stabilization of the thermocline. However, nutrient supply in the oligotrophic ocean is largely dependent on the recycling of organic matter. This involves hydrolytic processes catalyzed by extracellular enzymes released by bacteria, which temperature dependence has not yet been evaluated. Here, we report a global assessment of the temperature‐sensitivity, as represented by the activation energies (E_a), of extracellular β‐glucosidase (βG), leucine aminopeptidase (LAP) and alkaline phosphatase (AP) enzymatic activities, which enable the uptake by bacteria of substrates rich in carbon, nitrogen, and phosphorus, respectively. These E_a were calculated from two different approaches, temperature experimental manipulations and a space‐for‐time substitution approach, which generated congruent results. The three activities showed contrasting E_a in the subtropical and tropical ocean, with βG increasing the fastest with warming, followed by LAP, while AP showed the smallest increase. The estimated activation energies predict that the hydrolysis products under projected warming scenarios will have higher C:N, C:P and N:P molar ratios than those currently generated, and suggest that the warming of oceanic surface waters leads to a decline in the nutrient supply to the microbial heterotrophic community relative to that of carbon, particularly so for phosphorus, slowing down nutrient recycling and contributing to further ocean oligotrophication.     

Spatial distribution of protists in the presence of macroaggregates in a marine system

The spatial distribution of marine protistan communities in the presence of organic macroaggregates, formed from natural seawater, was studied in several microcosm experiments. The presence of macroaggregates had two main effects. First, the size of the communities of bacteria, flagellates and ciliates increased, as these communities were three orders of magnitude higher in the aggregates than in the microcosm water. In addition, it brought the diversification on the niches accessible to planktonic microorganisms, as three phases were formed: water, aggregates and aggregate-water interphase. Some of the detected protistan taxa were only found in the water, and therefore they can be considered as truly free-swimming protists. Others quickly colonised the aggregates, and finally, some of them showed a preference for the aggregate-water interphase. We discuss this spatial structuring of the protistan community on the basis of their feeding strategies and structural and behavioural characteristics.     

Factors affecting preference responses of the freshwater ciliate Uronema nigricans to bacterial prey

ABSTRACT. To enhance our understanding of the factors affecting feeding selectivity of bacterivorous protists in aquatic systems, we examined the preference responses of the freshwater ciliate Uronema nigricans towards three bacterial prey taxa, Pseudomonas luteola, Serratia rubidaea , and Aeromonas hydrophila . Potential factors influencing the predator–prey contact rate included the previous feeding history of the ciliate and physiological state of bacteria. Preference indexes were obtained from multiple-choice mazes in which ciliates moved preferentially towards alternative bacteria or the prey species on which they had been feeding. Uronema nigricans showed differential attraction towards the offered prey types, and these preferences varied as a function of the ciliate feeding history: U. nigricans growing on P. luteola showed lower preference responses towards the offered bacteria than U. nigricans growing on S. rubidaea . The bacteria in stationary phase elicited a higher degree of attraction than bacteria in exponential phase, probably due to a higher concentration of carbohydrates in the former. Therefore, this protist will preferentially swim towards bacteria in stationary growth phase, although the degree of this response will be affected by the recent feeding history of the ciliate.     

Influence of organic matter quality in the cleavage of polymers by marine bacterial communities

The influence of the quality of organic matter on the hydrolysisof polymers by marine bacteria was investigated in microcosmscontaining aggregates created in rolling tanks. Two types ofmicrocosms were analysed: microcosms type M1 from unalteredseawater and microcosms type M2 from phytoplankton cultures.Kinetics of aminopeptidase, -glucosidase and ß-glucosidasewere measured in the ambient water and the aggregates in thetwo types of microcosms. Bacteria attached to aggregates expressedenzymes with K_m values higher than those of the bacteria inthe ambient water for the three hydrolytic activities analysedin both types of microcosms. Attached bacteria showed higherrates of polymer hydrolysis than free-living bacteria only inmicrocosms M2 created from freshly produced phytoplanktonicmaterial, while free-living bacteria were more active than attachedbacteria in the microcosms M1 containing unaltered seawater.The ratio V_max/K_m, which describes the ability of enzymes tocompete at low substrate concentration, shows that free-livingbacteria are more efficient at dealing with low substrate concentrationsin microcosms derived from natural seawater, where the liquidphase may be depleted of utilizable dissolved organic matter,than in the microcosms derived from phytoplankton cultures.Our data suggest that the hydrolytic activities of both attachedand free-living bacteria are significantly influenced by thequality of the aggregates and the consequences of this influenceare discussed.     

Grazing rates of diverse morphotypes of bacterivorous ciliates feeding on four allochthonous bacteria

AIMS: The permanence in aquatic systems of allochthonous bacteria coming from sewage effluents is a risk for public health. This work aimed to analyse the elimination of the bacteria Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli and Klebsiella pneumoniae by a riverine ciliate community. METHODS AND RESULTS: The ciliates were characterized and identified on the basis of morphological and behavioural features and grouped in nine morphotypes. Differential grazing by each morphotype on the four allochthonous bacteria was carried out by adding fluorescently labelled bacteria to the water samples, and measuring their uptake along time. CONCLUSIONS: The nine morphotypes were present in all the samples but in different proportions, being the most abundant the small scuticociliates and hipotrichs. The smallest morphotypes showed grazing rates lower than 20 FLB ciliate(-1) h(-1), with a preference towards K. pneumoniae. The larger morphotypes showed in general the highest grazing rates, but the selectivity was hardly attributable to bacterial size or type of cell wall. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The elimination of allochthonous bacteria in an aquatic system may be highly different depending on the diversity of the ciliated protistan community in the system and on the nature of the bacterial prey.     

One step partial purification of β-D-galactosidase from Kluyveromyces marxianus CDB 002 using STREAMLINE-DEAE

The intracellular enzyme -D-galactosidase provides interesting applications in the dairy industry, which are able to solve problems related to product processing, or can alleviate lactose intolerance in some populations. In order to obtain a technical enzyme, yeast cells of Kluyveromyces marxianus CDB 002 were disrupted by high pressure homogenization and an innovative chromatographic technique was tested for the recovery of -D-galactosidase. A STREAMLINE 25 column, containing 65 ml STREAMLINE-DEAE was equilibrated with 50 mM potassium phosphate buffer pH 7.5 at an upward flow of 250 cmh^–1. 100–200 ml cell homogenate were applied onto the expanded gel. After unbound proteins and cellular debris were washed out, the bed was allowed to sediment and -D-galactosidase was eluted with a downward flow of 0.2 M NaCl in the same buffer. A 6-fold purification factor was achieved with 63% activity recovery, while removing cell debris at a single step, thus avoiding a centrifugation step. Concentration and volume of the applied sample affected purification and gel performance. The results presented show STREAMLINE-DEAE chromatography to be an interesting method for the production of -D-galactosidase as a technical enzyme, since it can also be applied on a large scale without much modification.