Showing papers on "Growth medium published in 2009"

The class IId bacteriocin thuricin-17 increases plant growth.

Abstract: The mechanisms by which many plant growth promoting rhizobacteria (PGPR) affect plants are unknown We recently isolated a rhizosphere bacterium (Bacillus thuringiensis NEB17), that promotes soybean growth and screened the liquid growth medium in which it grew for plant growth stimulating materials We have also shown that it produces a bacteriocin (named by us as thuricin-17 and a member of the recently described class IId bacteriocins) Here we show that application of this bacteriocin to leaves (spray) or roots (drench) directly stimulates the growth of both a C3 dicot (soybean) and a C4 monocot (corn) This growth stimulation is similar in nature to that previously seen when plants are treated with Nod factors Strain NEB17 contains three copies of the gene for thuricin 17 that code for identical amino acid sequences These two lines of evidence suggest that the dual functions of these proteins may have constrained their evolution This is the first report of direct plant growth enhancement by a bacteriocin

Effect of culture medium and cryoprotectants on the growth and survival of probiotic lactobacilli during freeze drying.

Abstract: Aims: To investigate the effects of the medium and cryoprotective agents used on the growth and survival of Lactobacillus plantarum and Lactobacillus rhamnosus GG during freeze drying. Methods and Results: A complex medium was developed consisting primarily of glucose, yeast extract and vegetable-derived peptone. Trehalose, sucrose and sorbitol were examined for their ability to protect the cells during freeze drying. Using standardized amount of cells and the optimized freeze drying media, the effect of the growth medium on cell survival during freeze drying was investigated. The results showed that glucose and yeast extract were the most important growth factors, while sucrose offered better protection than trehalose and sorbitol during freeze drying. When the cells were grown under carbon limiting conditions, their survival during freeze drying was significantly decreased. Conclusions: A clear relationship was observed between cell growth and the ability of the cells to survive during the freeze drying process. Significance and Impact of the Study: The survival of probiotic strains during freeze drying was shown to be dependent on the cryoprotectant used and the growth medium.

Catalase Overexpression Reduces Lactic Acid-Induced Oxidative Stress in Saccharomyces cerevisiae

Abstract: Industrial production of lactic acid with the current pyruvate decarboxylase-negative Saccharomyces cerevisiae strains requires aeration to allow for respiratory generation of ATP to facilitate growth and, even under nongrowing conditions, cellular maintenance. In the current study, we observed an inhibition of aerobic growth in the presence of lactic acid. Unexpectedly, the cyb2{Delta} reference strain, used to avoid aerobic consumption of lactic acid, had a specific growth rate of 0.25 h–1 in anaerobic batch cultures containing lactic acid but only 0.16 h–1 in identical aerobic cultures. Measurements of aerobic cultures of S. cerevisiae showed that the addition of lactic acid to the growth medium resulted in elevated levels of reactive oxygen species (ROS). To reduce the accumulation of lactic acid-induced ROS, cytosolic catalase (CTT1) was overexpressed by replacing the native promoter with the strong constitutive TPI1 promoter. Increased activity of catalase was confirmed and later correlated with decreased levels of ROS and increased specific growth rates in the presence of high lactic acid concentrations. The increased fitness of this genetically modified strain demonstrates the successful attenuation of additional stress that is derived from aerobic metabolism and may provide the basis for enhanced (micro)aerobic production of organic acids in S. cerevisiae.

Isolation of phosphate-solubilizing fungi from phosphate mines and their effect on wheat seedling growth.

Abstract: Three phosphate-solubilizing fungi, identified as Penicillium expansum, Mucor ramosissimus, and Candida krissii, were isolated from phosphate mines (Hubei, People’s Republic of China) and characterized. All the isolates demonstrated diverse levels of phosphate-solubilizing capability in National Botanical Research Institute’s phosphate growth medium containing rock phosphate as sole phosphate source. Acidification of culture medium seemed to be the main mechanism for rock phosphate solubilization. Indeed, citric acid, oxalic acid, and gluconic acid were shown to be present in the culture medium inoculated with these isolates. Moreover, the isolates produced acid and alkaline phosphatases in culture medium, which may also be helpful for RP solubilization. A strong negative correlation between content of soluble phosphorus and pH (r = – 0.89; p < 0.01) in culture medium was observed in this study. All the isolates promoted growth, soil available phosphorus, phosphorus, and nitrogen uptake of wheat seedling in field soil containing rock phosphate under pot culture conditions, thus demonstrating the capability of these isolates to convert insoluble form of phosphorus into plant available form from rock phosphate, and therefore hold great potential for development as biofertilizers to enhance soil fertility and promote plant growth.

Sphagnan – a pectin‐like polymer isolated from Sphagnum moss can inhibit the growth of some typical food spoilage and food poisoning bacteria by lowering the pH

Abstract: Aims: Investigate if the antibacterial effect of sphagnan, a pectin-like carbohydrate polymer extracted from Sphagnum moss, can be accounted for by its ability to lower the pH. Methods and Results: Antibacterial activity of sphagnan was assessed and compared to that of three other acids. Sphagnan in its acid form was able to inhibit growth of various food poisoning and spoilage bacteria on low-buffering solid growth medium, whereas sphagnan in its sodium form at neutral pH had no antibacterial activity. At similar acidic pH, sphagnan had comparable antibacterial activity to that of hydrochloric acid and a control rhamnogalacturonan pectin in its acid form. Conclusions: Sphagnan in its acid form is a weak macromolecular acid that can inhibit bacterial growth by lowering the pH of environments with a low buffering capacity. Significance and Impact of the Study: It has previously been suggested that sphagnan is an antimicrobial polysaccharide in the leaves of Sphagnum moss with a broad range of potential practical applications. Our results now show that sphagnan in its acid form can indeed inhibit bacterial growth, but only of acid-sensitive species. These findings represent increased knowledge towards our understanding on how sphagnan or Sphagnum moss might be used in practical applications.

The Escherichia coli CsrB and CsrC small RNAs are strongly induced during growth in nutrient-poor medium.

Abstract: The carbon storage regulatory (Csr) system is a complex network controlling various phenotypes in many eubacteria. So far, the external conditions by which the system is regulated are poorly understood. Here we show that the expression of the two noncoding small RNAs CsrB and CsrC in Escherichia coli is strongly increased in cultures grown in minimal medium. Addition of tryptone, casamino acids or a mixture of amino acids to a culture grown in minimal medium led to a rapid reduction in the levels of CsrB. Based on this we propose that the expression of the Csr sRNAs is controlled by the amino acid availability in the growth medium.

Analysis of the Pseudomonas putida CA-3 proteome during growth on styrene under nitrogen-limiting and non-limiting conditions.

Abstract: Pseudomonas putida CA-3 is a styrene-degrading bacterium capable of accumulating medium-chain-length polyhydroxyalkanoate (mclPHA) when exposed to limiting concentrations of a nitrogen source in the growth medium. Using shotgun proteomics we analysed global proteome expression in P. putida CA-3 supplied with styrene as the sole carbon and energy source under N-limiting (condition permissive for mclPHA synthesis) and non-limiting (condition non-permissive for mclPHA accumulation) growth conditions in order to provide insight into the molecular response of P. putida CA-3 to limitation of nitrogen when grown on styrene. A total of 1761 proteins were identified with high confidence and the detected proteins could be assigned to functional groups including styrene degradation, energy, nucleotide metabolism, protein synthesis, transport, stress response and motility. Proteins involved in the upper and lower styrene degradation pathway were expressed throughout the 48 h growth period under both nitrogen limitation and excess. Proteins involved in polyhydroxyalkanoate (PHA) biosynthesis, nitrogen assimilation and amino acid transport, and outer membrane proteins were upregulated under nitrogen limitation. PHA accumulation and biosynthesis were only expressed under nitrogen limitation. Nitrogen assimilation proteins were detected on average at twofold higher amounts under nitrogen limitation. Expression of the branched-chain amino acid ABC transporter was up to 16-fold higher under nitrogen-limiting conditions. Branched chain amino acid uptake by nitrogen-limited cultures was also higher than that by non-limited cultures. Outer membrane lipoproteins were expressed at twofold higher levels under nitrogen limitation. This was confirmed by Western blotting (immunochemical detection) of cells grown under nitrogen limitation. Our study provides the first global description of protein expression changes during growth of any organism on styrene and accumulating mclPHA (nitrogen-limited growth).

Abortiporus biennis tolerance to insoluble metal oxides: oxalate secretion, oxalate oxidase activity, and mycelial morphology.

Abstract: The ability of Abortiporus biennis to tolerate and solubilize toxic metal oxides (Cu2O, Al2O3, ZnO, CuFe2O4Zn, CdO, and MnO2) incorporated into agar media was investigated and the growth rate, oxalic acid secretion, and mycelial morphology were monitored. Among the tested metal oxides, formation of clear zones underneath the mycelium growing on Cu2O- and ZnO-amended plates was observed. ZnO, CdO and Cu2O caused the highest rate of fungal growth inhibition. An increased level of oxalic acid concentration was detected as a response of A. biennis to the presence of Cu2O, MnO2, ZnO and CuFe2O4Zn in growth medium. The oxalate oxidase (OXO) was found to be responsible for oxalic acid degradation in A. biennis cultivated in metal-amended media. An increased level of OXO was observed in media amended with Cu2O, ZnO and MnO2. Confocal microscopy used in this study revealed changes in mycelial morphology which appeared as increased hyphal branching, increased septation and increased spore number.

Culture nutrition and physiology impact the inhibitor tolerance of the yeast Pichia stipitis NRRL Y-7124.

Abstract: Pichia stipitis NRRL Y-7124 is one of the natural yeasts best able to utilize biomass because it is able to ferment hexoses and the pentose, xylose, to economically recoverable concentrations of ethanol. To test the impact of culture conditions on inhibitor tolerance, inhibitors were spiked to growing or stationary-phase P. stipitis supplied either glucose or xylose and varying nitrogen and mineral compositions; then the ensuing specific death rate response was measured. Resistance of glucose- or xylose-grown cells to inhibitors was generally greater in stationary-phase cells than log-phase cells, despite a greater exposure of stationary cells to ethanol. Consistent with this, the specific productivity of detoxification products, furan methanol or furan-2,5-dimethanol, from respective spikes of furfural or HMF increased as cultures progressed into stationary phase. However, when xylose was the substrate, ethanol resistance behaved uniquely and was greater for log- than stationary-phase cells. Amino acid enrichment of the growth medium significantly enhanced ethanol tolerance if xylose was the carbon source, but had no impact if glucose supplied carbon. Regardless of the carbon source, amino acid enrichment of the culture medium enhanced the ability of cells to resist furfural and HMF exposure. Mineral compositions tested had little impact on inhibitor resistance except stationary-phase xylose-grown cells were more susceptible to inhibitor exposure when magnesium sulfate was excessive. Observed tolerance optimization based on specific death rate as a function of culture physiological state, carbon source, nitrogen source and mineral composition provides new knowledge supporting process designs to convert biomass to ethanol using P. stipitis. Biotechnol. Bioeng. 2009; 102: 778–790. © 2008 Wiley Periodicals, Inc.

Yeast Strain for Production of Four Carbon Alcohols

Abstract: Yeast cells with a reduced general control response to amino acid starvation were found to have increased tolerance to butanol in the growth medium. The reduced response was engineered by genetic modification of a gene involved in the response, a GCN gene, to eliminate activity of the encoded protein. Yeast strains with an engineered butanol biosynthetic pathway and a genetic modification in a gene involved in the general control response to amino acid starvation, which have increased butanol tolerance, are useful for production of butanol.

Influence of growth medium on diagnostic characters of aspergillus and penicillium species

Abstract: Three fungal strains; namely, Aspergillus terreus, Penicillium janthinellum and Penicillium duclauxii were cultured on different growth media including yeast extract, malt extract, yeast-malt extract, Czapek's Dox, Sabourod's, Harrlod's, and potato dextrose. The growth and secondary metabolites of the three fungal strains were greatly affected by the growth medium. The colour of the culture and secondary metabolites were noticeably altered and changed according to the growth medium used.

Growth media and saprophytic use for pichia anomala

Abstract: A biologically pure culture of a yeast of the species Pichia anomala (WRL-076). The yeast is identified as NRRL Y-30842 and is applied to a site containing a deleterious microorganism. Further disclosed is a growth medium for increasing the viablility of yeast organisms.

Characterization of tricalcium phosphate solubilization by Stenotrophomonas maltophilia YC isolated from phosphate mines

Abstract: The phosphate solubilizing characteristics of a strain YC, which was isolated from phosphate mines (Hubei, China), were studied in National Botanical Research Institute’s phosphate (NBRIP) growth medium containing tricalcium phosphate (TCP) as sole phosphorus (P) source. The strain YC is identified as Stenotrophomonas maltophilia (S. maltophilia) based upon the results of morphologic, physiological and biochemical characteristics and 16S rRNA sequences analysis. The results show that the strain S. maltophilia YC can solubilize TCP and release soluble P in NBRIP growth medium. A positive correlation between concentration of soluble P and population of the isolate and a negative correlation between concentration of soluble P and pH in the culture medium are observed from statistical analysis results. Moreover, gluconic acid is detected in the culture medium by HPLC analysis. It indicates that the isolate can release gluconic acid during the solubilizing experiment, which causes acidification of the culture medium and then TCP solubilization. S. maltophilia YC has a maximal TCP solubilizing capability when using maltose as carbon source and ammonium nitrate as nitrogen source, respectively, in NBRIP growth medium.

Production of Alkaline Xylanase by an Alkalo-thermophilic Bacteria, Bacillus halodurans, MTCC 9512 isolated from Dung

Abstract: An alkalo-thermophilic bacteria from dung has been isolated using Emerson medium in the agar plates. The bacteria has growth at the pH 10 and temperature 55°C. The bacteria was screened for the xylanase activity using Congo red dye followed by wash out by 1 mM sodium chloride. A clear zone around the colony in the replica plate was considered to have xylanase activity. The suspected colony in another replica plate was grown in Emerson broth and extracellular xylanase enzyme activity was analyzed by the colorimetric method using dinitro salicylic acid for estimation of reducing power. The morphological study of the bacteria was done after Gram stain and using 40x amplification in the phase contrast microscope. The isolated bacteria retained violet color after washing with acetone. Therefore, it is gram positive. Further characterization using various morphological, physiological and biochemical tests confirmed the bacteria as Bacillus halodurans and was given Accession number MTCC 9512 by IMTECH, Chandigarh. Growth conditions for the bacteria were optimized for maximum production of xylanase. The maximum amount of xylanase activity was found at the pH 9.5 and temperature 55°C. The growth of the bacteria and enzyme production were monitored up to 52 hours and it was found that the bacteria grew logarithmically up to 30 hours. Different carbon sources viz. xylan, sucrose, glucose, starch individually at 0.5% concentration were used in the Emerson growth medium. Maximum biomass growth was found with xylan whereas xylanase was maximally produced with glucose as carbon source. Therefore, glucose was considered to be the best inducer followed by xylan among the various carbon sources used. The enzyme was enriched by using 0–80% ammonium sulfate precipitation followed by desalting through Sephadex-G-25 gel filtration. The results indicated inhibitory nature of ammonium sulfate.

Impact of stress conditions on the growth of Lactobacillus acidophilus IBB 801 and production of acidophilin 801.

Abstract: The influence of different stress conditions, such as low and high temperatures, the presence of salt or ethanol in the medium, on the growth of Lactobacillus acidophilus IBB801 and the production of acidophilin 801 was investigated in this study. The strain was able to grow at up at to 47°C, while higher temperatures were lethal. A slow growth was detected at 24°C, starting after 24 h of incubation, and the strain was able to survive at 10°C for more than 48 h of incubation. The protein profiles revealed by one dimensional SDS-PAGE showed at least four overexpressed and two repressed bands at low temperatures (10°C and 24°C) compared with the profiles at 37°C and 42°C. The bacteriocin activity was the highest when the producing strain was grown at optimum temperature. However, at 10°C, the inhibitory activity showed a slight increase compared with the one reached at 24°C and 47°C. The same increase in activity was observed in the presence of low amounts of salt (5 to 10 g/L). Higher concentrations of salt or ethanol addition to the growth medium had a negative effect on acidophilin biosynthesis.

Production of elastase by pathogenic and non-pathogenic fungi.

Abstract: Many yeasts, dermatophytes as well as moulds liberate hydrolytic enzymes into the growth medium. The enzymes may serve the fungus by different ways, e. g., they cause chemical and physical changes in the immediate surrounding of the fungus thus enhancing the living possibilities. The production of enzymes capable of digesting mammalian proteins would aid pathogenic fungi to penetrate the human epidermis and to spread in the dermis. Many of the dermatophytes are, in fact, capable of digesting keratin by the aid of keratinase, collagen by the aid of collagenase (RIPPON and LORINCZ, 1964) and even elastin by the aid of elastase (RIPPON and VARADI, 1968). Elastase and collagenase could serve the organism also by breaking the intercellular connection of the epidermal cells or by dissolving the basal membrane (KOTRAJARAS, 1965). The production of elastase, one of the most specific proteases by fungi has been the subject of only one report in which relatively few species were tested (RIPPON and VARADI, 1968). Further studies on the elastase producing ability of the pathogenic as well as non-pathogenic fungi might clarify the role of this enzyme in the pathogenicity of the fungi. They could also disclose whether tests on the elastase production could be of value in the routinly identification of fungi. Material and methods The production of elastase by growing colonies of yeasts, dermatophytes and moulds was tested by using two kinds of test agar plates. The basic growth medium was the same in both types of plates containing glucose 10.0 g, pepton aus casein (Merdc) 5.0 g, glycerol 5.0 g, sodium chloride 5.0 g, Standard I1 Nahrbouillon Merdc 15.0 g, agar 30.0 g, yeast extract 3.0 g and aqua destillata 1000 ml. One gram of elasdn powder (Sigma Chem. Comp.) was added to 100 ml sterilized agar which was poured on test plates thus following essentially the method of R~PPON and VARADI (1968). In these plates the grains of elastin appear gray against the yellowish badrground. Elastase activity was manifested as dissolution of the grains and thus as the appearance of a clear ring around the colony. In the second type of growth plates the elastin was replaced by elastin which was coupled with Remazolbrilliant Blue B according to RINDERKNECHT (1970). In these plates (incubated at 23' C) the grains are dark blue and after dissolution of the grains a clear ring is seen. The zones of cleraing around the fungi were recorded after 4, 7, and 14 days respectively. The species of fungi tested were from the collection of our laboratory (most of them are originally from Centraalbureau voor Schimmelcultures, Holland).

Degradation of 3-chloropropionic acid by escherichia coli JM109 expressing dehalogenase (deh) gene used as selection marker

Abstract: 3-Chloropropionic acid (3CP) in its carboxylate ionic form is a synthetic compound found in herbicide. The biodegradability of 3CP is not well documented but a microbe that has the ability to utilise 3CP as sole carbon and energy source has been isolated. The dehalogenase gene (deh) cloned from Rhodococcus sp. HJ1 could be used as a selection marker gene for vector in E. coli. Halogenated compound, especially 3CP inhibit the growth of some microorganisms. In current investigation, a 4 kb EcoR1 fragment of genomic DNA from Rhodococcus sp. HJ1 was cloned into pUC18 plasmid and transformed into an E. coli JM109 conferred 3CP resistance on them. Therefore, E. coli transformed with vector marked with deh could be easily selected on plates containing 3CP. The E. coli JM109 transformed with pTY096 (deh+) weakly expressed the deh gene as shown from its slow growth with cells doubling time of 22 h with minimal amount of chloride ion released in the growth medium

Improved yeast strain for production of four carbon alcohols

Abstract: Yeast cells with a reduced general control response to amino acid starvation were found to have increased tolerance to butanol in the growth medium. The reduced response was engineered by genetic modification of a gene involved in the response, a GCN gene, to eliminate activity of the encoded protein. Yeast strains with an engineered butanol biosynthetic pathway and a genetic modification in a gene involved in the general control response to amino acid starvation, which have increased butanol tolerance, are useful for production of butanol.

Secondary cell wall formation in Cryptococcus neoformans as a rescue mechanism against acid-induced autolysis.

Abstract: Growth of the opportunistic yeast pathogen Cryptococcus neoformans in a synthetic medium containing yeast nitrogen base and 1.0–3.0% glucose is accompanied by spontaneous acidification of the medium, with its pH decreasing from the initial 5.5 to around 2.5 in the stationary phase. During the transition from the late exponential to the stationary phase of growth, many cells died as a consequence of autolytic erosion of their cell walls. Simultaneously, there was an increase in an ecto-glucanase active towards β-1,3-glucan and having a pH optimum between pH 3.0 and 3.5. As a response to cell wall degradation, some cells developed an unusual survival strategy by forming ‘secondary’ cell walls underneath the original ones. Electron microscopy revealed that the secondary cell walls were thicker than the primary ones, exposing bundles of polysaccharide microfibrils only partially masked by an amorphous cell wall matrix on their surfaces. The cells bearing secondary cell walls had a three to five times higher content of the alkali-insoluble cell wall polysaccharides glucan and chitin, and their chitin/glucan ratio was about twofold higher than in cells from the logarithmic phase of growth. The cell lysis and the formation of the secondary cell walls could be suppressed by buffering the growth medium between pH 4.5 and 6.5.

Laminin-511 and fibronectin degradation with Candida yeast.

Abstract: Background: The invasion mechanism of Candida yeast is still partly unknown. In this study, we tested the ability of different commensal Candida yeast to degrade two basement membrane and extracellular matrix proteins: laminin-511 (Lm-511) and plasma fibronectin. Methods: Human Lm-511 was produced by an immortal keratinocyte cell line, labelled with 35S-methionine and immunoprecipitated from the growth medium with monoclonal antibodies. Human plasma fibronectin was purified from plasma samples of blood donors. Sonicated yeast cells and concentrated yeast cell growth media were incubated with Lm-511 in different pH values and the degradation was detected by fluorography. Fibronectin degradation by yeast was visualized by sodium dodecyl-sulphate polyacrylamide gel electrophoresis. Results: The reduced 220 kDa fibronectin monomers were found to be degraded at pH 7.8 by 10× concentrated growth media of most strains tested and at pH 3.0 the degradation was more pronounced. Sonicated cell fractions of C. tropicalis and C. parapsilosis caused degradation of plasma fibronectin at pH 7.8. Instead, none of the tested Candida cell fractions degraded Lm-511 under these conditions. Conclusions: It seems that cleavage of different laminin isoforms by Candida yeast is a laminin-specific process. The ability to cleave human plasma fibronectin is species- and pH-dependent but not hyphal-dependent and also this degradation may affect epithelial integrity.

Inducible expression of human angiostatin by AOXI promoter in P. pastoris using high-density cell culture.

Abstract: A high-density cell culture method was successfully established in P. pastoris with the alcohol oxidase I (AOXI) promoter in order to produce large quantities of recombinant human angiostatin (AS) which has been reported to have antiangiogenic activity. A preliminary study on fermentation conditions in shaking flasks indicated that adequacy of biomass is beneficial to obtain more products. The fermentation was carried out in a 10 l bioreactor with 5 l modified growth medium recommended by Invitrogen at 30°C. The cells were first grown in glycerol-PTM4 trace salts for 24 h. When the cell density reached A600 = 125, methanol-PTM4 trace salts was added to induce the expression of AS. During the fermentation, dissolved oxygen level was maintained at 20–30%, pH was controlled at 5 by the addition of 7 M NH4OH and the biomass was maintained at about A600 = 200. After 60 h of induction, the secreted AS was 153 mg/l. The recombinant AS inhibited the angiogenesis on CAM and suppressed the growth of B16 melanoma in C57BL/6J mice (P < 0.01).

The herbicide 2,4-dichlorophenoxyacetic acid. II. Triglyceride accumulation in L cells.

Abstract: L 929 cells in monolayer cultures exposed to 500 μg/ml of 2,4-dichlorophenoxyacetic acid have been found to accumulate triglycerides. Labelling experiments were performed with 3H-palmitate and 14C-acetate. The incorporation of 3H and 14C into total cell lipids of treated cells was about 3 times that in the control cells. The increase of 3H was mainly recovered in the triglyceride fractions both from whole cells and in isolated cytoplasmic particles. The specific activity of the triglycerides with regard to tritium remained similar, whereas the specific activity with regard to 14C was only one-fourth in the treated cells as compared to the control cells. This suggested that the fatty acids of the accumulated triglycerides were largely derived from the growth medium.

Green algae selection useful for phycoremediation of olive-mill wastewaters and increase of their lipid content by genetic engineering

Abstract: The main aim of this work was the selection and the characterization of algal strains useful for phycoremediation of olive-mill wastewaters (OMW). 100 algal strains from the ACUF collection of the Department of Biology of the University of Naples were screened for their capability to release enzymes with phenol-oxidase activity in the culture broth. The presence of enzymes with phenol-oxidase activity was revealed by ABTS enzymatic assay in 16 of the 100 analysed culture broths. Further enzymatic assays were carried out in order to: 1) evaluate the enzymatic activity of the culture broths in presence of ABTS and different phenolic compounds, as 2,6-DMP and syringaldazine; 2) evaluate the capability of the culture broths to decolourizate the azo dye RBBR. On the base of the results achieved with the enzymatic assays, two algal strains were selected in order to evaluate their capability to grow in, and to degrade the phenolic component of, OMW: Chlamydomonas pitschmannii and Scenedesmus vacuolatus. The two algae were grown for 21 days in OMW diluted with growth medium and the cell concentration and the total phenols amount were measured weakly. S. vacuolatus showed a capability to grow in presence of OMW, and to reduce the phenolic content, higher than C. pitschmannii. The second part of the PhD work was carried out at the Algae Biotechnology & Bioenergy research group of the University of Bielefeld; during this period the research was focused on the characterization of genes encoding for putative enzymes involved into the lipid biosynthetic pathway in the green alga Chlamydomonas reinhardtii. 5 genes encoding for putative diacylglycerol acyltransferases (CrDGAT1-5) were cloned into expression vector pGenD and used for the genetic manipulation of Chlamydomonas in order to obtain mutants able to overexpress these genes. For each gene, 1 to 3 mutants were able to over-express the new CrDGAT construct. The mutant with the highest expression level was selected by quantitative real time PCR analyses for phenotype characterization during the standard growth condition and during nitrogen and sulphur depletion. Number of cells, optical density and total amount of chlorophyll were evaluated in order to verify if the over-expression of these genes could affect the cell phenotype. The total lipid amount was measured by Nile Red fluorescence assay and compared with the wild type. During the normal growth condition no differences were noticed between mutants and wild type strain, demonstrating that the over-expression of these genes doesn’t affect the cell phenotype and suggesting the presence of a second bottleneck in the lipid biosynthetic pathway. When cultivated in nitrogen depletion, all cultures showed the highest lipid accumulation, however without differences between mutants and wild type strains. During the sulphur starvation, mutants D3-18 and D4-66 showed an increase in the lipid accumulation in average 2.0 and 1.8 times higher than the wild type.

Peptides containing 2-aminopimelic acid. Synthesis and study of in vitro effects on bacterial cells.

Abstract: Taking advantage of the peptide transport strategy, we have designed and synthesized several new peptides containing 2-aminopimelic acid (Apm), an inhibitor of the diaminopimelate pathway in bacteria: L-Lys-ambo-Apm, ambo-Apm-L-Lys, L-Lys-L-Ala-ambo-Apm, ambo-Apm-L-Ala-L-Lys, L-Ala(Cl)-ambo-Apm and ambo-Apm-L-Ala(Cl). In the two latter cases, Apm was associated with antibacterial amino acid beta-chloro-L-alanine [L-Ala(Cl)], an inhibitor of alanine racemase and transaminase B. The peptides displayed weak or no antibacterial activities; nevertheless, those containing L-Ala(Cl) had low MIC values in the presence of amino acids restoring protein synthesis. When tested on exponential phase Escherichia coli cells grown in minimal medium, the peptides were without effect or bacteriostatic, but important bacteriolytic effects could be observed, especially for the L-Ala(Cl)-containing peptides, when the growth medium was supplemented with specific amino acids. It was demonstrated that the weak or nil effect of the L-lysine-containing peptides was due to a poor uptake.

Effect of Fluoride on the Activity of Ornithine Decarboxylase in Normal and Fluoride Resistant LS Cells

Abstract: Renewal of growth medium caused an induction of ornithine decarboxylase (ODC) activity in LS cells grown in suspension culture. Addition of low concentrations of fluoride ions to the growth medium (up to 1.3 mM) resulted in a further increase in this induction of ODC-activity, whereas addition of 6 mM fluoride caused an inhibition of the induction and resulted in reduced ODC-activity as compared to controls. Since sodium fluoride had no stimulatory or inhibitory effect on the ODC-activity assay, it is likely that the effect is exerted on the regulation of ODC-activity in the cells. The effect of fluoride ions on the induction of ODC-activity upon renewal of the growth medium was markedly less pronounced in fluoride resistant LS cells.