Haloferax sp. D1227, a halophilic Archaeon capable of growth on aromatic compounds

A pink-pigmented halophilic Archaeon, Strain D1227, was isolated from soil contaminated with oil brine and shown to be a member of the genus Haloferax, based on: (1) its hybridization with a 16S rRNA probe universal for the Archaea; (2) its resistance to a broad spectrum of antibiotics that affect Bacteria; (3) its requirement for at least 0.86 M NaCl and 25 mM Mg²⁺ for growth; (4) its possession of C₅₀-carotenoids characteristic of the halophilic Arachaea; (5) the thin layer chromatographic pattern of its polar lipids, which was identical to that of other species of Haloferax; and (6) its pleomorphic cell morphology. However, in contrast to the known species of Archaea, Haloferax strain D1227 was able to use aromatic substrates (e.g., benzoate, cinnamate, and phenylpropanoate) as sole carbon and energy sources for growth. Physiologically similar organisms, such as Haloferax volcanii, Haloferax mediterrani, Haloarcula vallismortis, and Haloarcula hispanica, could not grow on these aromatic substrates. When grown on ¹⁴C-benzoate, strain D1227 mineralized 70% of the substrate and assimilated 19% of the ¹⁴C-label into cell biomass. In addition to growth on aromatic substrates, D1227 was also capable of growth on a variety of carbohydrates and organic acids. Optimum growth of strain D1227 occurred at 45°C in media containing 1.7–2.6 M NaCl and 100 mM Mg²⁺. Under optimum growth conditions, the cell shape varied from that of an oblate spheroid on mineral salts medium alone, to discshaped, irregular or triangular cells on the same medium amended with yeast extract and tryptone. To our knowledge, this is the first unequivocal demonstration of the ability of an Archacon to grow by mineralization of aromatic substrates, and it adds a new dimension to our appreciation of the physiological diversity of this group of prokaryotes.Abbreviations Ha. Haloarcula - Hf. Haloferax     

Obligately halophilicChromatium vinosum from Hamelin Pool,Shark Bay,Australia

A representative of the purple sulfur bacteria was isolated from organic-rich intertidal sediments of Hamelin Pool, Shark Bay, Australia. The isolate, strain HPC, is nutritionally versatile, being capable of photoheterotrophic growth in the absence of reduced sulfur sources and of dark microaerophilic growth, either heterotrophically or lithotrophically. Vitamins are not required. Nine organic carbon substrates, including the C₂–C₅ fatty acids, support photoheterotrophic growth. The isolate is an obligate halophile capable of growth over a wide salinity range (0.5%–8.5% NaCl). On the basis of its morphology, physiology, pigmentation, and DNA base ratio, strain HPC is considered to be an obligately halophilic representative ofChromatium vinosum.     

The growth of WI-38 cells in a serum-free,growth factor-free,medium with elevated calcium concentrations

Summary The growth of WI-38 cells in serum-free growth medium with and without hormone supplementation in the presence of elevated Ca²⁺ concentrations was investigated. At 5 mM CaCl₂, WI-38 cells seeded at low density without serum or hormone supplementation showed up to a 12-fold increased in cell number at saturation density over that obtained at day 1. Saturation densities were comparable when either 5 mM CaCl₂ or epidermal growth factor (1 mM CaCl₂) was used in the presence of insulin, dexamethasone and transferrin. Combining suboptimal doses of epidermal growth factor and CaCl₂ resulted in an additive effect on saturation density. Thus, nornal human diploid cells are capable of substantial growth in serum-free, hormone-free growth medium. In contrast, confluent cultures refed with the same medium are not responsive to elevated Ca²⁺ concentrations. In fact, elevated Ca²⁺ concentrations inhibited the proliferative response of confluent cultures to epidermal growth factor, but enhanced their response to the combined treatment of insulin, transferrin and dexamethasone. This work was supported by the United States Public Health Society grants T-32, CA09171 and AG-00378. Editor's Statement This paper rigorously dissects the interplay among external Ca²⁺ concentration, cell density and specific growth factors on fibroblast growth in defined medium. Wallace L. McKeehan     

Tolerance of extremely halophilic archaebacteria towards bromide

The tolerance of halophilic archaebacteria towards bromide was tested in view of the fact that bromide occurs in natural brines in concentrations of up to 66 mM. It was found that, while concentrations of up to 0.8–1M are tolerated well by all halobacterial types examined, great differences exist between species with respect to bromide tolerance. WhileHalobacterium (H. salinarium, H. halobium, andH. sodomense) andNatronobacterium species are only moderately tolerant,Haloarcula (H. vallismortis, H. marismortui), andHaloferax species (H. mediterranei, H. gibbonsii) tolerate higher concentrations.Haloferax volcanii proved extremely tolerant and showed growth in bromide media at very low chloride concentrations (below 50 mM). No correlation was found between bromide tolerance and the bromide concentration in the habitat from which the strains were isolated. Iodide proved much more toxic than bromide. Bromide-tolerant strains also proved relatively resistant to growth inhibition by iodide.     

Calcium carbonate precipitation by two groups of moderately halophilic microorganisms at different temperatures and salt concentrations

Carbonate crystal formation by 48 strains of moderately halophilic microorganisms currently assigned to the genusFlavobacterium andAcinetobacter has been investigated. Strains were grown at different salt concentrations (2.5%, 7.5%, and 20%, wt/vol, total salts) and temperatures (22°C and 32°C). All the strains tested were capable of precipitating calcium carbonate as calcite, but onlyAcinetobacter strains formed aragonite. High temperature and low ionic strength of medium favored crystal formation. The influence of species specificity on the type of crystal precipitated by moderately halophilic microorganisms and their possible role in active precipitation in nature are discussed.     

Influence of sulphur-containing compounds on the growth of Methanosarcina barkeri in a defined medium

Summary Optimal growth of Methanosarcina barkeri occurred in a defined medium containing methanol when 2.5–4 mM sodium sulphide was added giving a concentration of 0.04–0.06 mM dissolved sulphide (HS^–+S^2–. When the sulphide concentration was too low for optimal growth (e.g., 0.1 mM Na₂S added) the addition of the redox resin Serdoxit acted as a sulphide reservoir and caused a significant stimulation of growth. Furthermore it could be demonstrated that iron sulphide, zinc sulphide or L-methionine could also act as sulphur sources while the addition of sodium sulphate to sulphide-depleted media failed to restore growth. The amino acid L-cysteine (0.85 mM) stimulated growth but could not replace Na₂S.Under optimal cysteine-and sulphide concentrations the generation time of this strain was about 7–9 h during growth on methanol, giving a growth yield of about 0.14 g/g methanol consumed. Different M. barkeri strains were also able to grow under these conditions on acetate (30–50 h doubling time) without a significant lag-phase and with complete substrate consumption even though the inoculum was grown on methanol or H₂–CO₂. When methanol and acetate were present as a mixture in the medium both were used simultaneously.     

Investigation of mercaptans,organic sulfides,and inorganic sulfur compounds as sulfur sources for the growth of methanogenic bacteria

A variety of compounds were investigated for use as sulfur sources for the growth of methanogenic bacteria.Methanococcus (Mc.) deltae, Mc. maripaludis, Methanobacterium (Mb.) speciesGC-2B, GC-3B, andMMY, Methanobrevibacter (Mbr.) ruminantium, andMethanosarcina (Ms.) barkeri strain 227 grew well with sulfide, S^o, thiosulfate, or cysteine as sole sulfur source.Mbr. ruminatium was able to grow on SO ₄ ⁼ or SO ₃ ⁼ , andMs. barkeri strain 227 was able to grow on SO ₃ ⁼ , but not on SO ₄ ⁼ as a sole sulfur source.Mc. jannaschii grew with sulfide, S^o, thiosulfate or SO ₃ ⁼ , but not on cysteine or SO ₄ ⁼ as sole surface source.Mc. thermolithotrophicus, Mc. jannaschii, Mc. deltae, andMb. thermoautotrophicum strains Marburg and H were able to grow with methanethiol, ethanethiol,n-propanethiol,n-butanethiol, methyl sulfide, dimethyl sulfoxide, ethyl sulfide, or CS₂ as a sulfur source, when very low levels (20–30 M) of sulfide were present; no growth occurred on 5–100 M sulfide alone. Methanethiol, ethanethiol, and methyl sulfide-using cultures produced sulfide during growth.     

Two halophilic Ectothiorhodospira strains with unusual morphological,physiological and biochemical characters

Two strains belonging to the genus Ectothiorhodospira were isolated from enrichment cultures inoculated with sulfide-containing samples from the saltern of Trapani. Cells are motile short spirilla with internal stacks of membranes. During sulfide utilization they produce external globules of sulfur that are then completely oxidized to sulfate. These halophilic microorganisms need NaCl concentrations of 11% and 18% and a slightly alkaline pH. They are typical photoautotrophic bacteria, utilizing sulfide, sulfur and, only one of them, thiosulfate as photosynthetic electron donors; growth is stimulated by organic compounds. Neither of the two strains is capable of assimilatory sulfate reduction and neither grows in the dark. Pigments of the two strains are bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series subgroup 1B. Quinones are Q8 and MK8 in a strain and Q8 and MK7 in the other one: the latter situation, with quinone side chains of different lengths, is atypical within phototrophic bacteria. For morphological, physiological and biochemical characters, at least one of these strains clearly stays apart from the six Ectothiorhodospira species described until now.List of abbreviations HPLC high performance liquid chromatography - TLC thin layer chromatography - Qn ubiquinone—number of isoprenoid units of the side chain - MKn menaquinone—number of isoprenoid units of the side chain - R_t retention time - R_f retention factor Dedicated to the memory of Professor Gino Florenzano who inspired this work     

Utilization of Carbohydrates by Rhynchosporium secalis II. Absorption and Metabolism of Glucose, Galactose and Galacturonic acid

Glucose, galactose and galacturonic acid were taken up at different rates by the fungus Rhynchosporium secalis and were intracellularly converted to other forms of carbohydrate at different rates. These differences explain why, when there is only a single source of nutrient carbon in the growth medium, development of the fungus is greatest when glucose is present and least when galactose is present. Glucose and galactose were taken up by the same mechanism for their uptake showed a reciprocal competitive inhibition. Uptake mechanisms had a high affinity for glucose (apparent K_m 2.76 mM) and galacturonic acid (apparent K_m 3.10 mM) and a low affinity for galactose (apparent K_m 29.67 mM). After uptake, galactose accumulated in the mycelium, whereas glucose and galacturonic acid were rapidly converted to other soluble carbohydrates, principally trehalose and mannitol. The insoluble carbohydrates within the mycelium were little affected by the type of carbohydrate that was supplied to the fungus.     

Increasing NaCl and CaCl2 concentrations in the growth medium of quince leaves: II. Effects on shoot regeneration

Summary The effects of NaCl and CaCl₂ on shoot regeneration from quince (Cydonia oblonga BA L29 clone) leaves were investigated. Caulogenesis was induced on in vitro-grown leaves treated for 2d in liquid Murashige and Skoog (MS) medium with 11.3 μM 2,4-dichlorophenoxyacetic acid and cultured on MS gelled medium supplemented with 4.5 μM thidiazuron and 0.5 μM naphthaleneacetic acid. Three experiments were performed: in the first, we compared the effects of NaCl at 0, 25, 50, 100, and 200 mM in factorial combination with 3, 9, and 27 mM CaCl₂. In the second, NaCl was tested at 0, 5, 10, 20, 40, and 80 mM with CaCl₂ at 0.3, 1.0, and 3.0 mM. The third experiment was carried out with the same experimental design as the second one but replacing NaCl with Na₂SO₄. Shoot regeneration was evaluated after 50 d of culturing: 25 in darkness and 25 in white light. In the first experiment, shoot regeneration was very poor and was observed only at the lower salt concentrations. In the second experiment, the percentages of caulogenic leaves were much higher, but decreased with increasing NaCl concentration. The more pronounced negative effect of the highest NaCl concentrations appeared to be partly mitigated by CaCl₂ at 1 and 3 mM. The presence of 3 mM CaCl₂, in the experiment with Na₂SO₄, appeared to be even more effective in reducing the adverse effect of sodium stress on caulogenesis. This result was attributed to the lower Cl⁻ concentration in the growth medium, which resulted from replacing NaCl with Na₂SO₄. NaCl applied at low concentrations (5 and 10 mM) in combination with 3 mM CaCl₂ exerted a favorable effect on adventitious shoot regeneration. As regards the Na⁺ and Ca²⁺ interaction, when the Na⁺/Ca²⁺ ratio was below roughly 35 and 20, with NaCl and Na₂SO₄, respectively, at least 60% of leaves showed regenerating capacity, but optimal values of this ratio were not derived.     

Increasing NaCl and CaCl<Subscript>2</Subscript> concentrations in the growth medium of quince leaves: I. Effects on somatic embryo and root regeneration

Summary The effects of increasing concentrations of NaCl and CaCl₂ on quince (Cydonia oblonga Mill. BA 29 clone) somatic embryogenesis and adventitious root regeneration were investigated. Leaves collected from in vitro-grown shoots were used as explants and induced for 2d in liquid Murashige and Skoog medium containing 11.3 μM 2,4-dichlorophenoxyacetic acid. Explants were then cultured on semisolid Murashige and Skoog medium enriched with 4.7 μM kinetin and 0.5 μM naphthaleneacetic acid under red light for 25 d and under white light for another 25 d. Two experiments were performed: in the first, NaCl was used at 0,25, 50, 100, and 200 mM in factorial combination with CaCl₂ at 3, 9, and 27 mM; in the second, NaCl was applied at 0, 5, 10, 20, 40, and 80 mM in combination with CaCl₂ at 0.3, 1.0, and 3.0 mM. Quince leaves revealed the capacity to regenerate somatic embryos and/or adventitious roots. Quantitative and qualitative regeneration from leaves was affected by NaCl treatments: increasing NaCl concentrations, in combination with CaCl₂ at 1 mM, led to an increase in the proportion of leaves producing somatic embryos only, and to a decrease of both leaves regenerating roots only and leaves simultaneously producing somatic embryos and adventitious roots. This suggests a beneficial effect of salt stress on the embryogenic process. The regeneration response decreased with increasing salt concentrations and was almost totally inhibited above 50 mM NaCl and 9 mM CaCl₂. The presence of CaCl₂ in the culture medium apparently mitigated the effects of salt stress, but only when NaCl was applied at 40 mM. NaCl at 5 mM, in the presence of 0.3 or 1 mM CaCl₂, was favorable both to somatic embryo and root production. No value of the ratio Na⁺/Ca²⁺ was found to be optimal for the regeneration processes.     

Sources of Nitrogen Supporting Growth and Embryogenesis in Cultured Wild Carrot Tissue

This paper seeks to calarify conflicting reports on the nitrogen requirements for in vitro embryogenesis in Daucus carota. Tissue derived from petiole explants of the wild strain of this species were tested with a variety of sources of cellular nitrogen under conditions otherwise favorable for in vitro embryogenesis. The use of very small, sieved and well-washed inocula reduced the carry-over of soluble materials with the inoculum. Embryo yield was quantified by direct counting of samples. Nitrate at concentrations ranging from 5 to 95 mM KNO₃ supportes only weak growth and very low embryogenesis under the exacting conditions of these experiments. As little as 0.1 mM NH₄Cl added to a nitrate medium allows some embryogenesis and 10 mM NH₄Cl is near optimal when KNO₃ is in the range of 12 to 40 mM concentration. Glutamine, glutamic acid, urea and alanine can individually partially replace NH₄Cl as a supplement to KNO₃. Glutamine, alanine, and possibly glutamic acid can serve as sole sources of nitrogen supporting both good growth and embryogenesis. It was concluded that a reduced nitrogen source is required, at least as a supplement to nitrate, for rapid growth and for in vitro embryogenesis of cultured wild carrot tissue. The relationship of pH of the culture medium to growth and embryogenesis was explored and optima observed at approximately pH 5.4 for both processes.     

Soybean [Glycine max (L.) merrill] embryogenic cultures: The role of sucrose and total nitrogen content on proliferation

Summary To improve proliferation of soybean cultures in liquid medium, the effects of sucrose; total inorganic nitrogen; content of No₃ ⁻, NH₄ ⁺, Ca²⁺, PO₄ ³⁻, K⁺; NH₄ ⁺/NO₃ ⁻ ratio; and medium osmotic pressure were studied using cv. Jack. Sucrose concentration, osmotic pressure, total nitrogen content, and ammonium to nitrate ratio were found to be the major factors controlling proliferation of soybean embryogenic cultures. Growth decreased linearly as sucrose concentration increased from 29.7 mM to 175.3 mM. A sucrose concentration of 29.2 mM, a nitrogen content of 34.9 mM, at 1 to 4 ammonium to nitrate ratio were found to be optimal for the fastest proliferation of soybean embryogenic cultures. There was no significant effect on proliferation of cultures when concentrations of NH₄ ⁺, Ca²⁺, PO₄ ³⁻, and K⁺ were tested in the range of 3.50 to 10.50, 1.02 to 3.06, 0.68 to 2.04, and 22.30 to 36.70 mM, respectively. The relative proliferation of embryogenic cultures of four soybean genotypes was evaluated in Finer and Nagasawa medium and in the new medium formulation. Despite genotype-specific differences in growth, the genotypes tested showed a biomass increase in the new formulation equal to 278, 269, 170, and 251% for Chapman, F138, Jack, and Williams 82, respectively, relative to their growth on standard FN medium. Due to its lowered sucrose and nitrogen content, we are referring to the new medium as FN Lite.     

新疆艾丁湖中度嗜盐苯酚降解菌多样性研究

高盐含酚废水属于极难处理的废水之一,筛选具有生物学降解能力的嗜盐菌有助于解决这一难题。从新疆艾丁湖盐湖中分离筛选能够降解苯酚的中度嗜盐菌,了解盐湖中度嗜盐苯酚降解菌的多样性组成和降解能力。研究结果表明,10%(质量分数)的盐浓度条件下,分离得到166株嗜盐菌,通过以苯酚为唯一碳源的培养基进行降解活性筛选后得到45株阳性菌,根据细菌16S rRNA基因序列系统进化分析,这45株菌分别归类到3个门,5个科,9个属。其中拟诺卡氏菌属(Nocardiopsis)是优势菌,占总量的68.8%,其余菌分布于Bacillus、Gracilibacillus、Pontibacillus、Halobacillus、Marinococcus和Halomonas属。在含100 mg/L苯酚的液体培养基,经过10 d培养后,这45株菌降解效率为1%~17%。本研究为工业应用提供了嗜盐微生物种质资源,极具进一步发掘和研究价值。     

Intrasynaptosomal Free Mg2+ Concentration Measured with the Fluorescent Indicator Mag-Fura-2: Modulation by Na+ Gradient and by Extrasynaptosomal ATP

Abstract: Synaptosomes can be loaded with mag-fura-2 without significant perturbation of their ATP content by incubation for 10 min at 37°C with 10 µM mag-fura-2 acetoxymethyl ester in Hanks'-HEPES buffer (pH 7.45). The intrasynaptosomal free Mg²⁺ concentration ([Mg²⁺]_i) was found to be dependent on external Mg²⁺ concentration, increasing from 0.8 to 1.25 mM when the concentration of Mg²⁺ in the incubation medium increased from 1 to 8 mM. Dissipation of the Na⁺ gradient across the plasma membrane of synaptosomes by treatment with the Na⁺ ionophore monensin (0.2 mM) or with veratridine (0.2 mM) and ouabain (0.6 mM) produced a moderate increase of [Mg²⁺]_i, from 1.0 to 1.2–1.3 mM in an incubation medium containing 5 mM Mg²⁺. Plasma membrane depolarization by incubation of synaptosomes in a medium containing 68 mM KCl and 68 mM NaCl had no effect on [Mg²⁺]_i. Reversal of the Na⁺ gradient by incubation of synaptosomes in a medium in which external Na⁺ was replaced by choline increased [Mg²⁺]_i up to 1.6 and 2.2 mM for extrasynaptosomal Mg²⁺ concentrations of 1 and 8 mM, respectively. We conclude that a Na⁺/Mg²⁺ exchange operates in the plasma membrane of synaptosomes. In the presence of Mg²⁺ in the incubation medium, extrasynaptosomal ATP, but not ADP or adenosine, increased [Mg²⁺]_i from 1.1 ± 0.1 up to 1.6 ± 0.1 mM. The nonhydrolyzable ATP analogue adenosine 5′-(βγ-imido)triphosphate antagonized the effect of ATP, but had no effect by itself on [Mg²⁺]_i. It is concluded that Mg²⁺ transport across the plasma membrane of synaptosomes is modulated by the activity of an ecto-ATPase or an ecto-protein kinase.     

Balance between Cyanide-Sensitive and -Insensitive Respiration in Wheat Root Mitochondria, as Influenced by Salt Concentration in the Plant Growth Medium

Mitochondria were isolated from 7-day-old wheat roots (Triticum vulgare Vill. cv. Svenno Spring Wheat) grown in either a full-strength culture medium (100%) or in the same medium diluted 100 times (1%). Outer membrane integrity was assayed using the cytochrome c reduction assay. This indicated about 20% damage. Using an oxygen electrode the respiration of the mitochondria was measured with either malate or succinate as the substrate (both 40 mM). KCN (3 mM) and salicylhydroxamic acid (SHAM, 1 mM) were used as inhibitors. The properties of the isolated mitochondria (STATE 3 rate, ADP/O ratio, and KCN-sensitivity) depend upon the ionic concentration of the growth medium of the roots. In the mitochondria isolated from roots grown in the 1% medium (1% mitochondria) there is a synergistic effect of KCN and SHAM. This means that electrons can be shifted from one pathway to the other when only one of the inhibitors is added. This flexibility between the electron pathways is almost nil in the mitochondria isolated from roots grown in the 100% medium (100% mitochondria). The maximal capacity of the alternative electron pathway (= rate in the presence of KCN) is higher in 1% (40 nmol O₂ min^?1 (mg protein)^?1) than in 100% mitochondria (20 nmol O₂ min^?1 (mg protein)^?1. In 100% mitochondria the alternative pathway seems to be operating at maximal capacity in the absence of KCN with both substrates and in both STATES 3 and 4. In 1% mitochondria the alternative pathway functions at >50% of its capacity in the absence of KCN.     

Isolation and characterization of rat primary lung cells

Summary Lung cell culture may be useful as anin vitro alternative to study the susceptibility of the lung to various toxic agents. Lungs from female Wistar rats were enzymatically digested by recirculating perfusion through the pulmonary artery with a sequence of solutions containing deoxyribonuclease, chymopapain, pronase, collagenase, and elastase. Lung tissue was microdissected and resuspended and the cells obtained were washed by centrifugation. By this isolation method, 2×10⁸ cells per rat lung were obtained with an average viability of 97%. Lung cells cultured in medium containing antibiotics and serum maintained a viability of >70% for 5 d. Rat primary lung cells were exposed to various toxic agents and their viability was assessed by formazan production capacity after 18 h of incubation. Compared to rat and mouse hepatocyte cultures (EC⁵⁰=5.8 mM), rat primary lung cells were much more susceptible to hydrogen peroxide (EC₅₀=0.6 mM). All cell types were equally sensitive to the more potent toxicanttert-butylhydroperoxide (EC₅₀=0.1 mM). Paraquat was more toxic to lung cells (EC₅₀=0.03 mM) than to rat (EC₅₀=2.8 mM) and mouse (EC₅₀=0.2 mM) hepatocytes. In contrast, rat lung cells were less sensitive to sodium nitroprusside (EC₅₀=2.6 mM) compared to rat (EC₅₀=0.2 mM) and mouse (EC₅₀=0.03 mM) hepatocytes. Nitrofurantoin and menadione (at EC₅₀=0.04 mM and 0.006 mM, respectively) were more toxic to rat lung and liver cells than to murine hepatocytes (EC₅₀=0.2 mM and 0.04 mM, respectively). Our findings demonstrate the applicability of this rat primary lung cell culture for studying the effects of lung toxicants. Parts of the study had been presented orally at the meeting of the German Society of Toxicology and Pharmacology in Mainz (FRG), March 15–17, 1994.     

Ontogeny of somatic embryos in cucumber (<Emphasis Type="Italic">cucumis sativus</Emphasis> L.)

Summary Suspension culture of cucumber (Cucumis sativus L.) has been an inefficient method for production of somatic embryos owing to problems with embryo maturation and conversion. Embryogenic callus of cv. Green Long was induced on semisolid Murashige and Skoog (MS) medium containing 6.8 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.2 μM 6-benzylaminopurine (BA). A large number of globular somatic embryos were obtained on transfer of the callus to MS liquid medium supplemented with 87.6 mM sucrose, 1.1 μM 2,4-D, and improved by the addition of 342.4 μM l-glutamine. MS medium supplemented with 87.6 mM sucrose was more effective in somatic embryo production than other sugars. Subsequent development led to the formation of heart-and torpedo-shaped embryos. Maturation of somatic embryos occurred on plant growth regulator-free MS semi-solid medium containing 175.2 mM sucrose and 0.5 gl⁻¹ activated charcoal. Conversion of embryos into plants was achieved on half-strength MS semi-solid medium containing 87.6 mM sucrose and 1.4 μM gibberellic acid (GA₃) in a 16h photoperiod. Twenty-seven percent of embryos were converted into normal plants.     

Selection of Bradyrhizobium strains and provenances of Acacia mangium and Faidherbia albida: Relationship with their tolerance to acidity and aluminium

This work was designed to determine the role of the acidity and aluminium stress in the selection of partners in the Acacia symbioses with relevance to the persistence of the microsymbiont Bradyrhizobium in the soil and the growth and nodulation of the host plant respectively. Fifteen strains of Bradyrhizobium from Acacia mangium and Faidherbia albida formed a very homogenous acid tolerant group as indicated by their ability to grow better in a medium at pH 4.5 than in a medium at pH 6.8. By contrast, a growth experiment using an acid liquid media (pH 4.5), containing different concentrations of aluminium successfully identified strains sensitive to aluminium toxicity and those able to grow even in the presence of 100 M AlCl₃.Our results suggest that high amounts of aluminium in the soil rather than acidity (pH 4.5) were a major soil factor for selection of Bradyrhizobium strains capable of establishing a permanently high population under natural conditions.Unlike the behaviour of the microsymbiont, growth and nodulation of Acacia mangium and Faidherbia albida were not affected by aluminium, even at 100 M, but they might be significantly affected by medium acidity (pH 4.5) depending on plant provenances. It is therefore suggested that ability of the host plant to tolerate acidity stress should be taken into account first when screening effective Acacia-Bradyrhizobium combinations for use in afforestation trials.     

Characterization of and Ethanol Hyper-production by Clostridium thermocellum I-1-B

An ethanol hyper-producing clostridial strain, I-1-B, was isolated from Shibi hot spring, Kagoshima prefecture and identified as Clostridium thermocellum based on morphological and physiological proper­ ties. The carbohydrates used as energy sources were glucose, fructose, cellobiose, cellulose and esculin. Fermentation products were ethanol, lactate, acetate, formate, carbon dioxide, and hydrogen. The optimum, maximum, and minimum temperature for growth are about 60, 70, and 47°C, respectively. Optimum pH for growth is about 7.5, and growth occurs at starting pH between 6.0 and 9.0. I-1-B strain has strong tolerance for ethanol and hyper ethanol-productivity. Ethanol concentrations causing 50%. decrease of growth yield are 27 and 16g/liter for I-1-B and ATCC27405 of C. thermocellum, respectively. The organism was cultured on a medium containing 80 g/liter cellulose at 60°C for 156 h. The culture was fed with a vitamin mixture containing vitamin B₁₂ and mineral salts solution at intervals. In this culture the organism produced 23.6 g/liter (512mM) ethanol, 8.5 g/liter (94mM) lactate, 2.9 g/liter (48mM) acetate, and 0.9 g/liter (20mM) formate. The molar ratio of ethanol to total acidic products was 3.2. The ethanol productivity of the strain I-1-B is superior to any of the wild and mutant strains of C. thermocellum so far reported.