Growth yield increase linked to caffeate reduction in Acetobacterium woodii

Growth yields were determined with Acetobacterium woodii strain NZva 16 on hydrogen and CO₂, formate, methanol, vanillate, ferulate and fructose in mineral medium in the absence and presence of 0.05% yeast extract. Yeast extract was not essential for growth but enhanced growth yields by 25–100% depending on the substrate fermented. Comparison of yields on formate or methanol allowed calculation of an energy yield in the range of 1.5–2 mol ATP per mol acetate formed during homoacetate fermentation of A. woodii. In the presence of 6 mM caffeate, growth yields were determined with the substrates formate or methanol. Caffeate was reduced to hydrocaffeate and increased growth yields were obtained. An ATP yield of about 1 mol per mol of caffeate reduced was calculated. Cytochromes were not detectable in cell free extracts or membrane preparations.     

In vitro production studies with a wild-type Helicoverpa baculovirus

The potential use of a wild-type Helicoverpa baculovirus as a biopesticide, using insect cell culture for its production, has been investigated. A Helicoverpa zea cell line was adapted to grow in suspension culture using a serum-free medium, SF900II and serum supplemented SF900II. The serum supplemented cells were infected with a wild-type nuclear polyhedrosis virus of Helicoverpa armigera (HaNPV), at different stages of growth, in conditioned and tresh medium, to determine the effect of cell density on polyhedra production. Cultures infected at low cell densities, produced similar yields of virus (20–40 PIB/cell), irrespective of medium conditions. However, in infections which occurred at high cell densities, there was a 16-fold improvement in cell specific yields, when the spent medium was renewed with fresh medium prior to infection. Results indicated that only 60–70% of the viable cells in a culture produced polyhedra as a result of infections.     

Utilization of normal alkanes by yeasts

A stable mixed yeast culture designated as Culture 4, consisting of Candida intermedia and Candida lipolytica was investigated. The culture was judged stable based on uniformity of fermentation results and the nearly constant ratio of the two organisms at the completion of fermentations. However, the ratio of the two organisms at different times during the fermentation was not determined. The mixed culture grew more rapidly on n-alkanes than did C. intermedia; C. lipolytica did not grow on unsupplemented mineral salt–n-alkane medium. Solid n-alkanes were dissolved in 2,6,0,14-tetramethylpentadecane (pristane) for investigation as carbon sources. With Culture 4, on n-alkanes ranging from pentadecane (C₁₅) through octacosane (C₂₈), cell yields were 74.2–89.5%; generation times were 3.0–8.0 hr. during the exponential growth phase. The fastest growth rates and highest cell yields were obtained with docosane (C₂₂) as substrate. The cells obtained contained 6.75–8.81% nitrogen and 1.9–13.4% lipid. Crude protein yields were 34.4–47.6%. The oxidation of n-alkanes by C. intermedia was studied manometrically with resting whole cells. The alkaneoxidizing system of this organism appears to be constitutive and nonspecific for alkane substrates.     

Protoplasts formation inFusarium species

Formation of protoplasts from four species ofFusarium genus is described. Protoplasts were isolated from mycelium by enzymatic digestion of the cell wall in the presence of an osmotic stabilizer. The results obtained differed between the studied species. Best yields of protoplasts were obtained fromF. moniliforme (90 % cells as protoplasts).     

Rabies virus glycoprotein expression in Drosophila S2 cells: Influence of re-selection on protein expression

The aim of this study was to achieve expression of recombinant rabies virus glycoprotein (rRVGP) in Drosophila S2 cells. For this, a cDNA coding for the selection hygromycin antibiotic and the cDNA encoding the RVGP protein under the control of the constitutive actin promoter (Ac) were cloned in an expression plasmid, which was transfected into S2 cells. S2 cell populations (S2AcRVGPHy) showed rRVGP expression in cell lysates, attaining concentrations up to 1.5 μg/10⁷ cells (705 μg/L). Of the transfected cells, 20% were shown to express the rRVGP. Cell subpopulations selected by limiting dilution expressed higher rRVGP yields and 90% of the cells were shown to express the rRVGP. Cell populations re-selected by addition of hygromycin were shown to express 10 times higher rRVGP yields. The data presented here show that Drosophila S2 cells can be efficiently transfected with an expression/selection plasmid for rRVGP expression, allowing its synthesis with a high degree of physical and biological integrity. The importance of subpopulation selection was indicated by the increasing rRVGP yields during these procedures.     

Corecovery of lipids and fermentable sugars from Rhodosporidium toruloides using ionic liquid cosolvents: Application of recycle to batch fermentation

This work evaluates the ability of an ionic liquid‐methanol cosolvent system to extract lipids and recycle fermentable sugars recovered from oil‐bearing Rhodosporidium toruloides grown in batch culture on defined media using glucose and xylose as carbon sources. Growth on the recycled mixed carbon substrate was successful with glucose consumed before xylose and overall cell mass to lipid yields (Y_P/X) between 57% and 61% (w/w relative to whole dried cell mass) achieved. Enzymatic hydrolysis of the delipified carbohydrate fraction recovered approximately 9%–11% (w/w) of the whole dried cell mass as fermentable sugars, which were successfully recycled as carbon sources without further purification. In total, up to 70% (w/w) of the whole dried cell mass was recovered as lipids and fermentable sugars and the substrate to lipid yields (Y_P/S) was increased from 0.12 to 0.16 g lipid/g carbohydrate consumed, highlighting the promise of this approach to process lipid bearing cell biomass. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1239–1242, 2014     

Protoplast isolation and regeneration from <Emphasis Type="Italic">Gracilaria changii</Emphasis> (Gracilariales,Rhodophyta)

The tropical agarophyte Gracilaria changii has been much researched and documented by the Algae Research Laboratory, University of Malaya, especially with regards to its potential as a seaweed bioreactor for valuable compounds. Protoplast regeneration of this seaweed was developed following the optimization of protoplast isolation protocol. Effect of the concentration and combination of isolating enzymes, incubation period, temperature, enzyme solution pH, tissue source on the protoplast yields were used to optimize the isolation protocol. The enzyme mixture with 4% w/v cellulase Onozuka R-10, 2% w/v macerozyme R-10 and 1 unit mL^-1 agarase was found to produce the highest yield of protoplast at 28°C and 3 h incubation period. Thallus tips gave higher yields of protoplasts than middle segments. Freshly isolated G. changii protoplasts were cultured in MES medium. Regeneration of protoplast cell walls after 24 h was confirmed by calcofluor white M2R staining under UV fluorescence microscopy. The protoplasts with regenerated cell walls then underwent a series of cell division to produce callus-like cell masses in MES medium. Following this, juvenile plants of G. changii were obtained.     

Growth engineering of Propionibacterium freudenreichii shermanii for organic acids and other value-added products formation

Propionic acid production from glucose was studied using Propionibacterium freudenreichii shermanii. Conditions were optimized for high yields of propionic acid and total organic acids by sequential optimization of parameters like pH, inoculum age, inoculum volume and substrate concentration. Near-theoretical yield (0.54?±?0.023?g/g) was achieved for propionic acid with fermentation of 1% glucose using 20% (v/v) of 48?hr old P. shermanii at 30°C, pH maintained at 5.5. Total organic acid yield under these conditions was 0.74?±?0.06?g/g. The study resulted in achieving 98% and 95% theoretical yields of propionic acid and total organic acids, respectively. Under optimized conditions, along with organic acids, P. shermanii also produced vitamin B12 and trehalose intracellularly, showing its potential to be used as a cell factory.     

The Effect of Ultrasonificated Extracts of <Emphasis Type="Italic">Spirulina maxima</Emphasis> on the Anticancer Activity

The effect of ultrasonic extraction on extraction yields, cytotoxicity, and anticancer activity of Spirulina maxima was investigated in this study. Optimal extraction conditions were determined as 60 kHz frequency at 60°C for 30 min with 120 W intensity, which resulted in 19.3% of extraction yields and 19.1% of cytotoxicity on normal human cells. Yields from conventional water and ethanol extraction were 15.8% at 100°C and 8.3% at 80°C, respectively. It was found that the extracts obtained by ultrasonic extraction process selectively inhibited the digestive-related cancer cell lines, such as human stomach cancer cells, having 89% of the highest inhibition ratio and 4.5 of the highest selectivity. In adding 0.5 mg/mL of the extract, human promyelocytic leukemia cells' cell differentiation was increased 1.72 times over that of the control. Expression level of B cell lymphoma-2 from Hep3B cell was also effectively suppressed by the extract obtained at 60 kHz and 60°C, leading to the inhibition of the early step of carcinogenesis. This work suggests that anticancer activity of the extracts is due to water-soluble polysaccharides rather than proteins and is further supported by the result that the ultrasonification extraction process can efficiently extract relatively intact polysaccharides rather than digesting the proteins in S. maxima by matrix assisted laser desorption ionization-time of flight and high performance size exclusion chromatography chromatogram analyses. Therefore, ultrasonic extraction increases both extraction yield and the biological activity of S. maxima extracts, which might be useful as an alternative natural anticancer agent in the medical and food industries.     

Enzymatic synthesis of levan by Zymomonas mobilis levansucrase overexpressed in Escherichia coli

Summary Levansucrase gene from Zymomonas mobilis was expressed efficiently in Escherichia coli and the overproduced recombinant levansucrase amounted to 40% of the total cell protein. Using E. coli lysate, levan was synthesized in a sucrose-based medium enzymatically with the conversion yields of up to 46% from fructose liberated in 25 hrs of incubation. More levan was formed at lower temperatures in the reaction mixture, whereas higher temperatures were favoured for the accumulation of free fructose or short chain oligosaccharides.     

Video microscopic observations of living,isolated embryo sacs of Nicotiana and their component cells

Summary Living embryo sacs and megagametophytic cells of Nicotiana alata and Nicotiana tabacum were obtained using enzymatic maceration and microdissection. The yields of isolated embryo sacs, egg apparatus and central cells were up to 35%, 40% and 35%, respectively. Vectorial movement of organelles and undulations of tubular structures, presumably endoplasmic reticulum, were observed in eggs, synergids and central cells using video-enhanced microscopy. Despite evident viability using the fluorochromatic reaction, the egg displays much less organelle movement and therefore appears to be quiescent. The large vacuole of the central cell is traversed by mobile strands of cytoplasm through which organelles migrate. A polygonal network is located at the periphery of the central cell, which may contribute to anchorage of the cell with the embryo-sac wall. The observation of organelle movement provides direct evidence of the condition of the cell and may be a useful approach for assessing cell vigor.     

Extractability of polyhydroxyalkanoate synthesized by <Emphasis Type="Italic">Bacillus flexus</Emphasis> cultivated in organic and inorganic nutrient media

Bacillus flexus was isolated from local soil sample and identified by molecular methods. In inorganic nutrient medium (IM) containing sucrose as carbon source, yield of biomass and polyhydroxyalkanoate (PHA) were 2 g/l and 1 g/l (50% of biomass), respectively. Substitution of inorganic nitrogen by peptone, yeast extract or beef extract resulted in biomass yields of 4.1, 3.9 and 1.6 g/l, respectively. Corresponding yields of PHA in biomass was 30%, 40% and 44%. Cells subjected to change in nutrient condition from organic to inorganic, lacked diaminopimelic acid in the cell wall and the concentration of amino acids also decreased. Under these conditions the extractability of the polymer from the cells by hot chloroform or mild alkali hydrolysis was 86–100% compared to those grown in yeast extract or peptone (32–56%). The results demonstrated that growth, PHA production and the composition of cell wall of B. flexus are influenced by the organic or inorganic nutrients present in the growth medium. Cells grown in inorganic medium lysed easily and this can be further exploited for easier recovery of the intracellular PHA.     

Tissue degradation and enzymatic activity observed during protoplast isolation in two ornamental <Emphasis Type="Italic">Grevillea</Emphasis> species

Summary Degradative changes in tissue during protoplast isolation were a contributing factor to low protoplast yields in the saltsensitive Grevillea arenaria (R. Brown) and the salt-tolerant Grevillea ilicifolia (R. Brown). Protein and malondialdehyde content decreased significantly during the protoplast isolation procedure. Acid and neutral proteases were identified, and high acid protease activities were correlated to low protoplast yields. Acid phosphatase, catalase, polyphenol oxidase and lipoxygenase activities increased in both Grevillea species with cell wall digestion. High activities of catalase and low levels of polyphenol oxidase were correlated with high protoplast yields. Levels of acid phosphatase and lipoxygenase were not good indicators of final protoplast yields. The addition of the anti-oxidant, reduced glutathione, and the acid protease inhibitor, pepstatin A, significantly increased protoplast yields. Strategies were identified to minimize deleterious degradative effects during the isolation of protoplasts, including strict pH control, testing a number of cell wall digestion enzymes, and the addition of anti-oxidative metabolites and protease inhibitors.     

Growth yield of denitrifiers using nitrous oxide as a terminal electron acceptor

The molar yields (g cell/mol) forAlcaligenes faecalis, Pseudomonas stutzeri, Paracoccus denitrificans andPseudomonas perfectomarinus batch cultures, under nitrous oxide (N₂O) as the electron acceptor, were 11.2, 8.2, 6.1 and 4.4, respectively.Paracoccus denitrificans andPseudomonas perfectomarinus, which had the slowest growth rates, gave the lowest yields. Large maintenance energy costs may be partially responsible for this. The growth efficiencies ofA. faecalis andPs. perfectomarinus on N₂O indicate that the numbers of sites for oxidative phosphorylation in the electron transport system associated with N₂O reduction are about 49% and 39% of those in the electron transport system associated with O₂ respiration, respectively.     

Heterologous protein secretion from Aspergillus niger in phosphate-buffered batch culture

Summary Aspergillus niger was grown in batch culture containing various initial concentrations of sodium phosphate buffer (pH 6.5). A wild-type strain of A. niger and a transformed strain producing hen egg-white lysozyme were studied. The maximum cell yield was attained in medium not supplemented with phosphate. In those cultures acidification of the medium resulted in a minimum of pH 2.0 before reverting to near neutrality. Increasing the initial levels of phosphate buffer reduced the fall in pH but lowered cell yields. Secreted levels of lysozyme were maximal in the 50–100 mm range of added phosphate buffer although mycelial yields were reduced by one third of mycelial yields in medium unsupplemented with phosphate buffer. Offprint requests to: D. B. Archer     

Influence of bioreactor design on exopolysaccharide production byAureobasidium pullulans

Summary Exopolysaccharide yields byAureobasidium pullulans were affected by the bioreactor configuration in batch culture. However the yields obtained in the stirred-tank and airlift vessels were also dependent on the nitrogen source used, and independent of cell morphology.     

Growth yields of microorganisms on methanol and methane. A theoretical study

An attempt was made to calculate growth yields of microorganisms on methanol and methane on the basis of known biochemical pathways of C₁ metabolism. Since 3-phosphoglycerate is a key intermediate in the assimilation pathways of C₁ compounds, the calculations were based on the assumption that the synthesis of cell material from C₁ substrates can be regarded as a two step process. When Y_ATP on 3-phosphoglycerate was taken as 10.5, a maximal cell yield of organisms of the composition C₄H₈O₂N on methanol was found to be 0.73 g cells/g substrate. For growth on methane a value of 0.91 g cells/g substrate was calculated when a mixed function oxidase was implicated in methane oxidation. These yields were calculated on the basis of the ribulose phosphate pathway of formaldehyde fixation as the major pathway of C₁ assimilation. Yields calculated on the basis of the serine pathway were on an average 20% lower. The calculations disclosed that for growth on methane, at least for Methylococcus capsulatus, a reversed electron transport system is required when methane is oxidized by a mixed function oxidase. The theoretical cell yields on methanol and methane have been compared with experimentally obtained yields and the validity of the estimations of growth yields on the basis of the present calculations is discussed.     

Cellulase and protein production from mixed cultures of Trichoderma viride and a yeast

Fermentations with mixed cultures of the cellulolytic fungus Trichoderma viride and the yeast Saccharomyces cerevisiae or Candida utiliswere examined. The fermentations were carried out in an aerated 5 liter fermentor with NaOH treated barley straw as the cellulose source (2–4%). Yeast was inoculated 24–32 hr after the fungus and the growth of the two organisms was followed through the production of CO₂ and cell protein. In comparison with fermentations with T. viridealone, the production time for maximum yields of cellulases and cell protein was reduced by several days, depending on the straw concentrations. The protein content of the growth product was 21–22% and the amino acid composition of the product resembled that of T. viride alone.     

Direct extraction of astaxanthin from <Emphasis Type="Italic">Haematococcus</Emphasis> culture using vegetable oils

A green, downstream process using common vegetable oils was used for the direct extraction of astaxanthin from Haematococcus. The process consists of a single integrated unit to extract astaxanthin with subsequent separation of the astaxanthin-containing oil extract. Without a cell harvest process step, the culture broth was directly mixed with the vegetable oils; the astaxanthin inside the cell was extracted into the vegetable oil phase by hydrophobic interactions, with recovery yields of 88% and above. The oil extracts were simply separated from the culture medium containing cell debris by gravity settling only.     

Effects of marine algal proximate composition on methane yields

The suitability of differentGracilaria spp. and twoSargassum species for bioconversion to methane was determined through bioassays of methane yield.Gracilaria species and strains were excellent feedstocks for high methane yields, ranging from 0.28 to 0.40 m³ kg^–1 volatile solids added. These yields ranged from 58 to 95% of theoretical stoichiometric yields. Methane yields were highly correlated with acid soluble carbohydrate components of theGracilaria spp. BothSargassum fluitans andS. pteropleuron were poor feedstocks, with methane yields ranging from 0.12 to 0.19 m³ kg^–1 volatile solids added, 27 to 46% of theoretical stoichiometric yields, respectively. The various tissue types of theseSargassum species were also poor feedstocks for anaerobic digestion to methane. While there is no clear explanation for the low methane yields, the twoSargassum spp. appear to contain a high proportion of an insoluble, non-extractable component which may not be available as a substrate for bioconversion to methane.