Modelling the freezing response of baker's yeast prestressed cells: a statistical approach

Aims: To study the effect of prestress conditions on the freezing and thawing (FT) response of two baker’s yeast strains and the use of statistical analysis to optimize resistance to freezing. Methods and Results: Tolerance to FT of industrial strains of Saccharomyces cerevisiae was associated to their osmosensitivity and growth phase. Pretreatments with sublethal stresses [40°C, 0·5 mol l^?1 NaCl, 1·0 mol l^?1 sorbitol or 5% (v/v) ethanol] increased freeze tolerance. Temperature or hyperosmotic prestresses increased trehalose contents, nevertheless no clear correlation was found with improved FT tolerance. Plackett–Burman design and response surface methodology were applied to improve freeze tolerance of the more osmotolerant strain. Optimal prestress conditions found were: 0·779 mol l^?1 NaCl, 0·693% (v/v) ethanol and 32·15°C. Conclusions: Ethanol, saline, osmotic or heat prestresses increased freezing tolerance of two phenotypically distinct baker’s yeast strains. A relationship among prestresses, survival and trehalose content was not clear. It was possible to statistically find optimal combined prestress conditions to increase FT tolerance of the osmotolerant strain. Significance and Impact of the Study: Statistically designed combination of prestress conditions that can be applied during the production of baker’s yeast could represent a useful tool to increase baker’s yeast FT resistance.     

Molybdate reduction by Pseudomonas sp. strain DRY2

Aims: To isolate and characterize a potent molybdenum‐reducing bacterium. Methods and Results: A minimal salt medium supplemented with 10 mmol l^?1 molybdate, glucose (1·0%, w/v) as a carbon source and ammonium sulfate (0·3%, w/v) as a nitrogen source was used in the screening process. A molybdenum‐reducing bacterium was isolated and tentatively identified as Pseudomonas sp. strain DRY2 based on carbon utilization profiles using Biolog GN plates and partial 16S rDNA molecular phylogeny. Strain DRY2 produced 2·4, 3·2 and 6·2 times more molybdenum blue compared to Serratia marcescens strain DRY6, Enterobacter cloacae strain 48 and Eschericia coli K12, respectively. Molybdate reduction was optimum at 5 mmol l^?1 phosphate. The optimum molybdate concentration that supported molybdate reduction at 5 mmol l^?1 phosphate was between 15 and 25 mmol l^?1. Molybdate reduction was optimum at 40°C and at pH 6·0. Phosphate concentrations higher than 5 mmol l^?1 strongly inhibited molybdate reduction. Inhibitors of electron transport system such as antimycin A, rotenone, sodium azide and cyanide did not inhibit the molybdenum‐reducing enzyme activity. Chromium, copper, mercury and lead inhibited the molybdenum‐reducing activity. Conclusions: A novel molybdenum‐reducing bacterium with high molybdenum reduction capacity has been isolated. Significance and Impact of the Study: Molybdenum is an emerging global pollutant that is very toxic to ruminants. The characteristics of this bacterium suggest that it would be useful in the bioremediation of molybdenum pollutant.     

Comparison of supplements* to enhance recovery of heat‐injured Salmonella from egg albumen

Aims: The purpose of this study was to determine the proficiency of supplements to enhance the recovery of Salmonella from heat‐treated liquid egg albumen on solid agar media. Methods and Results: Salmonella‐inoculated albumen, heated at 53·3°C for 4 min, was plated on 39 combinations of solid media with or without the addition of 12 supplements. Greater numbers of Salmonella (P < 0·05) recovered with the addition of 1·0 g l^?1 ferrous sulfate (FeSO₄) than with any other supplements, except for 0·5 or 1·0 g l^?1 3′3′‐thiodipropionic acid (TDP), which recovered equivalent populations. Addition of 1·0 g l^?1 sodium pyruvate or 6·0 g l^?1 yeast extract plus 1·0 g l^?1 sodium pyruvate supported greater resuscitation than unsupplemented tryptic soy agar (TSA) or supplementing with 0·01 or 0·1 g l^?1 N‐propyl gallate, 10 g l^?1 activated charcoal, 0·1 g l^?1 KMnO₄ or 50 mg l^?1 ethoxyquin. The remaining supplements supported recovery of equivalent numbers of Salmonella, which were fewer cells than recovered with 1·0 g l^?1 FeSO₄, yet greater populations than recovered with 50 mg l^?1 ethoxyquin. Conclusion: Supplementation of plating media with FeSO₄, TDP or sodium pyruvate enhanced recovery of sublethally injured Salmonella from albumen. Significance and Impact of the Study: Pasteurizing albumen impedes recovery of pathogens. These results suggest that the addition of supplements to plating media may assist resuscitation and colony development of heat‐injured salmonellae.     

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.     

Isolation and characterization of butachlor‐catabolizing bacterial strain Stenotrophomonas acidaminiphila JS‐1 from soil and assessment of its biodegradation potential

Aims: Isolation, characterization and assessment of butachlor‐degrading potential of bacterial strain JS‐1 in soil. Methods and Results: Butachlor‐degrading bacteria were isolated using enrichment culture technique. The morphological, biochemical and genetic characteristics based on 16S rDNA sequence homology and phylogenetic analysis confirmed the isolate as Stenotrophomonas acidaminiphila strain JS‐1. The strain JS‐1 exhibited substantial growth in M9 mineral salt medium supplemented with 3·2 mmol l^?1 butachlor, as a sole source of carbon and energy. The HPLC analysis revealed almost complete disappearance of butachlor within 20 days in soil at a rate constant of 0·17 day^?1 and half‐life (t_½) of 4·0 days, following the first‐order rate kinetics. The strain JS‐1 in stationary phase of culture also produced 21·0 μg ml^?1 of growth hormone indole acetic acid (IAA) in the presence of 500 μg ml^?1 of tryptophan. The IAA production was stimulated at lower concentrations of butachlor, whereas higher concentrations above 0·8 mmol l^?1 were found inhibitory. Conclusions: The isolate JS‐1 characterized as Stenotrophomonas acidaminiphila was capable of utilizing butachlor as sole source of carbon and energy. Besides being an efficient butachlor degrader, it substantially produces IAA. Significance and Impact of the Study: The bacterial strain JS‐1 has a potential for butachlor remediation with a distinctive auxiliary attribute of plant growth stimulation.     

Study of laccase production by Pleurotus ostreatus in a 5 l bioreactor and application of the enzyme to determine the antioxidant concentration of human plasma

Aims: To achieve high laccase production from Pleurotus ostreatus in a bench top bioreactor and to utilize the enzyme for determination of the total antioxidant concentration (TAC) of human plasma. Methods and Results: Laccase production by P. ostreatus studied in a benchtop bioreactor was as high as, 874·0 U ml^?1 in presence of copper sulfate. The enzyme was used to replace metmyoglobin and hydrogen peroxide for the estimation of TAC in human plasma. The trolox equivalent antioxidant concentrations determined by the laccase‐based method and metmyoglobin method ranged from 1·63 ± 0·011 to 1·80 ± 0·006 mmol l^?1 and from 1·41 ± 0·004 to 1·51 ± 0·008 mmol l^?1 plasma, respectively. Conclusions: Pleurotus ostreatus produced high amount of extracellular laccase in a benchtop bioreactor. The enzyme can be used to assay TAC of blood plasma without the interference encountered with the hydrogen peroxide and metmyoglobin mediated assay method. Significance and Impact of the Study: Laccase production by P. ostreatus obtained in this study was the highest among all reported laccase producing white‐rot fungi. Moreover, an accurate laccase‐based assay method was developed for detection of TAC in human plasma.     

Biofilm formation by bacterial associations under various salinities and copper ion stress

Abstract

The study addresses the effect of abiotic (medium salinity and copper ions) and biotic (interactions between populations) factors on the formation of structured communities by binary associations consisting of halotolerant bacteria (Alcaligenes sp. 1‐1 or Acinetobacter sp. 1‐19) and a wild-type B. subtilis 2335 strain or a transgenic strain. The results showed that 250 mg l^?1 of copper ions inhibit formation of biofilms by monocultures of the tested strains. Binary associations of the strains were more resistant to high concentrations (250 mg l^?1) of copper ions. At the lowest NaCl concentration (0.05% and 2.5%) and in the presence of copper ions, bacilli seemed to help halotolerant bacteria survive. Under increased salinity and in the presence of copper ions, structured communities developed due to halotolerant bacteria. Coexistence under stressful conditions was beneficial for the both groups of bacteria.     

The effect of different kinds of electrolyte and non‐electrolyte solutions on the survival rate and morphology of zebrafish Danio rerio embryos

The effect of electrolyte and non‐electrolyte solutions on the survival and on the morphology of zebrafish Danio rerio embryos was investigated. Embryos in different ontogenetic stages were incubated in electrolyte (NaCl, KCl, MgCl₂ and CaCl₂) and non‐electrolyte solutions [sucrose and polyvinylalcohol (PVA)] of different concentrations for 5 – 15 min. The embryos were hatched to the long‐pec stage and the effective concentrations which caused a 50% decrease in embryo development (EC₅₀) were determined. The morphometric changes, which were caused by the test solutions, were measured. Ion channel blockers were used to see if active ion transport played a role for embryo survival. Finally, dechorionated embryos were exposed to the test solutions to get indications about the importance of chorion and perivitelline space. For 12 hours post fertilization (hpf) embryos and a 15 min exposure period, EC₅₀ was highest for MgCl₂ (1·60 mol l^?1), followed by sucrose (0·73 mol l^?1), NaCl (0·49 mol l^?1), KCl (0·44 mol l^?1), CaCl₂ (0·43 mol l^?1) and PVA [0·0005 mol l^?1 (2·2%)]. EC₅₀ were lower for early embryonic stages than for advanced stages for all solutions with exception of MgCl₂ and sucrose. At the EC₅₀, MgCl₂ and CaCl₂ solutions did not induce morphometric changes. NaCl and sucrose solutions induced reversible morphometric changes, which were compensated within 10 min. Only the EC₅₀ of KCl and PVA solutions induced permanent morphometric changes, which could not be compensated. Incubation of embryos in electrolyte and non‐electrolyte solutions together with ouabain (blocker of Na⁺– K⁺ ATPase), HgCl₃ (dose‐dependent inhibition of aquaporine channels), verapamil (inhibition of calcium and magnesium uptake) and amiloride (inhibition of sodium uptake) significantly decreased the per cent of embryos developing to the long‐pec stage in comparison to the same solutions without blockers. Ouabain and HgCl₃ also induced morphometric changes. For dechorionated embryos the survival rates in water and in the different test solutions were similar to untreated embryos.     

Use chemometric techniques in the optimization of a specific bioassay for betalactams in milk

Aims: A microbiological bioassay using Geoacillus stearothermophilus was optimized to detect betalactams at concentrations near to the Maximum Residue Limits (MRLs), with low cross‐specificity for tetracycline. Methods and Results: A factorial design (3 × 4) was used to evaluate the effects of concentration of spores (2·0 × 10⁶, 4·0 × 10⁶ and 8·0 × 10⁶ spores ml^?1) and incubation time (3·0, 3·5, 4·0 and 4·5 h) on the response of the bioassay. Then, desirability function to raise the detection capabilities (CC_β) of tetracyclines and increase sensitivity to betalactams was implemented. Significant effects of Log[S] and incubation time [It] on the CC_β of betalactams and tetracyclines were observed. Finally, high value of global desirability (D = 0·853), adequate betalactams CC_β (3·8 μg l^?1 of penicillin ‘G’, 27 μg l^?1 of oxacillin, 8·1 μg l^?1 of ampicillin, 48 μg l^?1 of cloxacillin) and high tetracyclines CC_β (5260 μg l^?1 chlortetracycline, 1550 μg l^?1 of oxytetracycline, 1070 μg l^?1 of tetracycline) were calculated. Conclusions: The application of chemometric tools allows the optimization of a bioassay that detects betalactam residues in milk. The more robust conditions have been achieved in Log[S] = 6·30 and [It] = 4·20 h. Significance and Impact of the Study: The logistic regression model and the desirability function are adequate chemometric techniques to improve the properties of the methods, because it is possible to increase sensitivity and decrease cross‐specificity simultaneously.     

Increased synthesis of α‐tocopherol,paramylon and tyrosine by Euglena gracilis under conditions of high biomass production

Aims: To analyse the production of different metabolites by dark‐grown Euglena gracilis under conditions found to render high cell growth. Methods and Results: The combination of glutamate (5 g l^?1), malate (2 g l^?1) and ethanol (10 ml l^?1) (GM + EtOH); glutamate (7·15 g l^?1) and ethanol (10 ml l^?1); or malate (8·16 g l^?1), glucose (10·6 g l^?1) and NH₄Cl (1·8 g l^?1) as carbon and nitrogen sources, promoted an increase of 5·6, 3·7 and 2·6‐fold, respectively, in biomass concentration in comparison with glutamate and malate (GM). In turn, the production of α‐tocopherol after 120 h identified by LC‐MS was 3·7 ± 0·2, 2·4 ± 0·1 and 2 ± 0·1 mg [g dry weight (DW)]^?1, respectively, while in the control medium (GM) it was 0·72 ± 0·1 mg (g DW)^?1. For paramylon synthesis, the addition of EtOH or glucose induced a higher production. Amino acids were assayed by RP‐HPLC; Tyr a tocopherol precursor and Ala an amino acid with antioxidant activity were the amino acids synthesized at higher concentration. Conclusions: Dark‐grown E. gracilis Z is a suitable source for the generation of the biotechnologically relevant metabolites tyrosine, α‐tocopherol and paramylon. Significance and Impact of the Study: By combining different carbon and nitrogen sources and inducing a tolerable stress to the cell by adding ethanol, it was possible to increase the production of biomass, paramylon, α‐tocopherol and some amino acids. The concentrations of α‐tocopherol achieved in this study are higher than others reported previously for Euglena, plant and algal systems. This work helps to understand the effect of different carbon sources on the synthesis of bio‐molecules by E. gracilis and can be used as a basis for future works to improve the production of different metabolites of biotechnological importance by this organism.     

Knocking out of tailoring genes eryK and eryG in an industrial erythromycin-producing strain of Saccharopolyspora erythraea leading to overproduction of erythromycin B, C and D at different conversion ratios

Aims: To overproduce erythromycin C, B or D and evaluate the effect of disruption of tailoring genes eryK and eryG in an industrial erythromycin producer. Methods and Results: The tailoring genes eryG and eryK were inactivated individually or simultaneously by targeted gene disruption in an industrial strain Saccharopolyspora erythraea HL3168 E3, resulting in the overproduction of erythromycin C (2·48 g l^?1), B (1·70 g l^?1) or D (2·15 g l^?1) in the mutant strain QL‐G, QL‐K or QL‐KG, respectively. Analysis of the erythromycin congeners throughout the fermentation indicated that, at the end of fermentation, comparatively large amount of erythromycin D (0·67 g l^?1) was accumulated in QL‐G, whereas only small amount of erythromycin D (0·10 g l^?1) was produced in QL‐K. Conclusions: Inactivation of tailoring genes eryG and eryK in the high producer did not affect the biosynthesis of erythromycin. However, erythromycin D could be more efficiently methylated by EryG than be hydroxylated by EryK. Significance and Impact of the Study: Development of the mutant strains provides a method for the economical large‐scale production of potent lead compounds. The information about the accumulation and conversion of erythromycins in the industrial strains may contribute to further improving erythromycin production.     

Ventilatory and behavioural responses of the marbled sole Pseudopleuronectes yokohamae to progressive hypoxia

This study identified ventilatory and behavioural responses in the marbled sole Pseudopleuronectes yokohamae under experimentally induced progressive decreases in dissolved oxygen (DO) levels. Ventilation frequency showed an increase with decreasing DO levels from normoxia to 2·75 mg O₂ l^?1, followed by a decrease in ventilation frequency at decreased DO levels from 2·00 to 0·75 mg O₂ l^?1. At DO levels below 2·00 mg l^?1, behaviours at the bottom were suppressed, whereas avoidance behaviours increased. A decrease in avoidance behaviours was observed from 1·00 to 0·75 mg O₂ l^?1. Upside‐down reversal and incapacitation at DO levels of 1·00–0·75 mg O₂ l^?1 suggested that sublethal effects on P. yokohamae were induced. The responses observed before the sublethal DO level could be interpreted as an effort to maintain oxygen uptake, reduce routine activities and facilitate avoidance. The observed DO level thresholds that induce behavioural responses, in addition to sublethal effects, indicate hypoxia‐tolerance that is important for understanding the effects of hypoxia on coastal ecosystems.     

Isolation of a naturally-occurring nickel resistance plasmid from a rare hypersaline estuary (Laguna Madre,Texas, USA) for potential use as a bio-indicator of metal contamination

Metal-resistant bacteria were isolated from sediments of the Laguna Madre, a rare hypersaline estuary impacted by many anthropogenic compounds, including various metals and metalloids. Bacteria were initially isolated on nutrient agar supplemented with NaCl; random isolates (n = 100) were tested for metal resistance toward zinc, nickel, chromium, and cadmium using a pour plate disc assay. Metal-resistant cultures were assayed for plasmids that contained naturally-occurring heavy metal resistance genes. Putative metal-resistance plasmids were tested for metal-resistance efficacy by transforming a metal-sensitive strain of Escherichia coli. Polymerase Chain Reaction (PCR) primers were designed to detect cnrA, part of a nickel–cobalt resistance gene cluster, and restriction endonuclease digests were performed to detect restriction sites within the plasmid. Results showed that many bacterial isolates tested were resistant toward most of the metals used in this study. Among tested bacteria cultures, 34 were resistant to zinc, 64 were resistant to chromium, and 51 resistant to cadmium. Only 8 cultures were resistant to nickel; however, most bacteria were found to be resistant to more than one metal. Several plasmids were found from the bacteria isolates. One plasmid, designated pDZ5, was isolated from a bacterium identified as Bacillus pumilus by 16S rRNA sequencing. Plasmid pDZ5 conferred nickel resistance to the metal-sensitive E. coli strain and was found to contain cnrA as confirmed by PCR amplification. Plasmid pDZ5 was successfully cut with restriction enzymes for potential ligation with reporter genes. The presence, abundance and expression of pDZ5 may prove to be a useful bio-indicator of metal contamination, specifically nickel pollution, in the Laguna Madre due to the fewer number of bacteria that were nickel-resistant compared to other metals.     

Use of liposomes in transforming yeast cells

Summary Plasmid DNAs of YEp13 and pMA56 were encapsulated in liposomes. Cells ofSaccharomyces cerevisiae strain SHY3 were protoplasted. After fusing membranes of the liposomes and the protoplasts, transformation of the regenerated yeast cells with the plasmids has been confirmed.     

Isolation and Characterization of Acidithiobacillus ferrooxidans Strain D3-2 Active in Copper Bioleaching from a Copper Mine in Chile

Acidithiobacillus ferrooxidans strain D3-2, which has a high copper bioleaching activity, was isolated from a low-grade sulfide ore dump in Chile. The amounts of Cu²⁺ solubilized from 1% chalcopyrite (CuFeS₂) concentrate medium (pH 2.5) by A. ferrooxidans strains D3-2, D3-6, and ATCC 23270 and 33020 were 1360, 1080, 650, and 600 mg·l ^?1·30 d^?1. The iron oxidase activities of D3-2, D3-6, and ATCC 23270 were 11.7, 13.2, and 27.9 μl O₂ uptake·mg protein^?1·min^?1. In contrast, the sulfite oxidase activities of strains D3-2, D3-6, and ATCC 23270 were 5.8, 2.9, and 1.0 μl O₂ uptake·mg protein^?1·min^?1. Both of cell growth and Cu-bioleaching activity of strains D3-6 and ATCC 23270, but not, of D3-2, in the chalcopyrite concentrate medium were completely inhibited in the presence of 5 mM sodium bisulfite. The sulfite oxidase of strain D3-2 was much more resistant to sulfite ion than that of strain ATCC 23270. Since sulfite ion is a highly toxic intermediate produced during sulfur oxidation that strongly inhibits iron oxidase activity, these results confirm that strain D3-2, with a unique sulfite resistant-sulfite oxidase, was able to solubilize more copper from chalcopyrite than strain ATCC 23270, with a sulfite-sensitive sulfite oxidase.     

Modelling the growth and ethanol production of Brettanomyces bruxellensis at different glucose concentrations

Aim: To study the effect of glucose concentrations on the growth by Brettanomyces bruxellensis yeast strain in batch experiments and develop a mathematical model for kinetic behaviour analysis of yeast growing in batch culture. Methods and Results: A Matlab algorithm was developed for the estimation of model parameters. Glucose fermentation by B. bruxellensis was studied by varying its concentration (5, 9·3, 13·8, 16·5, 17·6 and 21·4%). The increase in substrate concentration up to a certain limit was accompanied by an increase in ethanol and biomass production; at a substrate concentration of 50–138 g l^?1, the ethanol and biomass production were 24, 59 and 6·3, 11·4 g l^?1, respectively. However, an increase in glucose concentration to 165 g l^?1 led to a drastic decrease in product formation and substrate utilization. Conclusions: The model successfully simulated the batch kinetic observed in all cases. The confidence intervals were also estimated at each phase at a 0·95 probability level in a t‐Student distribution for f degrees of freedom. The maximum ethanol and biomass yields were obtained with an initial glucose concentration of 138 g l^?1. Significance and Impact of the Study: These experiments illustrate the importance of using a mathematical model applied to kinetic behaviour on glucose concentration by B. bruxellensis.     

Interactions between Pseudomonas putida UW4 and Gigaspora rosea BEG9 and their consequences for the growth of cucumber under salt-stress conditions

Aims: After the determination of the toxic but nonlethal concentration of NaCl for cucumber, we examined the interaction between an ACC (1‐aminocyclopropane‐1‐carboxylate) deaminase producing bacterial strain and an arbuscular mycorrhizal fungus (AMF) and their effects on cucumber growth under salinity. Methods and Results: In the first experiment, cucumber seedlings were exposed to 0·1, 50, 100 or 200 mmol l^?1 NaCl, and plant biomass and leaf area were measured. While seeds exposed to 200 mmol l^?1 NaCl did not germinate, plant growth and leaf size were reduced by 50 or 100 mmol l^?1 salt. The latter salt cancentration caused plant death in 1 month. In the second experiment, seeds were inoculated with the ACC deaminase‐producing strain Pseudomonas putida UW4 (AcdS⁺), its mutant unable to produce the enzyme (AcdS^?), or the AMF Gigaspora rosea BEG9, individually or in combination and exposed to 75 mmol l^?1 salt. Plant morphometric and root architectural parameters, mycorrhizal and bacterial colonization and the influence of each micro‐organism on the photosynthetic efficiency were evaluated. The AcdS⁺ strain or the AMF, inoculated alone, increased plant growth, affected root architecture and improved photosynthetic activity. Mycorrhizal colonization was inhibited by each bacterial strain. Conclusions: Salinity negatively affects cucumber growth and health, but root colonization by ACC deaminase‐producing bacteria or arbuscular mycorrhizal fungi can improve plant tolerance to such stressful condition. Significance and Impact of the Study: Arbuscular mycorrhizal fungus and bacterial ACC deaminase may ameliorate plant growth under stressful conditions. It was previously shown that, under optimal growth conditions, Ps. putida UW4 AcdS⁺ increases root colonization by Gi. rosea resulting in synergistic effects on cucumber growth. These results suggest that while in optimal conditions ACC deaminase is mainly involved in the bacteria/fungus interactions, while under stressful conditions this enzyme plays a role in plant/bacterium interactions. This finding is relevant from an ecological and an applicative point of view.     

Biodegradation of nicotine by a newly isolated Agrobacterium sp. strain S33

Aims: To isolate and characterize bacteria capable of degrading nicotine from the rhizospheric soil of a tobacco plant and to use them to degrade the nicotine in tobacco solid waste. Methods and Results: A bacterium, strain S33, was newly isolated from the rhizospheric soil of a tobacco plant, and identified as Agrobacterium sp. based on morphology, physiological tests, Biolog MicroLog3 4·20 system and 16S rRNA gene sequence. Using nicotine as the sole source of carbon and nitrogen in the medium, it grew optimally with 1·0 g l^?1 of nicotine at 30°C and pH 7·0, and nicotine was completely degraded within 6 h. The resting cells prepared from the glucose‐ammonium medium or LB medium could not degrade nicotine within 10 h, while those prepared from the nicotine medium could completely degrade 3 g l^?1 of nicotine in 1·5 h at a maximal rate of 1·23 g nicotine h^?1 g^?1 dry cell. Using the medium containing nicotine, glucose and ammonium simultaneously to cultivate strain S33, the resting cells could degrade 98·87% of nicotine in tobacco solid waste with the concentration as 30 mg nicotine g^?1 dry weight tobacco solid waste within 7 h at a maximal rate of 0·46 g nicotine h^?1 g^?1 dry cell. Conclusions: This is the first report that Agrobacterium sp. has the ability to degrade nicotine. Agrobacterium sp. S33 could use nicotine as the sole source of carbon and nitrogen. The use of resting cells of the strain S33 prepared from the nicotine–glucose–ammonium medium was an effective method to degrade nicotine and detoxify tobacco solid waste. Significance and Impact of the Study: Nicotine in tobacco wastes is both toxic and harmful to human health and the environment. This study showed that Agrobacterium sp. S33 may be suitable for the disposal of tobacco wastes and reducing the nicotine content in tobacco leaves.     

Ethanol synthesis from glycerol by Escherichia coli redox mutants expressing adhE from Leuconostoc mesenteroides

Aims: Analysis of the physiology and metabolism of Escherichia coli arcA and creC mutants expressing a bifunctional alcohol‐acetaldehyde dehydrogenase from Leuconostoc mesenteroides growing on glycerol under oxygen‐restricted conditions. The effect of an ldhA mutation and different growth medium modifications was also assessed. Methods and Results: Expression of adhE in E. coli CT1061 [arcA creC(Con)] resulted in a 1·4‐fold enhancement in ethanol synthesis. Significant amounts of lactate were produced during micro‐oxic cultures and strain CT1061LE, in which fermentative lactate dehydrogenase was deleted, produced up to 6·5 ± 0·3 g l^?1 ethanol in 48 h. Escherichia coli CT1061LE derivatives resistant to >25 g l^?1 ethanol were obtained by metabolic evolution. Pyruvate and acetaldehyde addition significantly increased both biomass and ethanol concentrations, probably by overcoming acetyl‐coenzyme A (CoA) shortage. Yeast extract also promoted growth and ethanol synthesis, and this positive effect was mainly attributable to its vitamin content. Two‐stage bioreactor cultures were conducted in a minimal medium containing 100 μg l^?1 calcium d ‐pantothenate to evaluate oxic acetyl‐CoA synthesis followed by a switch into fermentative conditions. Ethanol reached 15·4 ± 0·9 g l^?1 with a volumetric productivity of 0·34 ± 0·02 g l^?1 h^?1. Conclusions: Escherichia coli responded to adhE over‐expression by funnelling carbon and reducing equivalents into a highly reduced metabolite, ethanol. Acetyl‐CoA played a key role in micro‐oxic ethanol synthesis and growth. Significance and Impact of the Study: Insight into the micro‐oxic metabolism of E. coli growing on glycerol is essential for the development of efficient industrial processes for reduced biochemicals production from this substrate, with special relevance to biofuels synthesis.     

The effect of hexose ratios on metabolite production in Saccharomyces cerevisiae strains obtained from the spontaneous fermentation of mezcal

Mezcal from Tamaulipas (México) is produced by spontaneous alcoholic fermentation using Agave spp. musts, which are rich in fructose. In this study eight Saccharomyces cerevisiae isolates obtained at the final stage of fermentation from a traditional mezcal winery were analysed in three semi-synthetic media. Medium M1 had a sugar content of 100 g l^?1 and a glucose/fructose (G/F) of 9:1. Medium M2 had a sugar content of 100 g l^?1 and a G/F of 1:9. Medium M3 had a sugar content of 200 g l^?1 and a G/F of 1:1. In the three types of media tested, the highest ethanol yield was obtained from the glucophilic strain LCBG-3Y5, while strain LCBG-3Y8 was highly resistant to ethanol and the most fructophilic of the mezcal strains. Strain LCBG-3Y5 produced more glycerol (4.4 g l^?1) and acetic acid (1 g l^?1) in M2 than in M1 (1.7 and 0.5 g l^?1, respectively), and the ethanol yields were higher for all strains in M1 except for LCBG-3Y5, -3Y8 and the Fermichamp strain. In medium M3, only the Fermichamp strain was able to fully consume the 100 g of fructose l^?1 but left a residual 32 g of glucose l^?1. Regarding the hexose transporters, a high number of amino acid polymorphisms were found in the Hxt1p sequences. Strain LCBG-3Y8 exhibited eight unique amino acid changes, followed by the Fermichamp strain with three changes. In Hxt3p, we observed nine amino acid polymorphisms unique for the Fermichamp strain and five unique changes for the mezcal strains.