Hydrogen sulfide formation as well as ethanol production in different media by cysND- and/or cysIJ-inactivated mutant strains of Zymomonas mobilis ZM4

Many bacteria reduce inorganic sulfate to sulfide to satisfy their need for sulfur, one of the most important elements for biological life. But little is known about the metabolic pathways involving hydrogen sulfide (H₂S) in mesophilic bacteria. By genomic sequence analysis, a complete set of genes for the assimilatory sulfate reduction pathway has been identified in the ethanologen Zymomonas mobilis. In this study, the first ATP sulfurylase- and final sulfite reductase-encoding genes cysND and cysIJ, respectively, in the putative pathway from sulfate to sulfite in Z. mobilis ZM4 was singly or doubly inactivated by homologous recombination and a site-specific FLP-FRT recombination. The resultant mutants, ?cysND, ?cysIJ and ?cysND-cat?cysIJ, were unable to produce detectable H₂S in glucose or sucrose-containing rich medium and sweet sorghum juice, in which the wild-type ZM4 produced detectable H₂S. While adding sulfite (SO₃ ^2?) into media impaired the growth of the mutants and ZM4 to varying degrees, the sulfite restored the H₂S formation in the ?cysND in the above media, but not in the ?cysIJ and ?cysND-cat?cysIJ mutants. Although it seemed that the inactivation of cysND and cysIJ did not exert a significant negative effect on the cell growth at least in glucose or sucrose medium, the ethanol production of all mutants was inferior to that of ZM4 in sucrose medium and sweet sorghum juice. In addition, adding l-cysteine to glucose-containing rich media restored H₂S formation of all mutants, indicating the existence of another pathway for producing H₂S in Z. mobilis. All these results would help to further elucidate the metabolic pathways involving H₂S in Z. mobilis and exploit the biotechnological applications of this industrially important bacterium.     

Sorbitol production by Zymomonas mobilis

Summary High resolution ¹³C Nuclear Magnetic Resonance (NMR) spectroscopy has been employed to determine the chemical composition of the unknown major products in a sucrose or fructose plus glucose fermentation to ethanol by the bacterium Zymmonas mobilis. When grown on these sugars Z.mobilis was found to produce significant amounts of sorbitol, up to 43 g·l^-1 for strain ZM31 when grown on 250 g·l^-1 sucrose.The production of sorbitol and decrease of glucose, fructose, or sucrose was followed throughout batch fermentations by NMR and HPLC. Sorbitol was shown to be derived only from fructose by [¹⁴C]-feeding experiments. Additionally ³¹P NMR spectroscopy was utilized to determine the concentrations of both glucose 6-phosphate and fructose 6-phosphate relative to their respective concentrations in Z.mobilis cells fermenting glucose or fructose alone.It is suggested that free glucose inside the cell inhibits fructokinase. Free intracellular fructose may then be reduced to sorbitol via a dehydrogenase type enzyme. Attempts to grow Z.mobilis on sorbitol were unsuccessful, as were experiments to induce growth via mutagenesis.This work was supported in part by the National Energy Research, Development and Demonstration Council of Australia     

Levan production using mutant strains of Zymomonas mobilis in different culture conditions

Several levan hyperproducing mutants of Zymomonas mobilis strains were selected by mutagenesis with N-methyl-N-nitro-nitrosoguanidine and caffeine. Highest levan production (41 g l^–1) was obtained with a mutant strain HL 29 in a culture medium containing 200 g sucrose l^–1 and 0.5 g (NH₄)₂SO₄ l^–1 stored at 7 °C for 29 days. This is the first report describing the levan synthesis by Z. mobilis at 7 °C.     

Growth ofZymomonas on lactose: Gene cloning in combination with mutagenesis

Summary Wild-type strains ofZymomonas mobilis have a limited substrate range of glucose, fructose and sucrose. In order to expand this substrate range, transconjugants ofZ. mobilis containing Lac⁺ plasmids have been constructed. Although -galactosidase is expressed in such strains, they lack the ability to grow on lactose. We now report the development ofZ. mobilis strains capable of growth on lactose. This was achieved in two stages. First, a broad host range plasmid was constructed (pRUT102) which contained the lactose operon under the control of aZ. mobilis promoter plus genes for galactose utilization.Z. mobilis CP4.45 containing pRUT102 was then subjected to mutagenesis combined with continued selection pressure for growth on lactose. One strain,Z. mobilis SB6, produced a turbid culture that yielded 0.25% ethanol from 5% lactose (plus 2% yeast extract) in 15 days.     

Optimisation of submerged culture conditions for the production of mycelial biomass and exopolysaccharide byPleurotus nebrodensis

The optimisation of submerged culture conditions and nutritional requirements was studied for the production of exopolysaccharide (EPS) fromPleurotus nebrodensis. The optimal temperature and initial pH for both mycelial growth and EPS production in shake flask cultures were 25 °C and 8.0, respectively. Maltose was found the most suitable carbon source for both mycelial biomass and EPS production. Yeast extract was favourable nitrogen source for both mycelial biomass and EPS production. Optimum concentration of each medium component was determined using the orthogonal matrix method. The optimal combination of the media constituents for mycelial growth and EPS production was as follows: 200 g l^?1 bran, 25 g l^?1 maltose, 3 g l^?1 yeast extract, 1 g l^?1 KH₂PO₄, 1 g l^?1 MgSO₄ 7H₂O. Under the optimal conditions, the mycelial biomass (4.13 g l^?1) and EPS content (2.40 g l^?1) ofPleurotus nebrodensis was 2.3 and 3.6 times compared to the control with basal medium respectively.     

Evolution-based strategy to generate non-genetically modified organisms Saccharomyces cerevisiae strains impaired in sulfate assimilation pathway

Aims: An evolution‐based strategy was designed to screen novel yeast strains impaired in sulfate assimilation. Specifically, molybdate and chromate resistance was used as selectable phenotype to select sulfate permease–deficient variants that unable to produce sulfites and hydrogen sulfide (H₂S). Methods and Results: Four Saccharomyces cerevisiae parent strains were induced to sporulate. After tetrad digestion, spore suspensions were observed under the microscope to monitor the conjugation of gametes. Then, the cell suspension was inoculated in tubes containing YPD medium supplemented with ammonium molybdate or potassium chromate. Forty‐four resistant strains were obtained and then tested in microvinifications. Three strains with a low sulfite production (SO₂ <10 mg l^?1) and with an impaired H₂S production in grape must without added sulfites were selected. Conclusions: Our strategy enabled the selection of improved yeasts with desired oenological characteristics. Particularly, resistance to toxic analogues of sulfate allowed us to detect strains that unable to assimilate sulfates. Significance and Impact of the Study: This strategy that combines the sexual recombination of spores and application of a specific selective pressure provides a rapid screening method to generate genetic variants and select improved wine yeast strains with an impaired metabolism regarding the production of sulfites and H₂S.     

Effect of glucose concentration on the rate of fructose consumption in native strains isolated from the fermentation of Agave duranguensis

Studies on hexose consumption by Saccharomyces cerevisiae show that glucose is consumed faster than fructose when both are present (9:1 fructose to glucose) in the medium during the fermentation of Agave. The objective of this work was to select strains of S. cerevisiae that consume fructose equal to or faster than glucose at high fructose concentrations by analyzing the influence of different glucose concentrations on the fructose consumption rate. The optimal growth conditions were determined by a kinetics assay using high performance liquid chromatography (HPLC) using 50?g of glucose and 50?g of fructose per liter of synthetic medium containing peptone and yeast extract. Using the same substrate concentrations, strain ITD-00185 was shown to have a higher reaction rate for fructose over glucose. At 75?g of fructose and 25?g of glucose per liter, strain ITD-00185 had a productivity of 1.02 gL^?1?h^?1 after 40?h and a fructose rate constant of 0.071?h^?1. It was observed that glucose concentration positively influences fructose consumption when present in a 3:1 ratio of fructose to glucose. Therefore, adapted strains at high fructose concentrations could be used as an alternative to traditional fermentation processes.     

Bioethanol production in batch mode by a native strain of <Emphasis Type="Italic">Zymomonas mobilis</Emphasis>

Two wild strains of Zymomonas mobilis were isolated (named as ML1 and ML2) from sugar cane molasses obtained from different farms of Santander, Colombia. Initially, selection of the best ethanol-producer strains was carried out using ethanol production parameters obtained with a commercial strain Z. mobilis DSM 3580. Three isolated strains were cultivated in a culture medium containing yeast extract, peptone, glucose and salts, at pH 6 and 32°C with stirring rate of 65 rpm during 62 h. The best results of ethanol production were obtained with the native strain ML1, reaching a maximum ethanol concentration of 79.78 g l⁻¹. ML1 and ML2 strains were identified as Z. mobilis, according to the morphology, biochemical tests and molecular characterization by PCR of specific DNA sequences from Z. mobilis. Subsequently, the effect of different nitrogen sources on production of ethanol was evaluated. The best results were obtained using urea at a 0.73 g/l. In this case, maximum concentration of ethanol was 83.81 g l⁻¹, with kinetic parameters of yield of ethanol on biomass (Y_P/X) = 69.01(g g⁻¹), maximum volumetric productivity of ethanol (Qp_max) = 2.28 (g l⁻¹ h⁻¹), specific productivity of ethanol (q_P) = 3.54 (h⁻¹) and specific growth rate (μ) = 0.12 h⁻¹. Finally, we studied the effect of different culture conditions (pH, temperature, stirring, C/N ratio) with a Placket-Burman′s experimental design. This optimization indicated that the most significant variables were temperature and stirring. In the best culture conditions a significant increase in all variables of response was achieved, reaching a maximum ethanol concentration of 93.55 g l⁻¹.     

Rock phosphate solubilization by four yeast strains

The solubilization of rock phosphate (RP) by four yeast strains, Rhodotorula sp., Candida rugosa, Saccharomyces cerevisiae and Saccharomyces rouxii, which were isolated from wheat rhizospheric soils, was investigated in this study. The yeast isolates demonstrated diverse levels of soluble phosphate releasing abilities in modified Pikovskaya liquid medium containing RP as sole phosphate source. C. rugosa was the most effective solubilizer under different conditions, followed by Rhodotorula sp., S. rouxii and S. cerevisiae. Acidification of the broth seemed to be the major mechanism for RP solubilization by the yeast isolates, and the increase in soluble phosphate released was correlated significantly with an increase in titratable acidity and a drop in pH. The optimal composition for the solubilization of RP by the yeast isolates in the broth was 20 g L^?1 glucose, 1 g L^?1 yeast extract, 0.5 g L^?1 (NH₄)₂SO₄, and 5 g L^?1 RP, respectively. The yeast isolates were able to solubilize RP at wide range of temperature and initial pH, with the maximum percentage of soluble phosphate released being recorded at 30–35 °C and pH 5–6, respectively.     

Characterization of a Symbiotic Coculture of Clostridium thermohydrosulfuricum YM3 and Clostridium thermocellum YM4

Clostridium thermohydrosulfuricum YM3 and C. thermocellum YM4 were isolated from a coculture which was obtained from an enrichment culture inoculated with volcanic soil in Izu Peninsula, Japan. Strain YM3 had advantages over reported C. thermohydrosulfuricum strains in that it fermented inulin and could accumulate ethanol up to 1.3% (wt/vol). The highest ethanol yield obtained was 1.96 mol/mol of anhydroglucose unit in cellobiose. Strain YM4 had features different from those reported in C. thermocellum strains: it formed spores rarely (at a frequency of <10^-5), it required CO₂ and Na₂CO₃ for growth, and it fermented sucrose. Strain YM4 completely decomposed 1% Avicel within 25 h when the inoculum constituted 2% of the culture medium volume, and it produced 0.22 U of Avicelase and 2.21 U of carboxymethylcellulase per ml of the medium. The doubling times on Avicel, cellobiose, and glucose were 2.7, 1.1, and 1.6 h, respectively. Reconstructed cocultures of strains YM3 and YM4 were very stable and degraded Avicel more rapidly than did strain YM4 monoculture. Without yeast extract, neither microorganism was able to grow. However, the coculture grew on cellulose without yeast extract and produced ethanol in high yield. Moreover, cell-free spent culture broth of strain YM3 could replace yeast extract in supporting the growth of strain YM4. The symbiotic relationship of the two bacteria in cellulose fermentation is probably a case of mutualism.     

Effect of calcium and sodium salts on ethanol production in high sugar fermentation by free cells ofZymomonas mobilis

Summary Addition of calcium carbonate enhanced ethanol production byZymomonas mobilis ZM4 and a mutant (ZMI₂), especially at higher concentrations (200–400 g/L) of glucose and sucrose, as well as at higher temperature (42°C) by the mutant. Calcium and sodium carbonates neutralized the acid produced in the medium and enhanced the ethanol production. The Na salts were less effective in the parent strain and were not favourable for the mutant. Ca²⁺ ions played a direct role in augmenting ethanol production as evidenced by the effect of calcium chloride at controlled pH (5.5).     

Kinetic analysis of ethanol production by an acetate-resistant strain of recombinant Zymomonas mobilis

Zymomonas mobilis ZM4/Ac^R (pZB5), a mutant recombinant strain with increased acetate resistance, has been isolated following electroporation of Z. mobilis ZM4/Ac^R. This mutant strain showed enhanced kinetic characteristics in the presence of 12 g sodium acetate l^–1 at pH 5 in batch culture on 40 g glucose, 40 g xylose l^–1 medium when compared to ZM4 (pZB5). In continuous culture, there was evidence of increased maintenance energy requirements/uncoupling of metabolism for ZM4/Ac^R (pZB5) in the presence of sodium acetate; a result confirmed by analysis of the effect of acetate on other strains of Z. mobilis. Nomenclature m Cell maintenance energy coefficient (g g^–1 h^–1)Maximum overall specific growth rate (1 h^–1)Maximum specific ethanol production rate (g g^–1 h^–1)Maximum specific total sugar utilization rate (g g^–1 h^–1)Biomass yield per mole of ATP (g mole^–1 Ethanol yield on total sugars (g g^–1)Biomass yield on total sugars (g g^–1)True biomass yield on total sugars (g g^–1)     

Statistical optimization of simple culture conditions to produce biomass of an ochratoxigenic mould biocontrol yeast strain

Aim: To maximize biomass production of an ochratoxigenic mould–controlling strain of Lachancea thermotolerans employing response surface methodology (RSM). Methods and Results: Using Plackett–Burman screening designs (PBSD) and central composite designs (CCD), an optimized culture medium containing (g l^?1): fermentable sugars (FS), 139·2, provided by sugar cane molasses (CMz), (NH₄)₂HPO₄ (DAP), 9·0, and yeast extract (YE), 2·5, was formulated. Maximal cell concentration obtained after 24 h at 28°C was 24·2 g l^?1cell dry weight (CDW). The mathematical model obtained was validated in experiments performed in shaken‐flask cultures and also in aerated bioreactors. Maximum yield and productivity values achieved were, respectively, of 0·23 g CDW/g FS in a medium containing (g l^?1): FS, 87·0; DAP, 7·0; YE, 1·0; and of 0·96 g CDW l^?1 h^?1 in a medium containing (g l^?1): FS, 150·8 plus DAP, 6·9. Conclusions: Optimized culture conditions for maximizing yeast biomass production determined in flask cultures were applicable at a larger scale. The highest yield values were attained in media containing relatively low‐CMz concentrations supplemented with DAP and YE. Yeast extract would not be necessary if higher productivity is the aim. Significance and Impact of the Study: Cells of L. thermotolerans produced aerobically could be sustainably produced in a medium just containing cheap carbon, nitrogen and phosphorus sources. Response surface methodology allowed the fine‐tuning of cultural conditions.     

An improved synthetic medium for continuous cultivation of Zymomonas mobilis

Summary A synthetic medium for continuous cultivation of Zymomonas mobilis was developed using the chemostat pulse technique in appropriate experimental designs. Yeast extract could be replaced by a mixture of six mineral salts, Ca-pantothenate, l-as-partate, and l-serine. Kinetic data from continuous cultivations of strains ATCC 10988 and ZM4 are presented and compared with published data.     

Effect of sugar concentration on ginsenoside biosynthesis in hairy root cultures of Panax quinquefolium cultivated in shake flasks and nutrient sprinkle bioreactor

The study assessed the influence of sugar concentration (10, 20, 30, 50, 70, 100, 120 g l^?1) on growth and ginsenoside biosynthesis in Panax quinquefolium hairy roots cultivated in shake flasks and a nutrient sprinkle bioreactor. The highest growth rate was achieved in medium containing 3–5 % sucrose. More than 70 g l^?1 or less than 20 g l^?1 sugar content in the medium induces significant inhibition of root growth when cultivated in shake flasks. The saponin content was determined using HPLC. The maximum yield (above 9 mg g^?1 d.w.) of the sum of six examined ginsenosides (Rb₁, Rb₂, Rc, Rd, Re and Rg₁) in hairy roots cultivated in shake flasks was obtained with 30 g l^?1 sucrose in the medium. The sucrose concentration in the medium was found to correlate with saponin content in bioreactor-cultured specimens. A higher level of protopanaxadiol derivatives was found for lower (20 and 30 g l^?1) sucrose concentrations; higher sucrose concentrations (50 and 70 g l^?1) in the medium stimulated a higher level of Rg group saponins.     

Characterization of a superior thermotolerant mutant ofZymomonas mobilis ZM4 for ethanol production in glucose medium

Summary Nitrosoguanidine-induced, stable theromotolerant mutant (ZMI₂) ofZymomonas mobilis ZM4 was found to possess almost normal cell morphology, and a better ethanol tolerance at 42°C than the parent strain (ZM4). Its kinetic parameters, in converting different concentrations of glucose to ethanol, were comparable to ZM4 at 30°C, and significantly superior at 42°C. In a 200 g/L glucose medium in a pH-stat (5.0) at 42°C, the mutant yielded more ethanol (71.0 g/L) (improved to 73.7 g/L at pH 5.5) and alcohol dehydrogenase (ADH) than the parent strain. The ADH levels in both the strains were repressed, depending upon the increased level of sugar and degree of temperature.     

The intracellular sucrase (SacA) of Zymomonas mobilis is not involved in sucrose assimilation

The intracellular sucrase SacA from Zymomonas mobilis was purified to homogeneity from a recombinant E. coli strain containing the SacA gene under an expression system. The protein was monomeric with a molecular mass of 58 kDa. The sucrase activity was maximal at 25 °C and thermal stability of the purified protein was low (50% recovery after 30 min at 46 °C ). The activation energy was low at 33 kJ mol^–1. Maximum activity was at pH 6.5. Activity was strongly inhibited (>99%) by SH blocking reagents and reducing agents slightly (10–60%) increased the activity of purified SacA. The sucrase showed a low K _M (42 mM) and k _cat (125 s^–1) which indicated its very low efficiency for sucrose hydrolysis. A mutant strain of Z. mobilis not able to grow on sucrose was isolated. This strain (ZM4S) lacked the two sucrases SacB and SacC but SacA was present in the intracellular fraction. Therefore, SacA alone is unable to allow growth Z. mobilis on sucrose.     

Embryogenic protoplast cultures of orange jessamine (Murraya paniculata) and their regeneration into plants flowering in vitro

Embryogenic callus was induced from the hypocotyl region of seedlings germinated from immature embryos of orange jessamine (Murraya paniculata (L.) Jack) on Murashige & Tucker (1969) medium containing 50 g l^-1 sucrose, 5.0 mg l^-1 benzyladenine, 2.5 mg l^-1 2,4-dichlorophenoxyacetic acid and 600 mg l^-1 malt extract. Isolated protoplasts divided to produce callus on Murashige & Tucker (1969) medium containing 50 g l^-1 sucrose, 0.01 mg l^-1 gibberellin A₄₊₇ and 600 mg l^-1 malt extract. Callus developed to plantlets via somatic embryogenesis on Murashige & Tucker (1969) medium with 50 g l^-1 lactose but no plant growth regulators. These plantlets flowered in vitro on half strength Murashige & Tucker (1969) medium containing 50 g l^-1 sucrose after 2 months culture.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - FM full strength MT medium - FMG full strength MT medium +1 mg l^-1 GA₃ - GA₃ gibberellin A₃ - GA₄₊₇ gibberellin A₄₊₇ - HM half strength MT medium - HMG half strength MT medium +1 mg l^-1 GA₃ - MT Murashige & Tucker (1969)     

Recovery of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.) plants transformed with the antifungal plant defensin gene PDF1.2

Embryogenic avocado cultures derived from ‘Hass’ protoplasts were genetically transformed with the plant defensin gene (pdf1.2) driven by the CaMV 35S promoter in pGPTV with uidA as a reporter gene and bar, the gene for resistance to phosphinothricin, the active ingredient of the herbicide Finale® (Basta) (Bayer Environmental Science, Research Triangle Park, Durham, NC ). Transformation was mediated by Agrobacterium tumefaciens strain EHA105. Transformed cultures were selected in the presence of 3.0 mg l^?1 phosphinothricin in liquid maintenance medium for 3–4 mo. Liquid maintenance medium consisted of modified MS medium containing (per liter) 12 mg NH₄NO₃ and 30.3 mg KNO₃ and supplemented with 0.1 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 30 g l^?1 sucrose, 3.0 mg l^?1 phosphinothricin, and 0.41 μM picloram. Somatic embryo development from transformed cultures was initiated on MS medium supplemented with 45 g l^?1 sucrose, 4 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 10% (v/v) filter-sterilized coconut water, 3.0 mg l^?1 phosphinothricin, and 6.0 g l^?1 gellan gum. Limited plant recovery occurred from somatic embryos on semi-solid MS medium supplemented with 3.0 mg l^?1 phosphinothricin, 4.44 μM 6-benzylaminopurine (BA), and 2.89 μM GA₃; transformed shoots were micrografted on in vitro-grown seedling rootstocks. Approximately 1 yr after acclimatization in the greenhouse, transformed shoots were air-layered to recover transformed roots. Genetic transformation of embryogenic cultures, somatic embryos, and regenerated plants was confirmed by polymerase chain reaction (PCR), Southern blot hybridization, the XGLUC reaction for uidA, and application of the herbicide Finale® to regenerated plants.     

Symbiotic effectiveness and plant growh promoting traits in some Rhizobium strains isolated from Phaseolus vulgaris L.

The ability of Rhizobia to colonize roots of certain legumes and promote their growth has been proven previously. In this study the symbiotic efficiency of 47 Rhizobium strains with 6 common bean cultivars was evaluated under greenhouse condition. Fourteen strains showed the best symbiotic efficiency, whereas some isolates could not induce nodules on host plants. The ability of fourteen superior strains to solubilize phosphorus and zinc and to produce auxin, HCN and siderohores was evaluated in the laboratory assays. Rhizobium strain Rb102 produced the highest amount of auxin (14.2?mg?l^?1) in the medium containing l-tryptophan. None of the isolates were able to solubilize ZnO and ZnCO₃ on solid medium but in liquid medium some of them had negligible solubilization. The highest P solubility in liquid and solid medium was observed in strains Rb113 and Rb130, respectively. Strain Rb102 produced the highest amount of siderophores. None of the isolates were able to produce HCN. This study showed that there was a great diversity between the strains of Rhizobium in terms of their plant growth promoting traits symbiotic efficiency which supports the importance of screening rhizobia for selecting the most efficient strains. The genetic diversity of the isolates was analyzed by PCR–RFLP of the 16S rDNA. Our rhizobia were clustered into 10 groups showing high levels of diversity.