Lipid formation and γ-linolenic acid production by Mucorales fungi grown on sunflower oil

Lipid formation and γ-linolenic acid (GLA) production by 48 species of Mucorales fungi grown on sunflower oil (which consists of 70% linoleic acid ; LA) were studied. The strains accumulated 42·7–65·8% lipid in biomass (7·66–13·39 g l⁻¹). Eight cultures produced more than 200 mg GLA l⁻¹. Highest GLA yields exhibited Mucor mucedo CCF-1384 and Cunninghamella echinulata CCF-103 (379 and 373 mg l⁻¹, respectively). Mortierella alpina CCF-185 synthesized 465 mg l⁻¹ arachidonic acid. While the decrease of LA utilization index (ratio of LA content of cell lipid/LA content of oil source) was accompanied with growth of delipidized biomass and with reduction of lipid accumulation within the cells, high lipid yield was as a consequence of the direct oil source incorporation into intracellular lipid.     

Acetoin production in Saccharomyces cerevisiae wine yeasts

Abstract One hundred strains of Saccharomyces cerevisiae were examined for the capacity to produce acetoin in synthetic medium and in grape must. The low production of acetoin was found to be the more common pattern in this species. Most strains exhibited a similar distribution in both media, production ranging from non-detectable amounts to 12 mg 1⁻¹. Only four strains produced high quantities of acetoin, up to 29.5 mg l⁻¹ in synthetic medium and up to 194.6 mg l⁻¹ in grape must. This biometric study showed the existence of two phenotypes, "low and high acetoin production", that could be selected for conferring a desirable flavour of the final product.     

Response surface methodology to optimize the nutritional parameters for enhanced production of jasmonic acid by Lasiodiplodia theobromae

Aims:  To find out the cumulative effect of the nutritional parameters and to enhance the production of jasmonic acid (JA) in static fermentation by Lasiodiplodia theobromae using response surface methodology (RSM).
Method and Results:  Malt extract, sucrose, NaNO₃ and MgSO₄.7H₂O were analysed by a 30-trial central composite design using RSM for optimizing their concentrations in the medium and the effect of their mutual interaction on JA production. Sucrose and NaNO₃ were found highly significant in influencing the JA production. Malt extract and MgSO₄.7H₂O showed an effect on the JA production in interaction with other variables. When the optimum values of the parameters obtained through RSM (19·95 g l⁻¹ malt extract, 50 g l⁻¹ sucrose, 7·5 g l⁻¹ NaNO₃ and 3·51 g l⁻¹ MgSO₄.7H₂O) were applied, 32% increase in JA production (299 mg l⁻¹) was observed in comparison with 225 mg l⁻¹ of JA produced with same media components not analysed by RSM and subsequently validated the statistical model.
Conclusions:  Increase in JA production was achieved by optimizing the nutritional parameters.
Significance and Impact of the Study:  This is the first report of using RSM for optimizing a medium for JA production. It resulted in an increase in JA production without augmentation of costly additives.     

Optimization of D-ribose production with a transketolase-affected Bacillussubtilis mutant strain in glucose and gluconic acid-based media

When the transketolase-deficient and D -ribose-producing Bacillus subtilis strain ATCC 21951 was grown in a glucose (200 g l⁻¹)-based medium (Kintaka et al. 1986), only 11 g l⁻¹ D -ribose was synthesized, in addition to acetic acid (12 g l⁻¹) and acetoin plus 2,3-butanediol (24 g l⁻¹), within 1 week of fermentation. After optimizing the process conditions at 2 l fermentor scale (simplified medium composition, pH 5·0 or 6·0, highly oxidative (1000 rev min⁻¹, 3 vvm)), 40 g l⁻¹ D -ribose was obtained from 200 g l⁻¹ D -glucose, in addition to 14·5 g l⁻¹ acetoin, during 1 week of fermentation. By partially substituting D -glucose with D -gluconic acid (100 g l⁻¹ D -glucose plus 50 g l⁻¹ D -gluconic acid) under highly oxidative (1000 rev min⁻¹, 3 vvm) and pH-controlled (pH 6·5) conditions, D -ribose productivity increased (45 g l⁻¹) and acetoin formation (7·5 g l⁻¹) dropped, as did the fermentation time (3·5 d). The mixed carbon substrate procedure here developed provides an excellent alternative to the less efficient glucose-based processes described so far.     

The content of prostaglandins and their precursors in Mortierella and Cunninghamella species

Five strains of filamentous fungi belonging to the genera Mortierella and Cunninghamella were examined for the content of dihomo-γ-linolenic, arachidonic, eicosapentaenoic acids and prostaglandins (type E₂ and F _2α). Prostaglandins were detected using an ELISA method in mycelia of all tested strains (range 50–4800 ng g⁻¹ of PGE₂ and 6–30 ng g⁻¹ of PG F _2α). Several micro-organisms also produced prostaglandins in the culture medium (2·2–137·6 μg l⁻¹ for PGE₂ and 0·4–7·8 μg l⁻¹ for PG F _2α).     

Amines in fresh beef of normal pH and the role of bacteria in changes in concentration observed during storage in vacuum packs at chill temperatures

E dwards , R.A., D ainty , R.H., H ibbard , C.M. & R amantanis , S.V. 1987. Amines in fresh beef of normal pH and the role of bacteria in changes in concentration observed during storage in vacuum packs at chill temperatures.
The amine content of fresh and vacuum-packaged beef of normal pH stored at 1°C was evaluated by high performance liquid chromatography of dansyl derivatives. Fresh samples contained five amines, viz. putrescine, cadaverine, histamine, sperm-ine and spermidine. Development of a natural spoilage flora during storage led to increases in concentration of putrescine and cadaverine and the production of a sixth amine, tyramine. Pure culture meat inoculation experiments showed tyramine formation to be restricted to lactobacilli and to strains of Lactobacillus divergens and Lact. carnis in particular; strains of leuconostocs, Enterobacteriaceae, Pseudomonas spp. and Brochothrix thermosphacta were negative. Production of tyramine at cell densities >log₁₀ 6/cm² indicated its potential as an objective measure of acceptability/spoilage.     

Effects of feeding and induction strategy on the production of BmR1 antigen in recombinant E. coli

Aim:  To investigate the effects of feeding and induction strategies on the production of Bm R1 recombinant antigen.
Methods and Results:  Fed-batch fermentation was studied with respect to the specific growth rate and mode of induction to assess the growth potential of the bacteria in a bioreactor and to produce high yield of Bm R1 recombinant antigen. Cells were grown at a controlled specific growth rate (μ_set) during pre-induction, followed by constant feeding postinduction. The highest biomass (24·3 g l⁻¹) was obtained during fed-batch process operated at μ_set of 0·15 h⁻¹, whereby lower μ_set (0·075 h⁻¹) gave the highest protein production (9·82 mg l⁻¹). The yield of Bm R1 was increased by 1·2-fold upon induction with 1 mmol l⁻¹ IPTG (isopropyl-β- d -thiogalactoside) compared to using 5 mmol l⁻¹ and showed a further 3·5-fold increase when the culture was induced twice at the late log phase.
Conclusions:  Combination of feeding at a lower μ_set and twice induction with 1 mmol l⁻¹ IPTG yielded the best result of all variables tested, promising an improved method for Bm R1 production .
Significance and Impact of the Study:  This method can be used to increase the production scale of the Bm R1 recombinant antigen to meet the increasing demand for Brugia Rapid^™, a commercial diagnostic test for detection of brugian filariasis.     

Galacto-oligosaccharide production using a recycling cell culture of Sterigmatomyces elviae CBS8119

Addition of small amounts of Fe²⁺, Zn²⁺, Cu²⁺ and thiamine-HCl to the culture medium was required for promoting the galacto-oligosaccharide (Gal-OS)-producing activity of Sterigmatomyces elviae CBS8119, when the concentration of yeast extract in the medium was lowered to 0·1 g l⁻¹. Galacto-oligosaccharide production using a recycling cell culture was performed in a medium containing 360 mg ml⁻¹ of lactose supplemented with optimal concentrations of Fe²⁺ (1·5 mg l⁻¹ of FeSO₄.7H₂O), Zn²⁺ (15 mg l⁻¹ of ZnSO₄.7H₂O), Cu²⁺ (0·5 mg l⁻¹ of CuSO₄.5H₂O) and thiamine-HCl (1 mg l⁻¹ ) . Galacto-oligosaccharide production was maintained at high levels during six cycles of production, with the amount of Gal-OS produced in each cycle being more than 216 mg ml⁻¹ (weight yield of more than 60%).     

In vitro regeneration and genetic transformation of the berberine-producing plant,Thalictrum flavum ssp. glaucum

Protocols have been developed for the in vitro regeneration and Agrobacterium -mediated genetic transformation of meadow rue, Thalictrum flavum ssp. glaucum . Ten-day-old seedlings were bisected along the embryonic axis and the cotyledons were co-cultured with various Agrobacterium tumefaciens strains for 3 days. The cotyledons were cultured on a shoot induction medium (B5 salts and vitamins, 30 g l⁻¹ sucrose, 2 mg l⁻¹ kinetin, and 3 g l⁻¹ Gelrite) containing 25 mg l⁻¹ hygromycin B as the selection agent and 250 mg l⁻¹ timentin to facilitate the elimination of Agrobacterium . Only the oncogenic A. tumefaciens strains A281 and C58 produced transgenic T. flavum callus tissues. A281 was the most effective strain producing hygromycin-resistant callus on 85% of the explants. Transgenic callus was subcultured on the shoot induction medium every 2 weeks. After 12 weeks, hygromycin-resistant shoots that formed on explants exposed to strain A281 were transferred to a root induction medium (B5 salts and vitamins, 25 mg l⁻¹ hygromycin B, 250 mg l⁻¹ timentin, and 3 g l⁻¹ Gelrite). Detection of the β -glucuronidase ( GUS ) gene using a polymerase chain reaction assay, the high levels of GUS mRNA and enzyme activity, and the cytohistochemical localization of GUS activity confirmed the genetic transformation of callus cultures and regenerated plants. The transformation process did not alter the normal content of berberine in transgenic roots or cell cultures; thus, the reported protocol is valuable to study the molecular and metabolic regulation of protoberberine alkaloid biosynthesis.     

Phytoplankton production in relation to physico-chemical conditions in a small, oligotrophic subarctic lake in South Greenland

Phytoplankton production and physico-chemical parameters were measured during the summers of 1976 and 1977 in Lake 95 at 60°52'N, 46°01'W (area 8 ha, z 6.9 m). The duration of lake stratification during summer was weather dependent. Concentrations of total-CO₂ ranged from 2.04 mg l⁻¹ (surface) to 4.02 mg l⁻¹ (bottom during stratification). Nutrient concentrations were generally low, and systematic seasonal variation could not be detected with certainty. Vertical distribution of primary production typically showed a distinct maximum followed by a decline with depth. The low production, both per unit area (max. 60 mg C m⁻² d⁻¹) and per unit volume (max, 14.3 mg C m⁻³ d⁻¹), and the annual production of 4.7 g C m⁻² classified the lake as extremely oligotrophic.     

Research note: In vitro inhibition of Listeria monocytogenes growth by veillonellae cultures grown on tartrate media

Veillonellae cultures were grown on agar media supplemented with tartrate and examined for inhibitory effects on the growth of Listeria monocytogenes . Veillonellae cultures grown on media supplemented with 0 or 50 mmol l⁻¹ of tartrate did not inhibit the growth of L. monocytogenes ; however, veillonellae grown on media supplemented with 100, 150 or 200 mmol l⁻¹ of tartrate did inhibit the growth of L. monocytogenes . The inhibition of the growth of L. monocytogenes by the veillonellae was correlated with the increased production of acetate and propionate from tartrate by veillonellae and with the reduction of the pH of the media by L. monocytogenes .     

Photosynthesis, growth and structural characteristics of holm oak resprouts originated from plants grown under elevated CO2

The physiological characteristics of holm oak ( Quercus ilex L.) resprouts originated from plants grown under current CO₂ concentration (350 μl l⁻¹) (A-resprouts) were compared with those of resprouts originated from plants grown under elevated CO₂ (750 μl l⁻¹) (E-resprouts). At their respective CO₂ growth concentration, no differences were observed in photosynthesis and chlorophyll fluorescence parameters between the two kinds of resprout. E-resprouts appeared earlier and showed lower stomatal conductance, higher water-use efficiency and increased growth (higher leaf, stem and root biomass and increased height). Analyses of leaf chemical composition showed the effect of elevated [CO₂] on structural polysaccharide (higher cellulose content), but no accumulation of total non-structural carbohydrate on area or dry weight basis was seen. Four months after appearance, downregulation of photosynthesis and electron transport components was observed in E-resprouts: lower photosynthetic capacity, photosystem II quantum efficiency, photochemical quenching of fluorescence and relative electron transport rate. Reduction in ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) activity, deduced from the maximum carboxylation velocity of RuBisCo, accounts for the observed acclimation. Increased susceptibility of photosynthetic apparatus to increasing irradiance was detected in E-resprouts.     

The effect of osmotic shock and subsequent adaptation on the thermotolerance and cell morphology of Listeria monocytogenes

The relationship between the time of exposure to different levels of NaCl and the corresponding changes in thermotolerance and cell morphology of Listeria monocytogenes was investigated. The kinetics of the increase in thermotolerance, after an osmotic upshift, showed a very rapid initial response (<2 min) followed by a more gradual increase whereby cells, after 4 h exposure at 30°C, became nearly as heat resistant as those grown for 48 h under the same conditions. Cells grown in media with 0.09 mol l⁻¹ NaCl subjected to a short osmotic up-shock in media containing 0.5, 1.0 or 1.5 mol l⁻¹ NaCl showed a 1.3, 2.5 and 8-fold increase in thermotolerance, respectively. Osmotic adaptation, signified by growth at the higher NaCl concentration, however, resulted in a 2- to 3-fold additional increase in thermotolerance. An osmotic down-shock caused a very rapid loss of thermotolerance (<5 min). Osmotic shock and adaptation experiments were also performed in minced beef where similar changes in thermotolerance were observed. Cell morphology was markedly affected by the osmolarity of the growth medium. Cells grown in media containing 1.5 mol l⁻¹ NaCl became up to 50 times longer than cells grown in media with 0.09 mol l⁻¹ NaCl, but no direct link to thermotolerance could be made.     

Polymerase chain reaction detection and speciation of Campylobacter upsaliensis and C. helveticus in human faeces and comparison with culture techniques

A polymerase chain reaction (PCR) assay based on the 16S rRNA gene and an improved DNA extraction procedure were developed for the direct detection and differentiation of Campylobacter upsaliensis and C. helveticus in seeded human faeces. The PCR assay was compared with culture detection by a membrane filter (MF) technique and on selective agar (SA) containing 8 mg l⁻¹ cefoperazone. Both MF culture and the PCR assay detected 10⁵ colony-forming units (cfu) g⁻¹ faeces. Selective agar culture of some strains could detect as few as 10³ cfu g⁻¹ faeces. However, some strains were susceptible to cefoperazone and either failed to grow or were detected only with reduced sensitivity in the presence of the antibiotic. Detection by MF and SA both required 48–96 h incubation in a microaerobic atmosphere and did not specifically identify the isolate. By contrast, the PCR assay could be completed within 8 h and accurately identified the two phenotypically similar species, C. upsaliensis and C. helveticus.     

Competition between anoxygenic phototrophic bacteria and colorless sulfur bacteria in a microbial mat

Abstract The populations of chemolithoautotrophic (colorless) sulfur bacteria and anoxygenic phototrophic bacteria were enumerated in a marine microbial mat. The highest population densities were found in the 0–5 mm layer of the mat: 2.0 × 10⁹ cells cm⁻³ sediment, and 4.0 × 10⁷ cells cm⁻³ sediment for the colorless sulfur bacteria and phototrophs, respectively. Kinetic parameters for thiosulfate-limited growth were assessed for Thiobacillus thioparus T5 and Thiocapsa roseopersicina M1, both isolated from microbial mats. For Thiobacillus T5, growing at a constant oxygen concentration of 43 μmol l⁻¹, μ_max was 0.336 h⁻¹ and K _s 0.8 μmol l⁻¹. Phototrophically grown Thiocapsa strain M1 displayed a μ_max of 0.080 h⁻¹ and a K _s of 8 μmol l⁻¹ when anoxically grown under thiosulfate limitation. In a competition experiment with thiosulfate as electron donor, Thiocapsa became dominant during a 10-h oxic/14-h anoxic regimen at continuous illumination, despite the higher affinity for thiosulfate of Thiobacillus .     

Chronic toxicity of ammonia to juvenile gilthead seabream Sparus aurata and related histopathological effects

Effect of 20 days exposure of juvenile gilthead seabream ( Sparus aurata ) to elevated levels of ammonia on growth and survival was examined in a continuous flow system. Suppressed growth and reduced survival were observed at concentrations of 8.2 and 13 mg l⁻¹ total ammonia-N (0.5 and 0.7 mg l⁻¹ un-ionized ammonia-N, respectively) and higher. The maximum acceptable toxic concentration (MATC) for growth was between 4.8 to 8.2 mg l⁻¹ total ammonia-N (0.3 and 0.5 mg l⁻¹ un-ionized ammonia-N, respectively). Fish exposed to high ammonia levels (13 mg l⁻¹ total ammonia-N, 0.7 mg l⁻¹ un-ionized ammonia-N) displayed clear signs of liver pathology. Existing evidence suggests that S. aurata is less sensitive to ammonia than other reported marine and freshwater fish.
Under certain conditions ammonia concentration in the intensive fish ponds in Eilat may exceed the no observed effect concentration for S. aurata .     

The role of light and CO2 in optimising the conditions for shoot proliferation of Actinidia deliciosa in vitro

Proliferating cultures of Actinidia deliciosa A. Chev., C. F. Liang and A. R. Ferguson cv. Tomuri (♂) were grown under photosynthetic photon flux density (PPFD) rates ranging from 30 to 250 μmol m⁻² s⁻¹ in order to determine certain physiological parameters in vitro: CO₂ evolution, photosynthesis at three CO₂ atmospheric concentrations (330, 1450 and 4500 μl l⁻¹), fresh and dry matter accumulation and proliferation rate.
A proportional response in dry weight, dry/fresh weight ratios and PPFD was found. The proliferation rate increased up to 120 μmol m⁻² s⁻¹ but decreased at higher rates. At the highest PPFD, the CO² released from cultures and accumulated in the vessels reached 200 μl l⁻¹ of; at the lowest rate the CO₂ concentration reached 10500 μl l⁻¹ after 28 days of culture. The photosynthetic rate at 1450 and 4500 μl l⁻¹ of CO₂ was nearly 4 times higher than at the lowest concentration tested.     

Production of B-group vitamins by two Rhizobium strains in chemically defined media

Twenty-eight strains of Rhizobium spp. were tested for their ability to grow in chemically-defined medium lacking growth factors. Two strains, R. meliloti GR4B and Rhizobium spp. ( Acacia ) GRH28, were selected, on the basis of their good growth under the conditions imposed, for further quantification of the production of water-soluble vitamins (thiamine, niacin, riboflavin, pantothenic acid and biotin) in chemically defined media amended with different compounds (mannitol, glucose or sodium succinate) as sole carbon sources. Qualitative and quantitative production of vitamins in chemically-defined media was significantly affected by the use of C sources of a different nature and the age of the cultures. Strain GRH28 produced all the vitamins analysed, and high biological levels of biotin (14 ng ml^–1 culture) were detected after 6 d of culture in mineral medium amended with mannitol. Pantothenic acid was the vitamin detected in the highest amounts (up to 1 μg ml^–1 of culture) in culture supernatant fluids of strain GR4B grown for 6 d with succinate as sole carbon source.     

Effect of culturing processes and copper addition on laccase production by the white-rot fungus Fomes fomentarius MUCL 35117

Aim:  To produce high laccase activities from the white-rot fungus Fomes fomentarius .
Methods and Results:  Different culturing methods, viz, cell immobilization on stainless steel sponges and plastic material and solid-state fermentation (SSF) using wheat bran as substrate were used for laccase production by the white-rot fungus F. fomentarius . The SSF study expresses the highest laccase activities, nearly to 6400 U l⁻¹ after 13 days of laboratory flasks cultivation. When the wheat bran medium was supplemented with 2 mmol l⁻¹ copper sulfate, laccase activity increased by threefold in comparison to control cultures, reaching 27 864 U l⁻¹. With the medium thus optimized, further experiments were performed in a 3 l fixed-bed bioreactor (working volume 1·5 l) leading to a laccase activity of about 6230 U l⁻¹ on day 13.
Conclusions:  The results obtained clearly showed the superiority of wheat bran for laccase production over stainless steel sponges and plastic material. Supplementing the wheat bran solid medium with 2 mmol l⁻¹ copper sulfate allowed obtaining high activities at flask scale. The system was scaled to fixed-bed laboratory reactor.
Significance and Impact of the Study:  The high enzyme production along with the low-cost of the substrate, showed the suitability of the system F. fomentarius – SSF for industrial purposes.     

Itaconic acid production by Aspergillus terreus on raw starchy materials

Starchy materials such as corn starch, soft wheat flour, potato flour, cassava flour, sorghum starch, sweet potato and industrial potato flours, either acid or enzymatically hydrolysed, were used as substrates for itaconic acid production by Aspergillus terreus NRRL 1960. Both production and yield were highest on corn starch (18·4 g l⁻¹ and 34·0%, respectively). The degree of hydrolysis had a great influence on acid production which was highest when corn starch was saccharified at 85 DE (dextrose equivalent). In a 3 litre benchtop fermenter, itaconic acid production and productivity were 19·8 g l⁻¹ and 0·13 g l⁻¹ h⁻¹, respectively.