Appl Microbiol Biotechnol (2000) 54: 319325

Springer-Verlag 2000

ORIGINAL PAPER

F. F. Hezayen B. H. A. Rehm R. Eberhardt A. Steinbuchel

Polymer production by two newly isolated extremely halophilic archaea: application of a novel corrosion-resistant bioreactor

Received: 3 December 1999 / Received revision: 22 February 2000 / Accepted: 25 February 2000

Abstract A novel corrosion-resistant bioreactor composed of polyetherether ketone (PEEK), tech glass and silicium nitrite ceramics was constructed and applied for the cultivation of two newly isolated, extremely halophilic archaea producing poly(c-glutamic acid) (PGA), or poly(b-hydroxy butyric acid) (PHB), respectively. These bacteria were isolated from hypersaline soil close to Aswan (Egypt). The isolate strain 40, which is related to the genus Natrialba, produced large amounts of PGA when cultivated on solid medium. Culture conditions were optimised applying the corrosion-resistant bioreactor. PGA production was dependent on NaCl concentration and occurred about at 20% (w/v) NaCl in the medium. A maximum cell density of about 1.6 g cell dry matter/l was obtained when the bioreactor was stirred and aerated in a batch fermentation process using proteose-peptone medium. The supernatant was monitored with respect to PGA formation, and after 90 h a maximum of 470 mg/l culture volume was detected by HPLC analysis. Culture conditions were optimized for the isolate 56, which accumulated PHB as intracellular granules. Batch fermentations in the stirred and aerated bioreactor applying acetate and n-butyric acid as carbon sources led to cell density of 2.28 g cell dry matter/l and a maximum PHB accumulation contributing to about 53% of cellular dry weight. About 4.6 g PHB were isolated from 10.6 g dried cells of strain 56, which exhibited a weight average molar mass of 2.3 105 g mol)1 and a polydispersity of about 1.4.

Introduction
Aerobic extremely halophilic archaebacteria require at least 10% (2 M) NaCl for growth and are classied within the family Halobacteriaceae (Kamekura 1998). One member of this family, Haloferax mediterranei, has been reported to produce an extracellular polysaccharide, which results in mucous colonies on solid medium (Rodriguez-Valera et al. 1983). This polymer was isolated from the supernatant of liquid cultures, and yields up to 3 g/l were detected. The polymer was composed of mannose, glucose, galactose and another unidentied sugar as well as amino sugars, uronic acids and considerable amounts of sulphate, which contributed to the acidic nature of this polymer (Anton et al. 1988). The structure of the repeating unit was revealed: 4)-b-DGlcpNAcA-(1 6)-a-D-Manp-(1 4)-b-D-GlcpNAcA3-O-SO -(1 (Parolis et al. 1996). This exopolymer 3 might be used as a resistant thickening agent in various applications. L-Glutamate has been identied as a constituent of the cell wall of the archaebacterium Natronococcus occultus (Niemetz et al. 1997). The extremely halophilic strain 40, which has been recently isolated (Hezayen et al., submitted), is the rst described archaebacterium which produces extracellular poly-c-D-(glutamate) (PGA). Various species of the genus Bacillus, e.g. B. subtilis and B. anthracis, produce large amounts of extracellular PGA (Cheng et al. 1989). Most of the fermentation studies and the molecular characterisation of PGA biosynthesis were conducted with B. subtilis IFO3335, and PGA can be used as a biodegradable thickener, humectant, sustained-release material or drug carrier in the elds of food, cosmetics, or medicine (Kunioka 1997). In contrast to the above-mentioned exopolymers, polyhdroxyalkanoates (PHAs) are accumulated as intracellular bacterial storage compounds. These water-insoluble biopolymers exhibit interesting physical properties which, for some PHAs, resemble those of polyethylene and polypropylene (Steinbuchel et al. 1997;

F. F. Hezayen B. H. A. Rehm A. Steinbuchel (&) Institut fur Mikrobiologie, Westfalische Wilhelms-Universitat Munster, Corrensstrae 3, 48149 Munster, Germany e-mail: steinbu@uni-muenster.de Tel.: +49-251-8339821 Fax: +49-251-833-8388 R. Eberhardt FairMen Tec GmbH, Berliner Strasse 1, 37073 Gottingen, Germany

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Anderson and Dawes 1990). Various PHAs are synthesised by a variety of microorganisms employing different metabolic routes and PHA synthases with dierent substrate specicities (Steinbuchel and Valentin 1995; Steinbuchel and Fuchtenbusch 1998; Rehm and Steinbuchel 1999). Some members of the family Halo bactericeae have been shown to accumulate large amounts of poly(3-hydroxybutyrate), PHB, under conditions of nitrogen limitation and abundant carbon source (Fernandez-Castillo et al. 1986). However, only the carbohydrate-utilising species H. mediterranei accumulates large amounts of PHB, contributing to 60% of cellular dry weight (CDW), respectively. Moreover, PHB production of H. mediterranei was strongly enhanced to a maximum of about 6.0 g/l using phosphate limitation and starch as carbon source (Lillo and Rodriguez-Valera 1990). The potential use of extremely halophilic archaea in biotechnological processes is mainly based on their extreme salt tolerance, which enables cultivation without sterile precautions. This obviously reduces production costs. Therefore, we recently screened a variety of extremely halophilic bacteria isolated from a hypersaline soil sample close to Aswan (Egypt) with respect to exopolymer formation and PHB accumulation (Hezayen et al., in preparation). One new isolate, strain 40, which is related to the genus Natrialba, is capable of producing copious amounts of PGA when cultivated on solid medium. Another isolate, strain 56, accumulated large amounts of PHB. In this study, we optimised the cultivation conditions for these newly isolated and extremely halophilic archaea in a novel, corrosion-resistant, stirred tank bioreactor with the goal of production of the biopolymers PGA and PHB.

Cultivations in the corrosion-resistant, stirred tank bioreactor Batch cultivations were performed in a novel, corrosion-resistant, stirred and aerated 8-l tank bioreactor (FairMenTec, Gottingen, Germany). The bioreactor was made of corrosion-resistant polyetherether ketone (PEEK), tech glass and silicium nitrite ceramics, which was used to construct the wear- and corrosion-resistant ball bearings; stirring was performed via corrosion-resistant magnetic coupling (Fig. 1). The cultivation vessel was surrounded by a second thermostatted vessel (Fig. 1). The bioreactor was equipped with a digitised feedback regulation system, enabling control of the pH and monitoring of oxygen saturation. Both strains were cultivated in 6 l of the optimised medium, which was composed of basic medium plus carbon sources when indicated, and the pH value was kept constant at pH 7.2. The incubation temperature was 40 C, and the dissolved oxygen concentration was maintained above 15% of air saturation. PGA isolation and analysis The cells were grown on solidied basic medium containing 1% (w/v) casamino acids or protease peptone, harvested after 3 days from agar plates and suspended in distilled water. Cells were sedimented by 60 min ultracentrifugation at 40,000 g, and the polymer in the supernatant was precipitated by addition of one volume of ethanol. The polymer was collected, resuspended in water and lyophilised to a ne powder. It was then hydrolysed in 6 N HCl (1 mg/100 ml) for 12 h at 95 C, lyophilised again and dissolved in an equal volume of distilled water. The polymer sample was then subjected to HPLC analysis after ortho-phthaldialdehyde (OPA) (ortho-phthaldialdehyde) derivatisation as follows and according to the manual provided by Merck (Darmstadt, Germany): 460 ll hydrolysed sample were mixed with 200 ll 0.5 M borate buer, pH 9.5, and 100 ll OPA reagent. The mixture was incubated at room temperature for 200 s. Fifty microlitres of 0.75 N HCl were added to adjust the pH to 7.07.5, which stopped the reaction. Then, 100 ll of the OPA derivatised sample were mixed with 400 ll start eluent (26:74 MeOH:Na-acetate); 20 ll were subjected to HPLC analysis, and amino acids were identied by retention time using appropriate standards. The D-glutamate from hydrolysed PGA was quantitated based on calibration curves done with dierent concentrations of D-glutamate. PHB isolation and analysis Poly(b-hydroxy butyric acid) was extracted from lyophilised cells by chloroform in a Soxhlet apparatus and subsequently precipitated in ten volumes of ethanol. The precipitate was dissolved in chloroform and was again precipitated in ethanol in order to obtain highly puried PHB. PHA was qualitatively and quantitatively analysed by gas chromatography. Liquid cultures were centrifuged at 10,000 g for 30 min, and the cells were lyophilised overnight. Then, 35 mg lyophilised cell material was subjected to methanolysis in the presence of 15% (v/v) sulfuric acid. The resulting methyl esters of the constituent 3-hydroxyalkanoic acids were assayed by gas chromatography according to Brandl et al. (1988) and as described in detail recently (Timm and Steinbuchel 1990). Gas chromatog raphy analysis was performed by injecting 3 ll of sample into a Perkin-Elmer 8420 gas chromatograph using a 0.5-lm-diameter Permaphase PEG 25 Mx capillary column 60 m in length (Uberlingen, Germany). In addition, the puried polymer was also analysed by gas chromatography-mass spectrometry. For this, the polyester was dissolved in chloroform at an approximate concentration of 2 mg/ ml and after methanolysis 3 ll were injected into a Hewlett-Packard 6890 gas chromatograph/mass spectrometer (Palo Alto, USA). The same column as used for the gas chromatography analysis was applied using a temperature prole as described previously (Timm and Steinbuchel 1990).

Materials and methods

Bacterial strains Strain 40 (DSM13077), which is related to the genus Natrialba, and strain 56 (DSM 13151) were recently isolated from a soil sample collected from the surface of hypersaline soil in Aswan city (Egypt). Both isolates were related to archaebacteria based on partial 16S rDNA sequencing and on ether lipid analysis in addition to various physiological properties (Hezayen et al., in preparation). Media and growth conditions Strains 40 and 56 were isolated on S-G medium (Sehgal and Gibbons 1960), which contained 25% (w/v) NaCl, 2% (w/v) MgSO4 7H2O, 0.2% (w/v) KCl, 3% (w/v) trisodium citrate, 1% (w/v) yeast extract (Difco), and 0.75% (w/v) casamino acids. The pH was adjusted to 7.2 with NaOH. The organisms were grown and maintained on proteose peptone-salt medium, containing 25% (w/v) NaCl, 0.2% (w/v) KCl, 0.5% (w/v) MgSO4 7H2O, 0.75% (w/v) proteose peptone and 0.5% (w/v) yeast extract. The pH was adjusted to 7.2 with NaOH. To prepare agar plates, the media were solidied with 2% (w/v) of agar. The media were sterilised by autoclaving. The basic medium contained 22.5% (w/v) NaCl, 0.2% (w/v) KCl, 0.5% (w/v) MgSO4 7H2O, and 0.1% (w/v) yeast extract and was used to analyse the dierent activities of strains 40 and 56.

321 Fig. 1 The novel corrosion-resistant bioreactor with a detailed description of the stirring apparatus

Gel permeation chromatography analysis (GPC) Molecular weight analysis was conducted with puried PHA, which was dissolved in chloroform (510 mg/ml) and subjected to a Waters (Milford, USA) gel permeation chromatography system applying four sequentially arranged Styragel HR3-6 columns for separation and a model 410 dierential refractometer for detection. Polystyrene molecular weight standards with narrow polydispersities were employed for calibration.

Results
Construction of a new, corrosion-resistant, stirred tank bioreactor A novel corrosion-resistant bioreactor was constructed in order to enable cultivation of extremely halophilic archaebacteria, which require high salt concentrations for growth (10%, w/v, NaCl) and which show optimum growth at about 25% (w/v) NaCl. Additionally, an ecient aeration system was constructed, which is needed to provide suciently high dissolved-oxygen concentrations for cultivation in high-salt media (Fig. 1). The reaction system was made of PEEK and teon containing as gas release device a distributory disk, which was made of porous teon ending in a nozzle

with 100 holes. This porous teon disk in combination with the nozzle mediates the release of very small air bubbles that strongly enhances the dissolved oxygen concentration. These cultivation conditions particularly require the use of corrosion- and wear-resistant material. The novel bioreactor was basically described in the Materials and methods section and is outlined in Fig. 1. PEEK, which was obtained from Ensinger GmbH & Co (Anrochte, Germany) is a non-ammable, sterilizable (134 C, 2 bar, 20 min) thermoplastic material exhibiting very durable properties (Bottenbruch 1994). It does not show signicant changes even after 1000 h incubation in steam/water at 250 C, after 2000 h in mineral oil at 200 C, is insoluble in almost all common solvents and is inert to most acids and alkaline compounds and shows no reactions with and little or no absorption of saturated sodium chloride and many other inorganic salt solutions even at elevated temperatures. The two recently isolated archaebacterial strains 40 and 56 were cultivated in batch-processes to test the applicability of the new stirred tank bioreactor. Both strains showed growth rates comparable to cultivations in agitated asks (data not shown), but with strain 56 a strong increase of PHB production was obtained (see below).

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PGA production by strain 40 under various cultivation conditions The production of PGA by strain 40, which was recently discovered on solid medium by the appearance of highly mucous colonies, was studied on agar plates as well as in liquid medium applying baed asks. The cultivation conditions and the medium composition were changed in order to optimize the production of PGA. Incubation temperatures ranged from 30 C to 50 C were applied, and at 40 C the maximum PGA production was obtained. The eect of glutamate, glutamine and casein as carbon source on PGA formation was investigated, but none of these carbon sources enhanced PGA formation. Moreover, glutamine showed a 4-fold weaker PGA production than with casamino acids as carbon source, but the NH formation was about 5-fold higher (data 4 not shown). The salt concentration had severe inuence on the formation of PGA, and strong PGA formation occurred only at NaCl concentrations of 25% (w/v) and above (Fig. 2). The maximum PGA yield of 0.42 g/g CDW was obtained on solid medium containing 30% (w/v) NaCl (Fig. 2). The exopolymer, which was isolated after cultivation on solid medium, showed a high PGA content, about 85% (w/v). At NaCl concentrations below 20% (w/v), colonies did not show a slimy morphology. Cultivations in liquid medium using baed asks did not reveal a strong production of PGA, and Dglutamic acid constituted only 55% (w/v) of the isolated exopolymer. A maximum PGA yield of 0.2 g/g CDW was obtained when 30% (w/v) NaCl were applied. Furthermore, isolation of PGA from liquid cultures required cross-ow ltration (cut-o Mw of 30,000 g/mol) in order to remove NaCl, which strongly interferred with ethanol precipitation.

PGA production by strain 40 using the new stirred tank bioreactor Six litres of basic medium plus 1% (w/v) casamino acids was inoculated with a 0.5l preculture of strain 40, which was incubated for 3 days at 40 C in the same medium. The initial pH was adjusted to 7.2 which continuously increased during the time course of the fermentation and was then kept constant at pH 8.0. The incubation temperature was 40 C, and the oxygen saturation was kept at 15% using a stirring speed of 200 rpm. The optical density, NH concentration and PGA content were moni4 tored during fermentation. After 90 h incubation the PGA production reached its maximum of 470 mg/l cellfree supernatant, and a cell mass of about 1.6 g CDW/l was obtained (Fig. 3). After 100 h incubation the amount of PGA in the supernatant decreased to 447 mg/l. The NH concentration was continuously increasing from 4 about 0 g/l to about 2.8 g/l after 100 h of incubation. PHB production by strain 56 using various carbon sources and cultivation conditions Since the phosphate concentration had a strong impact on PHB production by H. mediterranei (Lillo and Rodriguez-Valera 1990), we applied various phosphate concentrations in order to investigate the inuence of the phosphate concentration on PHB synthesis by strain 56. The phosphate concentration did not show any eect on PHB synthesis by strain 56. Various carbon sources were analysed with respect to their eect on PHB accumulation in asks, that were incubated on a shaker. Casamino acids, proteose peptone, beef extract or yeast extract, when used as complex carbon source did not result in PHB accumulation (Fig. 4). However, when sodium pyruvate and n-butyric acid were used as carbon source resulted in

Fig. 2 Eect of NaCl concentration on the formation of poly(cglutamic acid) plus 1% (w/v) casamino acids (PGA) by strain 40. Cultivations were performed in basic medium using liquid cultivations in asks (j) or on solid medium using agar plates (r)

Fig. 3 Cultivation of strain 40 in the novel corrosion-resistant bioreactor using basic medium plus 1% (w/v) casamino acids. The optical density (OD) was monitored at 600 nm wavelength (m). In addition, the poly(c-glutamic acid) (PGA) formation (d) and the NH concentration (j) in the culture supernatants were analysed 4

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strong accumulation of PHB, contributing to about 25% and 18.5% of CDW, respectively. A combination of the two carbon sources sodium acetate and n-butyric acid (each 1%, w/v) resulted in an even stronger PHB accumulation contributing to about 34% of CDW (Fig. 4). To further improve PHB production, the eect of various concentrations of yeast extract, n-butyric acid and sodium acetate was studied with respect to cell growth and PHB accumulation (Fig. 5). The best cell growth and PHB accumulation were obtained using 0.1% (w/v) yeast extract, 1% (v/v) n-butyric acid and 1% (w/v) sodium acetate (Fig. 5). Fed batch cultivations were conducted feeding every 24 h 0.2% (w/v) of either n-butyric acid or sodium acetate. In contrast to batch cultivation using 0.2% (w/v) of either carbon source, fed batch cultivation resulted in a 2-fold increase of CDW/l and a 2-fold decrease in PHB accumulation after a 9 d incubation period (data not shown). Various incubation temperatures were analysed, and the optimum growth and PHB accumulation was observed at 40 C (Fig. 5). Time-dependent monitoring of the PHB accumulation revealed that PHB accumulation continuously increased up to the 8th day of incubation. Thereafter the PHB content decreased slowly, and after 12 days of incubation PHB contributed only 50% of the CDW (data not shown). The NaCl concentration had strong inuence on growth and PHB accumulation. At a low salt concentration of 10% (w/v), strong PHB accumulation occurred, contributing to about 44% of the CDW, whereas growth was impaired and only about 50% of the CDW/l compared to maximum cell growth was obtained. Best
Fig. 4 The eect of various carbon sources on the poly(bhydroxy butyric acid) (PHB) production by strain 56. Cultivations were performed in asks using basic medium, which contained 1% (w/v) of the respective carbon source. The PHB content was analysed after an incubation period of 8 days; CDW, cellular dry weight

Fig. 5 The eect of various concentrations of yeast extract (r), nbutyric acid (j) and sodium acetate (m) on the poly(b-hydroxy butyric acid) (PHB) production by strain 56. Cultivations were performed in asks using basic medium, which contained 1% (w/v) of the respective carbon source. The PHB content was analysed after an incubation period of 8 days; CDW, cellular dry weight

growth was obtained at a salt concentration of 22.5% (w/v), but PHB was found approximately 50% as compared to cells grown in the presence of 10% NaCl. PHB production by strain 56 using the new, corrosion-resistant, stirred tank bioreactor. Taking the results of the cultivation experiments in asks into account, strain 56 was now cultivated in the stirred

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Fig. 6 Cultivation of strain 56 in the novel corrosion-resistant bioreactor using basic medium containing 1% (v/v) n-butyric acid and 1% (w/v) sodium acetate as carbon sources. The cell density (d), the cellular dry weight (CDW) per liter medium (m) and the the poly(b-hydroxy butyric acid) (PHB) content (j) were monitored. The optical density (OD) was obtained using a wavelength of 600 nm

tank bioreactor. Six litres of basic medium containing 1% (w/v) n-butyric acid and 1% (w/v) sodium acetate as carbon sources were inoculated with 0.5 l of a preculture of strain 56, which was incubated for 3 days in proteose peptone-salt medium. The pH was kept constant at 7.2, the incubation temperature was 40 C, and the stirrer speed was adjusted to 200 rpm maintaining the concentration of dissolved oxygen at 15% saturation. The optical density, cell density and the PHB content of the cells were monitored during fermentation. After 11 days of fermentation, the maximum cell density of 2.28 g CDW/l and a maximum PHB content contributing to about 53% of the CDW was reached (Fig. 6). The cultivation was terminated after 12 d, because cell mass and PHB content decreased to 2.1 g CDW/l and 49% of CDW, respectively (Fig. 6). The cells from 5.2 l of culture volume were harvested and lyophilised leading to a total of 10.6 g of CDW. The dried cell material was extracted with chloroform for 3 d in a Soxhlet apparatus, and after precipitation in 10 vol of ethanol, 4.6 g of puried PHB were obtained, thus recovering approximately 87% of the accumulated polyester. Due to the long incubation period, the volumetric productivity was only 4.5 mg PHB l)1h)1 of PHB by strain 56. GC/MS analysis of the isolated polyester revealed that it was solely composed of 3-hydroxybutyric acid and GPC analysis showed a weight average molar mass of about 2.3 105 with a polydispersity of about 1.4.

cannot be done, because of the highly corrosive potential of the high-salt medium. Thus all parts of the bioreactor, which are exposed to the medium, should be replaced by alternative materials as mentioned above. A novel corrosion-resistant stirred tank bioreactor was constructed. Due to the chemical, physical and mechanical properties of PEEK and of the other materials used for construction of the bioreactor, and since PEEK is certied by the FDA (21 CFR 177.2415), since it belongs to class VI of the USB XXII and follows the EG directive 90/128/EWG as well as the cytotoxicity tests according to DIN/EN/ISO 309935, this bioreactor will not be only suitable for the cultivation of extremely halophilic archaea, but also for the cultivation of temperature and pH extremophiles and the production of food stus or ingredients and products for medical or pharmaceutical applications. In this study, this bioreactor was used for the cultivation of two new isolates of extremely halophilic archaea. The cultivations were basically monitored with respect to cell mass and polymer production. The use of the bioreactor, employing a 6 l culture volume provided an ecient tool to scale up the cultivation of the two strains of extremely halophilic archaea. Cell mass and polymer yields were at least comparable to ask cultivation. Since the bioreactor equipment enabled on-line adjustment of pH and on-line monitoring of oxygen saturation, the variation of these factors facilitated optimization of cultivation conditions of extremely halophilic archaea in media containing at least 25% (w/v) NaCl. PGA production by strain 40 The production of PGA by strain 40 was two times less in liquid cultures than on solid media. Furthermore, PGA produced from solid medium was an almost pure homopolymer, whereas the polymer retained from liquid cultivation in asks or in the bioreactor contained in addition to D-glutamic acid also other constituents, since D-glutamic acid made up only about 55% (w/w) of the isolated polymer. The isolation of PGA from liquid cultivation required cross-ow ltration in order to remove media components as well as NaCl, which interfered with ethanol precipitation of the PGA. Since casamino acids served as carbon source during cultivation and were not solely used as precurser for PGA production, the ammonia concentration increased during growth (Fig. 3). Whereas many PGA-producing bacteria such as B. subtilis IFO 3335 require L-glutamic acid or L-glutamine as precursor for PGA synthesis for ecient PGA production (Thorne et al. 1953, 1954; Kunioka 1997), these two amino acids did not enhance PGA production of strain 40. However, B. licheniforms 9945a showed the strongest volumetric PGA productivity of about 0.12 g l)1 h)1 and after 48 h96 h cultivation, a volumetric yield of about 11 g l)1 was obtained when L-glutamic acid, citric acid and glycerol were applied as carbon sources (Birrer et al. 1994). This volumetric yield is about 20 times higher than that was achieved with

Discussion
Performance of the new corrosion-resistant bioreactor Cultivation of extremely halophilic bacteria in bioreactors that contain parts manufactured from stainless steel

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strain 40 after 90 h cultivation. Interestingly, PGA production on solid as well as in liquid media depended on the salt concentration; PGA formation began at about 20% (w/v), NaCl and reached its maximum at NaCl saturation. These data suggested that PGA plays a role in the protection of the microorganism against dehydration. Since, based on its anionic character, PGA shows a strong water-binding capacity, this polymer might support the hydration status of the microorganism even under strong dehydrating conditions, as it occurs in the presence of high concentrations of NaCl. This functional role has also been suggested for the extracellular anionic polymer alginate, the production of which is induced and enhanced under dehydrating conditions (Rehm and Valla 1997). PHB production by strain 56 Various growth conditions were changed in order to optimize PHB production by strain 56. The phosphate concentration, which showed a strong inuence on PHB synthesis by H. mediterranei (Lillo and Rodriguez-Valera 1990), did not aect the accumulation of PHB in strain 56. The strongest eect on PHB production was exerted by the carbon source. Glucose and starch were the best carbon sources for PHB production by H. mediterranei. During optimized cultivation conditions, H. mediterranei produced about 6 g PHB/l, and PHB contributed to about 60% of the CDW providing a yield over the carbon source of 0.3 g/g (Lillo and Rodriguez-Valera 1990). In contrast, the best PHB production by strain 56 during cultivation in the corrosion-resistant bioreactor was obtained when n-butyric acid and sodium acetate were used as carbon sources. In such cells, PHB accumulation contributed to 53% of the CDW. Strain 56 showed only a low volumetric productivity of 4.6 mg PHB l)1h)1, which is about 1000 times less when compared with the high volumetric PHB productivity of Alcaligenes latus (Wang and Lee 1997). The theoretical PHB yield over carbon source was 0.54 g/g and a yield over carbon source of 0.45 g/g was obtained based on the amount of PHB that was isolated from the cells. This indicated an ecient conversion of the carbon source into PHB. Strain 56 is the second extremely halophilic archaebacterium, the physiology and culture conditions of which were evaluated in view of the production of PHB.
Acknowledgements This study was supported by a fellowship provided by the Egyptian Government to Francis F. Hezayen.

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