Hydrogen production by the hyperthermophilic bacterium Thermotoga maritima Part II: modeling and experimental approaches for hydrogen production.

BACKGROUND: Thermotoga maritima is a hyperthermophilic bacterium known to produce hydrogen from a large variety of substrates. The aim of the present study is to propose a mathematical model incorporating kinetics of growth, consumption of substrates, product formations, and inhibition by hydrogen in order to predict hydrogen production depending on defined culture conditions. RESULTS: Our mathematical model, incorporating data concerning growth, substrates, and products, was developed to predict hydrogen production from batch fermentations of the hyperthermophilic bacterium, T. maritima. It includes the inhibition by hydrogen and the liquid-to-gas mass transfer of H2, CO2, and H2S. Most kinetic parameters of the model were obtained from batch experiments without any fitting. The mathematical model is adequate for glucose, yeast extract, and thiosulfate concentrations ranging from 2.5 to 20 mmol/L, 0.2-0.5 g/L, or 0.01-0.06 mmol/L, respectively, corresponding to one of these compounds being the growth-limiting factor of T. maritima. When glucose, yeast extract, and thiosulfate concentrations are all higher than these ranges, the model overestimates all the variables. In the window of the model validity, predictions of the model show that the combination of both variables (increase in limiting factor concentration and in inlet gas stream) leads up to a twofold increase of the maximum H2-specific productivity with the lowest inhibition. CONCLUSIONS: A mathematical model predicting H2 production in T. maritima was successfully designed and confirmed in this study. However, it shows the limit of validity of such mathematical models. Their limit of applicability must take into account the range of validity in which the parameters were established.

Hydrogen production by the hyperthermophilic bacterium Thermotoga maritima part I: effects of sulfured nutriments, with thiosulfate as model, on hydrogen production and growth.

BACKGROUND: Thermotoga maritima and T. neapolitana are hyperthermophile bacteria chosen by many research teams to produce bio-hydrogen because of their potential to ferment a wide variety of sugars with the highest theoretical H2/glucose yields. However, to develop economically sustainable bio-processes, the culture medium formulation remained to be optimized. The main aim of this study was to quantify accurately and specifically the effect of thiosulfate, used as sulfured nutriment model, on T. maritima growth, yields and productivities of hydrogen. The results were obtained from batch cultures, performed into a bioreactor, carefully controlled, and specifically designed to prevent the back-inhibition by hydrogen. RESULTS: Among sulfured nutriments tested, thiosulfate, cysteine, and sulfide were found to be the most efficient to stimulate T. maritima growth and hydrogen production. In particular, under our experimental conditions (glucose 60 mmol L-1 and yeast extract 1 g L-1), the cellular growth was limited by thiosulfate concentrations lower than 0.06 mmol L-1. Under these conditions, the cellular yield on thiosulfate (Y X/Thio) could be determined at 3617 mg mmol-1. In addition, it has been shown that the limitations of T. maritima growth by thiosulfate lead to metabolic stress marked by a significant metabolic shift of glucose towards the production of extracellular polysaccharides (EPS). Finally, it has been estimated that the presence of thiosulfate in the T. maritima culture medium significantly increased the cellular and hydrogen productivities by a factor 6 without detectable sulfide production. CONCLUSIONS: The stimulant effects of thiosulfate at very low concentrations on T. maritima growth have forced us to reconsider its role in this species and more probably also in all thiosulfato-reducer hyperthermophiles. Henceforth, thiosulfate should be considered in T. maritima as (1) an essential sulfur source for cellular materials when it is present at low concentrations (about 0.3 mmol g-1 of cells), and (2) as both sulfur source and detoxifying agent for H2 when thiosulfate is present at higher concentrations and, when, simultaneously, the pH2 is high. Finally, to improve the hydrogen production in bio-processes using Thermotoga species, it should be recommended to incorporate thiosulfate in the culture medium.

Mobilisporobacter senegalensis gen. nov., sp. nov., an anaerobic bacterium isolated from tropical shea cake.

A Gram-stain positive, endospore-forming, strictly anaerobic bacterium, designated strain Gal1T, was isolated from shea cake, a waste material from the production of shea butter, originating from Saraya, Senegal. The cells were rod-shaped, slightly curved, and motile with peritrichous flagella. The strain was oxidase-negative and catalase-negative. Growth was observed at temperatures ranging from 15 to 45 °C (optimum 30 °C) and at pH 6.5-9.3 (optimum pH 7.8). The salinity range for growth was 0-3.5 % NaCl (optimum 1 %). Yeast extract was required for growth. Strain Gal1T fermented various carbohydrates such as mannose, mannitol, arabinose, cellobiose, fructose, glucose, maltose, sucrose, trehalose and lactose and the major end-products were ethanol and acetate. The only major cellular fatty acid was C16 : 0 (19.6 %). The DNA base G+C content of strain Gal1T was 33.8 mol%. Analysis of the 16S rRNA gene sequence of the isolate indicated that this strain was related to Mobilitalea sibirica DSM 26468T with 94.27 % similarity, Clostridium populeti ATTC 35295T with 93.94 % similarity, and Clostridium aminovalericum DSM 1283T and Anaerosporobacter mobilis DSM 15930T with 93.63 % similarity. On the basis of phenotypic characteristics, phylogenetic analysis and the results of biochemical and physiological tests, strain Gal1T was clearly distinguished from closely related genera, and strain Gal1T can be assigned to a novel species of a new genus for which the name Mobilisporobacter senegalensis gen. nov., sp. nov. is proposed. The type strain is Gal1T ( = DSM 26537T = JCM 18753T).

Thermanaeromonas burensis sp. nov., a thermophilic anaerobe isolated from a subterranean clay environment.

A strictly anaerobic, thermophilic and halotolerant strain, designated IA106T, was isolated from the seepage water collected in a metal biocorrosion test at a depth of 490 m, in a 130-160 m thick, subterranean Callovo-Oxfordian clay formation (158-152 million years old) in northern France. This geological formation has been selected as the potential host rock for the French high-level nuclear waste repository. Cells of strain IA106T stained Gram-positive and were non-motile, spore-forming, straight rods (0.5 × 2-6 µm). The five major fatty acids were C16 : 0 (15.9 %), C18 : 0 (15.4 %), iso-C17 : 1 I and/or anteiso-C17 : 1 B(14.8 %), iso-C17 : 0 (14.7 %) and iso-C15 : 0 (13.0 %). Growth was observed at temperatures ranging from 55 to 70 °C and at pH 5.5-9. The salinity range for growth was 0-20 g NaCl 1- 1. Yeast extract was required for growth. Strain IA106T was able to grow on lactate and various sugars in the presence of thiosulfate as electron acceptor. Sulfate, sulfite, elemental sulfur, fumarate, nitrate and nitrite were not reduced. The DNA G+C content was 60.2 mol%. 16S rRNA gene sequence analysis indicated that strain IA106T belonged to the family Thermoanaerobacteraceae, class Clostridia, phylum Firmicutes, and was most closely related to Thermanaeromonas toyohensis DSM 14490T (95.16 % 16S rRNA gene sequence similarity). On the basis of 16S rRNA gene sequence comparisons and physiological characteristics, strain IA106T represents a novel species of the genus Thermanaeromonas, for which the name Thermanaeromonas burensis sp. nov. is proposed. The type strain is IA106T ( = DSM 26576T = JCM 18718T).

Biochemical, transcriptional and translational evidences of the phenol-meta-degradation pathway by the hyperthermophilic Sulfolobus solfataricus 98/2.

Phenol is a widespread pollutant and a model molecule to study the biodegradation of monoaromatic compounds. After a first oxidation step leading to catechol in mesophilic and thermophilic microorganisms, two main routes have been identified depending on the cleavage of the aromatic ring: ortho involving a catechol 1,2 dioxygenase (C12D) and meta involving a catechol 2,3 dioxygenase (C23D). Our work aimed at elucidating the phenol-degradation pathway in the hyperthermophilic archaea Sulfolobus solfataricus 98/2. For this purpose, the strain was cultivated in a fermentor under different substrate and oxygenation conditions. Indeed, reducing dissolved-oxygen concentration allowed slowing down phenol catabolism (specific growth and phenol-consumption rates dropped 55% and 39%, respectively) and thus, evidencing intermediate accumulations in the broth. HPLC/Diode Array Detector and LC-MS analyses on culture samples at low dissolved-oxygen concentration (DOC  =  0.06 mg x L(-1)) suggested, apart for catechol, the presence of 2-hydroxymuconic acid, 4-oxalocrotonate and 4-hydroxy-2-oxovalerate, three intermediates of the meta route. RT-PCR analysis on oxygenase-coding genes of S. solfataricus 98/2 showed that the gene coding for the C23D was expressed only on phenol. In 2D-DIGE/MALDI-TOF analysis, the C23D was found and identified only on phenol. This set of results allowed us concluding that S. solfataricus 98/2 degrade phenol through the meta route.

Effects of hydrostatic pressure on growth and luminescence of a moderately-piezophilic luminous bacteria Photobacterium phosphoreum ANT-2200.

Bacterial bioluminescence is commonly found in the deep sea and depends on environmental conditions. Photobacterium phosphoreum ANT-2200 has been isolated from the NW Mediterranean Sea at 2200-m depth (in situ temperature of 13°C) close to the ANTARES neutrino telescope. The effects of hydrostatic pressure on its growth and luminescence have been investigated under controlled laboratory conditions, using a specifically developed high-pressure bioluminescence system. The growth rate and the maximum population density of the strain were determined at different temperatures (from 4 to 37°C) and pressures (from 0.1 to 40 MPa), using the logistic model to define these two growth parameters. Indeed, using the growth rate only, no optimal temperature and pressure could be determined. However, when both growth rate and maximum population density were jointly taken into account, a cross coefficient was calculated. By this way, the optimum growth conditions for P. phosphoreum ANT-2200 were found to be 30°C and, 10 MPa defining this strain as mesophile and moderately piezophile. Moreover, the ratio of unsaturated vs. saturated cellular fatty acids was found higher at 22 MPa, in agreement with previously described piezophile strains. P. phosphoreum ANT-2200 also appeared to respond to high pressure by forming cell aggregates. Its maximum population density was 1.2 times higher, with a similar growth rate, than at 0.1 MPa. Strain ANT-2200 grown at 22 MPa produced 3 times more bioluminescence. The proposed approach, mimicking, as close as possible, the in situ conditions, could help studying deep-sea bacterial bioluminescence and validating hypotheses concerning its role into the carbon cycle in the deep ocean.

Methanogenic octadecene degradation by syntrophic enrichment culture from brackish sediments.

A microbial enrichment culture from brackish sediments was able to grow on octadec-1-ene (an unsaturated aliphatic hydrocarbon) as sole source of carbon and energy, under methanogenic conditions. Octadecene degradation is stopped either when bromoethanesulfonic acid, a selective inhibitor of methanogenesis is introduced, or when hydrogen is introduced. In the presence of bromoethanesulfonic acid, the degradation is restored by the addition of a hydrogenotrophic sulfate-reducing microorganism with sulfate. Results of molecular biodiversity, which revealed the presence of bacteria as well as of acetoclastic and hydrogenotrophic methanogens, are consistent with a syntrophic degradation involving Bacteria and Archaea. This is the first demonstration of syntrophic alkene degradation by microbial communities, showing that syntrophy is more widespread than we could have thought so far. These results highlight the need for a better understanding of microbial interactions and their role in the organic-matter degradation in polluted environments.

Effect of O2 concentrations on Sulfolobus solfataricus P2.

Sulfolobus solfataricus P2 was grown aerobically at various O(2) concentrations. Based on growth parameters in microcosms, four types of behavior could be distinguished. At 35% O(2) (v/v; gas phase), the cultures did not grow, indicating a lethal dose of oxygen. For 26-32% O(2), the growth was significantly affected compared with the reference (21%), suggesting a moderate toxicity by O(2). For 16-24% O(2), standard growth was observed. For 1.5-15% O(2), growth was comparable with the reference, but the yield on O(2) indicated a more efficient use of oxygen. These results indicate that S. solfataricus P2 grows optimally in the range of 1.5-24% O(2), most likely by adjusting its energy-transducing machinery. To gain some insight into control of the respiratory system, transcriptomes of the strain cultivated at different O(2) concentrations, corresponding to each behavior (1.5%, 21% and 26%), were compared using a DNA microarray approach. It showed differential expression of several genes encoding terminal oxidases, indicating an adaptation of the strain's respiratory system in response to fluctuating oxygen concentrations.

Desulfovibrio tunisiensis sp. nov., a novel weakly halotolerant, sulfate-reducing bacterium isolated from exhaust water of a Tunisian oil refinery.

A novel weakly halotolerant, sulfate-reducing bacterium, designated strain RB22(T), was isolated from exhaust water of a Tunisian oil refinery. Cells of strain RB22(T) were Gram-negative, motile, vibrio-shaped or sigmoid and non-spore-forming, and occurred singly or in chains. Strain RB22(T) grew between 15 and 45 degrees C (optimum, 37 degrees C) and at pH 4.5 to 9 (optimum, pH 7). NaCl was not required for growth, but the strain tolerated high NaCl concentrations (up to 70 g l(-1)) with an optimum of 40 g l(-1). Sulfate, thiosulfate, sulfite and elemental sulfur served as electron acceptors, but not fumarate. Nitrate and nitrite were not reduced. Strain RB22(T) utilized lactate, formate, fumarate, succinate, glycerol, H(2)+CO(2) and methanol as substrates. The DNA G+C content was found to be 59.6 mol%. Phylogenetic analysis based on the 16S rRNA gene revealed that the isolate was a member of the genus Desulfovibrio, with no close relatives at the species level (16S rRNA gene sequence similarity of less than 95 %). Strain RB22(T) exhibited levels of 16S rRNA gene sequence similarity of 94.6 and 94.12 % to the type strains of the closely related species Desulfovibrio aespoeensis and Desulfovibrio dechloracetivorans, respectively. On the basis of genotypic and phylogenetic characteristics, and significant phenotypic differences, we suggest that strain RB22(T) represents a novel species, for which the name Desulfovibrio tunisiensis sp. nov. is proposed. The type strain is RB22(T) (=NCIMB 14400(T)=JCM 15076(T)=DSM 19275(T)).

Bioconversion of tyrosol into hydroxytyrosol and 3,4-dihydroxyphenylacetic acid under hypersaline conditions by the new Halomonas sp. strain HTB24.

This paper reports the characterization of a Halomonas sp. strain (named HTB24) isolated from olive-mill wastewater and capable of transforming tyrosol into hydroxytyrosol (HT) and 3,4-dihydroxyphenylacetic acid (DHPA) in hypersaline conditions. This is the first time that a halophile has been shown to perform such reactions. The potent natural antioxidant HT was obtained through a C3 hydroxylation on the ring cycle, whereas DHPA was synthesized via the 4-hydroxyphenylacetic acid (HPA) pathway, which has been well described from other bacterial sources. HT was produced first, and then DHPA was detected in the medium accompanied by traces of HPA. HPA involved another pathway resulting from the activity of an aryl-dehydrogenase, which is suggested to be responsible for both tyrosol and hydroxytyrosol oxidation. Maximal HT content (2.30 mM) and maximal DHPA (5.15+/-0.42 mM) were obtained from a culture inoculated in the presence of 20 mM tyrosol and 0.5 g L(-1) yeast extract. Following this, DHPA was quickly degraded into 5-carboxymethyl-2-hydroxymuconic semialdehyde by a 2,3-dioxygenase, finally resulting in succinate and pyruvate. Phylogenetic analysis of the 16S rRNA gene revealed that this isolate was a member of the genus Halomonas. Strain HTB24, with a G+C content of 55.3 mol%, is closely related to Halomonas neptunia DSM 15720(T), 'Halomonas alkaliantarctica' DSM 15686(T) and Halomonas boliviensis DSM 15516(T).

Modicisalibacter tunisiensis gen. nov., sp. nov., an aerobic, moderately halophilic bacterium isolated from an oilfield-water injection sample, and emended description of the family Halomonadaceae Franzmann et al. 1989 emend Dobson and Franzmann 1996 emend. Ntougias et al. 2007.

An aerobic, moderately halophilic, Gram-negative, motile, non-sporulating bacterium, strain LIT2(T), was isolated from an oilfield-water injection after enrichment on crude oil. Strain LIT2(T) grew between 15 and 45 degrees C and optimally at 37 degrees C. It grew in the presence of 1-25 % (w/v) NaCl, with an optimum at 10 % (w/v) NaCl. Predominant fatty acids were C(16 : 0) (26.9 %), C(18 : 1)omega7c (22.6 %), C(16 : 1)omega7c (20.4 %) C(19 : 0) cyclo omega8c (10.9 %) and C(17 : 0) (8 %). Interestingly, the relative percentages of these last two fatty acids were intermediate compared with most species among the family Halomonadaceae for which fatty acid composition has been determined. The DNA G+C content was 53.7 mol%, which is very low among the family Halomonadaceae. Strain LIT2(T) exhibited 16S rRNA gene sequence similarity values of 94.06-95.15 % to members of the genus Chromohalobacter, 94.21-94.65 % to members of the genus Halomonas and 93.57 % with the single species representative of the genus Cobetia. Based on the phylogenetic and phenotypic evidence presented in this paper, we propose the name Modicisalibacter tunisiensis gen. nov., sp. nov. to accommodate strain LIT2(T). The type strain of Modicisalibacter tunisiensis is LIT2(T) (=CCUG 52917(T) =CIP 109206(T)). A reassignment of the descriptive 16S rRNA signature characteristics of the family Halomonadaceae permitted placement of the new genus Modicisalibacter into the family.

Isolation of sulfate-reducing bacteria from Tunisian marine sediments and description of Desulfovibrio bizertensis sp. nov.

Several strains of sulfate-reducing bacteria were isolated from marine sediments recovered near Tunis, Korbous and Bizerte, Tunisia. They all showed characteristics consistent with members of the genus Desulfovibrio. One of these strains, designated MB3(T), was characterized further. Cells of strain MB3(T) were slender, curved, vibrio-shaped, motile, Gram-negative, non-spore-forming rods. They were positive for desulfoviridin as bisulfite reductase. Strain MB3(T) grew at temperatures of 15-45 degrees C (optimum 40 degrees C) and at pH 6.0-8.1 (optimum pH 7.0). NaCl was required for growth (optimum 20 g NaCl l(-1)). Strain MB3(T) utilized H(2) in the presence of acetate with sulfate as electron acceptor. It also utilized lactate, ethanol, pyruvate, malate, fumarate, succinate, butanol and propanol as electron donors. Lactate was oxidized incompletely to acetate. Strain MB3(T) fermented pyruvate and fumarate (poorly). Electron acceptors utilized included sulfate, sulfite, thiosulfate, elemental sulfur and fumarate, but not nitrate or nitrite. The G+C content of the genomic DNA was 51 mol%. On the basis of genotypic, phenotypic and phylogenetic characteristics, strain MB3(T) (=DSM 18034(T)=NCIMB 14199(T)) is proposed as the type strain of a novel species, Desulfovibrio bizertensis sp. nov.

Marinobacter vinifirmus sp. nov., a moderately halophilic bacterium isolated from a wine-barrel-decalcification wastewater.

A halophilic, Gram-negative, motile, non-sporulating bacterium designated strain FB1T was isolated from a wine-barrel-decalcification wastewater. The organism comprises straight rods and has a strictly respiratory metabolism with O2. Strain FB1T grows optimally at 20-30 degrees C and 5-6% NaCl. The predominant fatty acids were found to be C18:1omega9c (30.4%), C16:0 (25.7%), C12:0 3-OH (10.3%), C16:1omega9c (9.7%) and C16:1omega7c (8.4%). A phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain forms a coherent cluster within the genus Marinobacter. The highest level of 16S rRNA gene sequence similarity (97.9%) exhibited by strain FB1T was with the type strain of Marinobacter excellens. However, the level of DNA-DNA relatedness between the novel strain and M. excellens CIP 107,686T was only 31.2%. The DNA G+C content of strain FB1T was 58.7 mol%. On the basis of phenotypic and genotypic characteristics, and also phylogenetic evidence, strain FB1T is considered to represent a novel species of the genus Marinobacter, for which the name Marinobacter vinifirmus sp. nov. is proposed. The type strain is FB1T (=DSM 17747T=CCUG 52119T).

Bioconversion of ferulic acid to vanillic acid by Halomonas elongata isolated from table-olive fermentation.

Halomonas elongata strain Mar (=CCUG 52759) isolated from table-olive fermentation is the first halophilic bacterium to be shown to transform ferulic acid to vanillic acid under hypersaline conditions. During growth on ferulic acid, this strain was capable of promoting the formation of a significant amount of vanillic acid and trace quantities of vanillin. The products were confirmed by high-performance liquid chromatography and gas chromatography-mass spectrometry analyses. Based on the different metabolites identified, an oxidative side chain degradation pathway of ferulic acid bioconversion to vanillic acid was suggested. Phylogenetic analysis of 16S rRNA gene revealed that this isolated strain Mar was identified as H. elongata. To increase the formation of vanillic acid, a resting cell method using H. elongata strain Mar was performed. The optimal yield of vanillic acid (86%) was obtained after a 6 h reaction using 5 mM of ferulic acid and 4 g of dry weight of cells L(-1) pregrown on ferulic acid and harvested at the end of the exponential phase.

[Detection and kinetic characterization of Candida tropicalis contamination during fodder yeast production]. / Detección y caracterización cinética de una contaminación por Candida tropicalis durante la producción de levadura forrajera.

Candida tropicalis was identified as the etiologic agent of a severe contamination detected on an industrial fodder yeast production at the Cuban eastern region. After a detailed diagnostic task on raw material carried out on different factory sections, protocols to identify the contamination source and to isolate the microorganism were proposed. The identification was by comparison of the internal transcribed spacers ITS1 and ITS4 from 5.8S ribosomal DNA nucleotide sequences. In parallel, propagation of production strain, Candida utilis NRRL Y-660, at lab scale (2.5 l) was performed. Similar results to those observed in the factory concerning to its kinetic behavior in aerobic propagation with contaminated molasses, were detected at this level. The identification and primary kinetic characterization led to the implementation of sanitary and technological measures to bring production at its normal operational conditions as well as the application of prophylactic surveillance methodologies to avoid future contaminations.

Detecciòn y caracterizaciòn cìnètica en una contaminacion por Càndida tropicalis durante la producciòn de levadura forrajera / Detection and kinetic characterization of Candida tropicalis contamination during fodder yeast production

Se identificò a Candida tropicalis como el agente causal de una severa contaminaciòn detectada en la producciòn de levadura forrajera en una fàbrica de la regiòn oriental de Cuba. Posteriormente a un detallado diagnòstico en las materia primas y diferentes secciones de la fàbrica con el objetivo de identifcar el foco y aislar el microorganismo contaminante se procediò a su identificaciòn mediante comparaiòn de sustancias nucleòtidas de las regiones adyacentes no codificadas...(AU)

Candida tropicalis was identified as the etiologic agent of a severe contamination detected on an industrial fodder yeast production at the Cuban eastern region. After a detailed diagnostic task on raw material carried out on different factory sections, protocols to identify the contamination source and to isolate the microorganism were proposed...(AU)

Study of a hexane-degrading consortium in a biofilter and in liquid culture: biodiversity, kinetics and characterization of degrading strains.

A gasoline-degrading consortium, originating from a Mexican soil, was used to study its hexane-degradation kinetics in liquid culture and in a biofilter with mineral support. The biodiversity of the consortium depending on the culture conditions and electron and energy source (gasoline, hexane in liquid or hexane in the biofilter) was analyzed using a 16S rRNA-based approach. Significant differences between the populations were observed, indicating a probable adaptation to the substrate. Two strains, named SP2B and SP72-3, isolated from the consortium, belonged to Actinomycetes and demonstrated a high metabolic potential in hexane degradation. Even though the SP2B strain was related to Rhodococcus ruber DSM 43338(T) by phylogenetic studies, it displayed enlarged metabolic properties in hexane and other short-alkane degradation compared with the collection strain.

Isolation and characterization of Halomonas sp. strain IMPC, a p-coumaric acid-metabolizing bacterium that decarboxylates other cinnamic acids under hypersaline conditions.

A moderately halophilic, mesophilic, Gram-negative, motile, nonsporulating bacterium, designated strain IMPC, was isolated from a table-olive fermentation rich in aromatic compounds, after enrichment on p-coumaric acid under halophilic conditions. Strain IMPC was able to degrade p-coumaric acid. p-hydroxybenzaldehyde and p-hydroxybenzoic acid were detected as breakdown products from p-coumaric acid. Protocatechuic acid was identified as the final aromatic product of p-coumaric acid catabolism before ring fission. Strain IMPC transformed various cinnamic acids with substituent H, OH, CH(3) or OCH(3) in the para- and/or meta-position of the aromatic ring to the corresponding benzoic acids, indicating a specific selection. A beta-oxidation pathway was proposed for these transformations. Phylogenetic analysis of the 16S rRNA gene revealed that this isolate was a member of the genus Halomonas. Strain IMPC was closely related to Halomonas elongata ATCC 33173(T)and Halomonas eurihalina ATCC 49336(T).

Cloning and characterization of a tyrosinase gene from the white-rot fungus Pycnoporus sanguineus, and overproduction of the recombinant protein in Aspergillus niger.

A new tyrosinase-encoding gene (2,204 bp) and the corresponding cDNA (1,857 nucleotides) from the white-rot fungus Pycnoporus sanguineus BRFM49 were cloned. This gene consisted of seven exons and six introns and encoded a predicted protein of 68 kDa, exceeding the mature tyrosinase by 23 kDa. P. sanguineus tyrosinase cDNA was over-expressed in Aspergillus niger, a particularly suitable fungus for heterologous expression of proteins of biotechnological interest, under the control of the glyceraldehyde-3-phosphate-dehydrogenase promoter as strong and constitutive promoter. The glucoamylase preprosequence of A. niger was used to target the secretion. This construction enabled the production of recombinant tyrosinase in the extracellular medium of A. niger. The identity of the purified recombinant protein was confirmed by N-terminal amino acid sequencing. The maturation process was shown to be effective in A. niger, and the recombinant enzyme was fully active, with a molecular mass of 45 kDa. The best transformant obtained, A. niger D15#26-e, produced extracellular tyrosinase activities of 534 and 1,668 U l(-1) for monophenolase and diphenolase, respectively, which corresponded to a protein yield of ca. 20 mg l(-1).

Isolation and characterization of a novel Bacillus sp., strain YAS1, capable of transforming tyrosol under hypersaline conditions.

A moderately halotolerant, Gram-positive, aerobic, motile, spore-forming bacterium, designated as strain YAS1, was isolated from an olive-brine fermentation rich in aromatic compounds, after enrichment on tyrosol. Strain YAS1 grew between 25 and 45 degrees C and optimally at 37 degrees C. It grew in the presence of 0-15% (v/w) NaCl, with an optimum of 3-6% (v/w) NaCl. The DNA G+C content was found to be 49.9 mol%. Phylogenetic analysis of the 16S rRNA gene revealed that this isolate was a member of the genus Bacillus. The newly isolated strain YAS1 represents the first moderately halotolerant bacterium transforming tyrosol to p-hydroxyphenylacetic acid (PHPA) in the presence of yeast extract.