Purification and characterization of thermostable pectate-lyases from a newly isolated thermophilic bacterium, Thermoanaerobacter italicus sp. nov.

A novel thermophilic spore-forming anaerobic microorganism (strain Ab9) able to grow on citrus pectin and polygalacturonic acid (pectate) was isolated from a thermal spa in Italy. The newly isolated strain grows optimally at 70°C with a growth rate of 0.23 h⁻¹ with pectin and 0.12 h⁻¹ with pectate as substrates. Xylan, starch, and glycogen are also utilized as carbon sources and thermoactive xylanolytic (highest activity at 70°–75°C), amylolytic as well as pullulolytic enzymes (highest activity at 80°–85°C) are formed. Two thermoactive pectate lyases were isolated from the supernatant of a 300-l culture of isolate Ab9 after growth on citrus pectin. The two enzymes (lyases a and b) were purified to homogeneity by ammonium sulfate treatment, anion exchange chromatography, hydrophobic chromatography and finally by preparative gel electrophoresis. After sodium dodecylsulfate (SDS) gel electrophoresis, lyase a appeared as a single polypeptide with a molecular mass of 135 000 Da whereas lyase b consisted of two subunits with molecular masses of 93 000 Da and 158 000 Da. Both enzymes displayed similar catalytic properties with optimal activity at pH 9.0 and 80°C. The enzymes were very stable at 70°C and at 80°C with a half-life of more than 60 min. The maximal activity of the purified lyases was observed with orange pectate (100%) and pectate-sodium salt (90%), whereas pectin was attacked to a much lesser extent (50%). The K _m values of both lyases for pectate and citrus pectin were 0.5 g·l⁻¹ and 5.0 g·l⁻¹, respectively. After incubation with polygalacturonic acid, mono-, di-, and tri-galacturonate were detected as final products. A 2.5-fold increase of activity was obtained when pectate lyases were incubated in the presence of 1 mM Ca²⁺. The addition of 1 mM ethylenediaminetetraacetic acid (EDTA) resulted in complete inhibition of the enzymes. These heat-stable enzymes represent the first pectate-lyases isolated and characterized from a thermophilic anaerobic bacterium. On the basis of the results of the 16S rRNA sequence comparisons and the observed phenotypic differences, we propose strain Ab9 as a new species of Thermoanaerobacter, namely Thermoanaerobacter italicus sp. nov. Received: May 25, 1997 / Accepted: June 5, 1997     

Characterization of amylolytic and pullulytic enzymes from thermophilic archaea and from a newFervidobacterium species

Nine extremely thermophilic archaea and one novel thermophilic bacterium were screened for their ability to produce amylolytic and pullulytic enzymes. Cultivation of these micro-organisms was performed in the absence of elemental sulphur with starch as the major carbon source. Enzymatic activity was mainly detected in two archaea belonging to the order Thermoproteales,Desulfurococcus mucosus andStaphylothermus marinus, in two archaea belonging to the order Thermococcales,Thermococcus celer andT. litoralis and in two novel archaeal strains, TYS and TY previously isolated from the Guaymas Basin in the Gulf of California. Both amylolytic and pullulytic activities were also detected in a newly isolated thermophilic bacterium belonging to the order Thermotogales and previously described asFervidobacterium pennavorans. Best yields for enzyme production were obtained in 1–1 batch cultures with the strains TYS (13 units U/1 of amylase, 6 U/1 of pullulanase),F. pennavorans (2.5 U/l of amylase, 4.5 U/l of pullulanase) andT. litoralis (3.0 U/l of amylase). Enzymes were in general characterized by temperature optima around 90–100°C, pH optima around 5.5–6.5 and a high degree of thermostability. Due to the remarkable properties of these enzymes, they are of interest for biotechnological applications.     

Note: susceptibility to chlorine of Aeromonas hydrophila strains

The susceptibility of five Aeromonas hydrophila strains and one Escherichia coli strain to chlorine was studied under carefully controlled laboratory conditions. Of the Aer. hydrophila strains, two were from untreated water, two from tap water (immediately downstream of a water treatment plant) and one from the DSM collection. The study included disinfectant concentration (0.1, 0.2 and 0.5 mg l-1), pH (6, 7 and 8) and temperature (4, 21 and 32 degrees C) as controlled variables. The results indicated that the untreated water strains, the DSM strain and the E. coli strain were inactivated within 1 min of chlorine treatment. The strains from chlorinated water (TW11 and TW27) showed a different susceptibility to chlorine disinfection, the rate of inactivation being greater at pH6 than at pH8 for both strains. Under the standard conditions of temperature 21 degrees C, pH7 and chlorine concentration 0.2 mg l-1, an increase or decrease of approximately 1 log unit in the number of bacteria did not affect the kill rate of the strains TW11 and TW27.     

Rhamnolipid production by a novel thermophilic hydrocarbon-degrading Pseudomonas aeruginosa AP02-1

Thermophilic bacterial cultures were isolated from a hot spring environment on hydrocarbon containing mineral salts media. One strain identified as Pseudomonas aeruginosa AP02-1 was tested for the ability to utilize a range of hydrocarbons both n-alkanes and polycyclic aromatic hydrocarbons as sole carbon source. Strain AP02-1 had an optimum growth temperature of 45°C and degraded 99% of crude oil 1% (v/v) and diesel oil 2% (v/v) when added to a basal mineral medium within 7 days of incubation. Surface activity measurements indicated that biosurfactants, mainly glycolipid in nature, were produced during the microbial growth on hydrocarbons as well as on both water-soluble and insoluble substrates. Mass spectrometry analysis showed different types of rhamnolipid production depending on the carbon substrate and culture conditions. Grown on glycerol, P. aeruginosa AP02-1 produced a mixture of ten rhamnolipid homologues, of which Rha-Rha-C₁₀-C₁₀ and Rha-C₁₀-C₁₀ were predominant. Rhamnolipid-containing culture broths reduced the surface tension to ≈28 mN and gave stable emulsions with a number of hydrocarbons and remained effective after sterilization. Microscopic observations of the emulsions suggested that hydrophobic cells acted as emulsion-stabilizing agents.     

Survival of Enterobacter cloacae and Pseudomonas paucimobilis in yoghurts manufactured from cow's milk and soymilk during storage at different temperatures.

The survival of two microbial contaminants, Enterobacter cloacae and Pseudomonas paucimobilis, in yoghurts manufactured from cow's milk and soymilk was investigated during storage for 45 days at 4 and 12 degrees C. Sensory panel tests performed before microbiological investigation, showed that the flavor of soy-yoghurts made with cocoa powder or malt added did not have the beany taste of soy beans. Both contaminants were significantly resistant to low pH values during storage for 32 days at 4 degrees C. The survival at 4 degrees C was remarkable in both plain and flavored yoghurts and a population close to 10(2) C.F.U./ml was observed after 38 days of storage. Experiments performed with soymilk yoghurts showed an enhanced survival of P. paucimobilis at 4 degrees C compared to the storage in cow's milk yoghurts; microbial values were close to 7-8 x 10(6) C.F.U./ml after 16 days. Soymilk exhibited a protective effect on L. delbrueckii subsp. bulgaricus and S. thermophilus at 12 degrees C and, compared to the survival in cow's milk yoghurts, a larger number of viable cells of both probiotic microorganisms (10(6) and 10(8) C.F.U./ml, respectively) were observed after 36 days of storage.     

Susceptibility to chlorine of Aeromonas hydrophila strains

The susceptibility of five Aeromonas hydrophila strains and one Escherichia coli strain to chlorine was studied under carefully controlled laboratory conditions. Of the Aer. hydrophila strains, two were from untreated water, two from tap water (immediately downstream of a water treatment plant) and one from the DSM collection. The study included disinfectant concentration (0·1, 0·2 and 0·5 mg l⁻¹), pH (6, 7 and 8) and temperature (4, 21 and 32 °C) as controlled variables. The results indicated that the untreated water strains, the DSM strain and the E. coli strain were inactivated within 1 min of chlorine treatment. The strains from chlorinated water (TW11 and TW27) showed a different susceptibility to chlorine disinfection, the rate of inactivation being greater at pH 6 than at pH 8 for both strains. Under the standard conditions of temperature 21 °C, pH 7 and chlorine concentration 0·2 mg l⁻¹, an increase or decrease of approximately 1 log unit in the number of bacteria did not affect the kill rate of the strains TW11 and TW27.     

Purification and Properties of a Thermostable Pullulanase from a Newly Isolated Thermophilic Anaerobic Bacterium, Fervidobacterium pennavorans Ven5

Extremely thermophilic anaerobic fermentative bacteria growing at temperatures between 50 and 80(deg)C (optimum, 65 to 70(deg)C) were isolated from mud samples collected at Abano Terme spa (Italy). The cells were gram-negative motile rods, about 1.8 (mu)m in length and 0.6 (mu)m in width, occurring singly and in pairs. Cells commonly formed spheroids at one end similar to Fervidobacterium islandicum and Fervidobacterium nodosum. The new isolate differs from F. nodosum by the 7% higher G+C content of its DNA (40.6 mol%) but is similar to Fervidobacterium pennavorans and F. islandicum in its G+C content and phenotypic properties. The phylogenetic dendrogram indicates that strain Ven5 belongs to the order Thermotogales and shows the highest 16S ribosomal DNA sequence similarity to F. pennavorans, F. islandicum, and F. nodosum, with similarities of 99.0, 98.6, and 96.0%, respectively. During growth on starch the strain produced a thermostable pullulanase of type I which preferentially hydrolyzed (alpha)-1,6 glucosidic linkages. The enzyme was purified 65-fold by anion-exchange, gel permeation, and hydrophobic chromatography. The native pullulanase has a molecular mass of 240,000 Da and is composed of three subunits, each with a molecular mass of 77,600 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Optimal conditions for the activity and stability of the purified pullulanase were pH 6.0 and 85(deg)C. At pH 6.0, the half-life of the enzyme was over 2 h at 80(deg)C and 5 min at 90(deg)C. This is the first report on the presence of pullulanase type I in an anaerobic bacterium.     

Microbial ecology of submerged marine caves and holes characterized by high levels of hydrogen sulphide

Submarine caves, cavities and niches characterised by H₂S elevated concentrations are particularly interesting for their inhabiting microflora as well as for the overall chemical, geological and biological parameters. These ecosystems are usually populated by well adapted living forms, physically distributed following the in situ concentration and gradient of micronutrients, O₂ and H₂S, and also according to the values of temperature and pH. The biota is primarily characterised by prokaryotes (both autotrophic and heterotrophic) adapted to anoxic and/or microaerophilic condition and capable to form extensive biofilms on the rocky surfaces and even on the bottom sediment. These habitats can be defined as extreme, because the scarcity or absence of solar irradiation, the chemo-physical traits and the fact that specialised prokaryotes are often the only inhabitants. This review is focused on the microbial ecology of marine caves and holes characterised by high levels of hydrogen sulphide. Ecological and geological data are already available but very few insights as far as regard microbiology were achieved in order to describe these fascinating habitats. The autochthonous mesophilic and thermotolerant microorganisms living in these caves may have interesting physiological traits and eventually may lead to potential application in biotechnological processes.     

Effects of hydrostatic pressure and temperature on physiological traits of Thermococcus guaymasensis and Thermococcus aggregans growing on starch

The effects of temperature and hydrostatic pressure on growth of two novel Thermococcus species, T. guaymasensis and T. aggregans, were investigated. These archaea, isolated from the Guaymas Basin hydrothermal vent site at 2000 meters depth, are able to grow on starch in sulfur-depleted medium producing significant amounts of amylases and pullulanases. At 85 degrees C, T. guaymasensis exhibited a barophilic response at 20 and 35 MPa but inhibition of growth occurred at 50 MPa; at 50 MPa, cell replication was repressed, the mean cell size increased, and production of starch-hydrolysing enzymes was significantly stimulated. Barophily was also expressed by T. guaymasensis under 20 MPa at sub-optimal temperature (70 C) but morphological alterations of cells were observed earlier (35 MPa). No barophily was exhibited by T. aggregans at 85 degrees C. In this case, cell replication was repressed at 20 MPa and remarkable inhibition of growth occurred at 50 MPa. Only when T. aggregans was cultivated at 75 degrees C, a significant barophilic response was exhibited at 20 MPa, as shown by the rate of replication and metabolism. These results show that Thermococcus species, although isolated from the same ecosystem, differ with regard to the effects of pressure and temperature on cell physiology. The metabolic responses and their significance for potential biotechnological applications are also discussed.     

Effects of micronutrients on growth and starch hydrolysis of Thermococcus guaymasensis and Thermococcus aggregans

The effects of micronutrients on growth of Thermococcus guaymasensis and Thenrmococcus aggregans in a starch-containing medium were investigated. A trace minerals solution, a vitamins solution and calcium chloride were omitted from the medium or added in different amounts. The growth rates of both species were not affected over a significant range of concentrations of these compounds, but appreciable inhibition of growth was observed after the addition of elemental sulfur to the medium. T. guaymasensis exhibited a significant tolerance to high amounts of trace element and vitamin solutions but growth was inhibited by the omission of these compounds from the medium. Moreover, both amylolytic and pullulytic activities increased in the presence of 6-fold higher amounts of trace element and vitamin solutions, compared to the concentrations used in the usual medium. In T. aggregans, both enzymatic activities were enhanced in the presence of either increased (4-fold) amounts of trace element and vitamin solutions, or after the addition of elemental sulfur to the medium. Furthermore, larger activities of starch-hydrolysing enzymes were detected with a 10-fold higher concentration of calcium chloride, compared to the usual medium, in the absence of trace element and vitamin solutions. When both Thermococcus species were tested for the tolerance to specific cations and oxyanions, T. guaymasensis exhibited higher tolerance compared to T. aggregans, the former strain being capable to grow in the presence of 6 mM Ni2+, 4mM Cu2+, 1.5 mM SeO4(2-), and 1.5 mM MoO4(2-). The content of total cell proteins followed the pattern of starch-hydrolysing enzymes and an over-expression of proteins in the range of 35, 50 and 70 kDa was observed.