Dense autotrophic cultures of Alcaligenes eutrophus.

Alcaligenes eutrophus was grown autotrophically in 23-liter batch cultures in a controlled H2-O2-CO2 atmosphere. It was demonstrated that the need for periodic supplements of individual nutrients could be anticipated before cell growth depleted these nutrients to the point of becoming growth rate limiting. As a result, exponential growth was extended to optical densities of 44, with doubling times maintained at 2 h. Cultures having an initial optical density of 0.040 to 0.70 reached the final optical density of 60 in about 25 h. The final viable count was 1.2 X 10(11) cells per ml, and the dry weight was 25 g/liter.     

Anaerobic digestates are useful nutrient sources for microalgae cultivation: functional coupling of energy and biomass production

We investigated the extent to which nitrogenous and phosphorus nutrients from liquid anaerobic digestates could be recycled for photosynthetic growth of a microalga, Scenedesmus sp. AMDD. Digestates recovered from the anaerobic digestion of cow manure and swine manure and a co-digestion of swine manure and algal biomass were diluted in distilled water and used for algal growth with and without supplemental CO₂ addition. Nutrient assimilation and final biomass yield were retarded in all but the swine manure/algae co-digestate cultures supplemented with high CO₂. Swine manure digestate cultures supplemented with the typical complement of micronutrients normally added with a commonly used growth medium or with Fe/EDTA failed to grow any better than unamended controls. When the culture medium was prepared by blending swine manure digestate with 25 or 50 % algal biomass digestate, diluting it with lake water or by supplementing with magnesium, nutrient assimilation and final algal biomass yields were maximized, indicating that magnesium was critically limiting for algal growth in swine manure digestates. Magnesium amendment thus appears to be essential if nutrients from swine manure digestates are recycled for algal growth. No such requirement is necessary for recycling nutrients from digestates generated wholly or in part from algal biomass.     

Enzymatic digestion of spent yeast cells for nutrient recycling in inulase production

Summary An enzyme complex capable of lysing yeast cells was produced byArthrobacter sp. in a medium containing live cells ofKluyveromyces fragilis as the sole source of nutrients. The enzyme complex caused a 90% reduction in the optical density of viable yeast cells in 6 h at 25°C. This yeast cell hydrolysate can be used as a source of nitrogen, vitamins and minerals for subsequent growth of yeast cells (8.3 mg/ml) and further production of inulase (167 U/ml) representing 88 and 87% yield respectively, compared to cells grown on a standard yeast extract (1%) and sucrose (2%) medium.     

矿质元素对毛头鬼伞菌丝体生长的影响

研究了4种矿质元素对栽培食用菌毛头鬼伞菌丝体生长的影响。4种矿质单元素添加试验结果表明,能使毛头鬼伞菌丝体旺盛生长的最佳浓度分别是CaCl20.1 mmol/L、MgSO48 mmol/L、KCl 1.22 mmol/L、NaH2PO413 mmol/L。4种元素协同作用试验结果表明,单元素浓度的最佳生长量与混合施用时的最佳生长量一致。以毛头鬼伞的主要栽培料之一的棉子壳估算,原料中的4种矿质元素浓度均偏低,必须补充矿质元素才能满足毛头鬼伞菌丝体的旺盛生长。     

Secreted pitfall-trap fluid of carnivorous Nepenthes plants is unsuitable for microbial growth

Background and Aims

Carnivorous plants of the genus Nepenthes possess modified leaves that form pitfall traps in order to capture prey, mainly arthropods, to make additional nutrients available for the plant. These pitchers contain a digestive fluid due to the presence of hydrolytic enzymes. In this study, the composition of the digestive fluid was further analysed with regard to mineral nutrients and low molecular-weight compounds. A potential contribution of microbes to the composition of pitcher fluid was investigated.

Methods

Fluids from closed pitchers were harvested and analysed for mineral nutrients using analytical techniques based on ion-chromatography and inductively coupled plasma–optical emission spectroscopy. Secondary metabolites were identified by a combination of LC-MS and NMR. The presence of bacteria in the pitcher fluid was investigated by PCR of 16S-rRNA genes. Growth analyses of bacteria and yeast were performed in vitro with harvested pitcher fluid and in vivo within pitchers with injected microbes.

Key Results

The pitcher fluid from closed pitchers was found to be primarily an approx. 25-mm KCl solution, which is free of bacteria and unsuitable for microbial growth probably due to the lack of essential mineral nutrients such as phosphate and inorganic nitrogen. The fluid also contained antimicrobial naphthoquinones, plumbagin and 7-methyl-juglone, and defensive proteins such as the thaumatin-like protein. Challenging with bacteria or yeast caused bactericide as well as fungistatic properties in the fluid. Our results reveal that Nepenthes pitcher fluids represent a dynamic system that is able to react to the presence of microbes.

Conclusions

The secreted liquid of closed and freshly opened Nepenthes pitchers is exclusively plant-derived. It is unsuitable to serve as an environment for microbial growth. Thus, Nepenthes plants can avoid and control, at least to some extent, the microbial colonization of their pitfall traps and, thereby, reduce the need to vie with microbes for the prey-derived nutrients.     

Influence of nutrients on growth and bacteriocin production by Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442

The aim of this study was to investigate the effect of complex nutrients on microbial growth and bacteriocin production, in order to improve bacteriocin synthesis during the growth cycle of Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442. The fermentations were conducted at the optimum pH and temperature for bacteriocin production (pH 5.5+/-0.1 and temperature 25+/-0.1 degrees C). Because of their association with the final biomass, conditions favouring the increase of the produced biomass resulted in the increase of bacteriocin activity in the growth medium. Since the produced final biomass and the final concentration of the bacteriocins were associated with the amount of the carbon (glucose) and nitrogen source, better growth of the lactic acid bacterial strains favoured the increase of the specific bacteriocin production. Additionally, the bacteriocin production was influenced by carbon/nitrogen ratio.     

Optimum conditions for initiation of filamentation in Candida albicans.

A medium that initially produced filaments from almost all of its Candida albicans blastospore inoculum contained 1% mycological peptone and 0.2% glucose, final pH 7.4-7.5. The medium was inoculated to 10-6 cells/ml and incubated at 40 degrees C. Reversion to secondary blastospores began at a mean of 2.4 h after inoculation. The patterns of utilization of growth nutrients during optimal mycelial growth showed no correlation with the events of filamentation.     

Single-Cell Protein Production in Alkaline Mesquite Wood Extracts

A nine-member, mixed, cellulolytic, bacterial culture was used to evaluate the effects of sodium hydroxide normality, length and temperature of treatment, and the ratio of volume of alkali to mesquite wood on the suitability of alkali mesquite wood extractives as nutrients for bacterial growth. The presence or absence of air during the extraction process did not significantly affect results. The amount of lignin extracted and the total loss in weight of the wood during extraction were correlated to both alkali concentration and temperature. Neutralized extracts supported bacterial growth; growth was inversely related to the final salt concentration of the neutralized extract. Deionized extracts were superior to acid-neutralized extracts for the support of bacterial growth. The optimum conditions for extraction were 2.5 N NaOH at 30°C for 12 h. The study demonstrated that nutrients as well as growth inhibitory compounds are released from wood by alkali treatment. This study of alkali wood extracts and the previous study of washed alkali treated wood residues provide a data base for the optimization of alkali treatments of hard woods that are to be used as nutrients for the growth of cellulolytic cultures.     

Effects of micro-nutrients and major nutrients on natural phytoplankton populations

Six experiments were carried out with natural phytoplanktonpopulations grown under simulated natural conditions, in anattempt to assess the gross effects of major nutrient and micronutrientperturbations upon the growth and species composition of thesepopulations. Water was collected from Yaquina Bay, Oregon, andfiltered into twenty-litre growth containers. Some of this waterwas treated with activated carbon to remove a portion of thetrace metals and trace organics. Various combinations of majornutrients, micro-nutrients, and chelators were added to thesecultures. Inoculation was with unfiltered bay water, at a ratioof 1:1000. Incubation was in outdoor tanks of water circulatingfrom the bay, under natural sunlight. The major nutrients andmicro-nutrients appeared to have fundamentally different effectson the growth and species composition of phytoplankton. Themicro-nutrients had marked effects on the species compositionof the phytoplankton populations, while major nutrients hadminimal effects on species composition. The micro-nutrientsalso had substantial effects on population growth rates, lagperiods, and final yields. The major nutrients had a substantialeffect only on final yields.     

Pollution control of swine manure and straw by conversion to Chaetomium cellulolyticum SCP feed

Swine manure has a very high pollution potential and obnoxious odor. Large farms particularly are confronted with a manure disposal problem since environmentally acceptable solutions are now required by government regulations. Swine manure was found to be a good source of supplementary nutrients to ferment wheat straw into single-cell protein (SCP) with Chaetomium cellulolyticum when 0.13g (NH₄)₂SO₄/g solid was used as an additional source of N. In batch fermentations, inhibitory effects, possibly due to soluble released from the straw during alkali or acid pretreatment, were overcome by starting the fermentation at about pH 7.0 and then reducing it to 5.0 during growth. An overall protein productivity of up to 66 mg/L h was obtained from a slurry mixture of 1% w/v solids of manure and straw. This compares favorably with 99 mg/L h when manure was fermented with glucose instead of straw as the main carbon source. A high protein productivity of 200 mg/L h was obtained from a slurry mixture containing anaerobically prefermented swine manure liquor and 1.5% w/v solids from straw. The final products of the manure and straw fermentations contained 25–30% DW crude protein and 6–20% DW cellulose and the materials were free of the original obnoxious odor and undesirable microbial contamination.     

Seedling growth of four fire-following Mediterranean plant species deprived of single mineral nutrients

1. Anthyllis vulneraria, Cistus creticus, Hippocrepis unisiliquosa and Pinus brutia are frequent post-fire colonizers whose seedlings are subjected to the mineral conditions imposed by ash in the early stages of growth. We test the hypothesis that the effective internal supply of mineral elements in the seeds of these species may complement the external availability of specific nutrients.
2. Newly germinated seedlings were grown in nutrient solutions, each deficient in one of the following elements: nitrogen, phosphorus or potassium. Control treatments consisted of full nutrients and distilled water. Seedling growth was monitored over 12weeks. The final dry masses of the seedlings were taken as a measure of the availability of each element from the seeds' own stored reserves.
3. For the two legume species nitrogen was the most limiting element followed by phosphorus. Potassium deficiency had no effect on the final legume dry biomass. For Cistus, the order was nitrogen followed by potassium, while phosphorus deprivation had no effect on growth during the 12weeks of the experiment. Deprivation of single minerals had no significant effect on seedling growth of Pinus over the same period. The two legumes and Cistus when denied nitrogen had relatively greater relative root ratios than control plants.
4. The relative seedling requirements of each species for early external supplies of the various nutrients for establishment in the field are discussed with reference to the adaptation of the four species to post-fire regeneration.     

CO Metabolism in the Acetogen Acetobacterium woodii

The Wood-Ljungdahl pathway allows acetogenic bacteria to grow on a number of one-carbon substrates, such as carbon dioxide, formate, methyl groups, or even carbon monoxide. Since carbon monoxide alone or in combination with hydrogen and carbon dioxide (synthesis gas) is an increasingly important feedstock for third-generation biotechnology, we studied CO metabolism in the model acetogen Acetobacterium woodii. When cells grew on H₂-CO₂, addition of 5 to 15% CO led to higher final optical densities, indicating the utilization of CO as a cosubstrate. However, the growth rate was decreased by the presence of small amounts of CO, which correlated with an inhibition of H₂ consumption. Experiments with resting cells revealed that the degree of inhibition of H₂ consumption was a function of the CO concentration. Since the hydrogen-dependent CO₂ reductase (HDCR) of A. woodii is known to be very sensitive to CO, we speculated that cells may be more tolerant toward CO when growing on formate, the product of the HDCR reaction. Indeed, addition of up to 25% CO did not influence growth rates on formate, while the final optical densities and the production of acetate increased. Higher concentrations (75 and 100%) led to a slight inhibition of growth and to decreasing rates of formate and CO consumption. Experiments with resting cells revealed that the HDCR is a site of CO inhibition. In contrast, A. woodii was not able to grow on CO as a sole carbon and energy source, and growth on fructose-CO or methanol-CO was not observed.     

Leaf traits, shoot growth and seed production in mature Fagus sylvatica trees after 8 years of CO2 enrichment

Background and Aims

Masting, i.e. synchronous but highly variable interannual seed production, is a strong sink for carbon and nutrients. It may, therefore, compete with vegetative growth. It is currently unknown whether increased atmospheric CO₂ concentrations will affect the carbon balance (or that of other nutrients) between reproduction and vegetative growth of forest species. In this study, reproduction and vegetative growth of shoots of mature beech (Fagus sylvatica) trees grown at ambient and elevated atmospheric CO₂ concentrations were quantified. It was hypothesized that within a shoot, fruiting has a negative effect on vegetative growth, and that this effect is ameliorated at increased CO₂ concentrations.

Methods

Reproduction and its competition with leaf and shoot production were examined during two masting events (in 2007 and 2009) in F. sylvatica trees that had been exposed to either ambient or elevated CO₂ concentrations (530 µmol mol⁻¹) for eight consecutive years, between 2000 and 2008.

Key Results

The number of leaves per shoot and the length of terminal shoots was smaller or shorter in the two masting years compared with the one non-masting year (2008) investigated, but they were unaffected by elevated CO₂ concentrations. The dry mass of terminal shoots was approx. 2-fold lower in the masting year (2007) than in the non-masting year in trees growing at ambient CO₂ concentrations, but this decline was not observed in trees exposed to elevated CO₂ concentrations. In both the CO₂ treatments, fruiting significantly decreased nitrogen concentration by 25 % in leaves and xylem tissue of 1- to 3-year-old branches in 2009.

Conclusions

Our findings indicate that there is competition for resources between reproduction and shoot growth. Elevated CO₂ concentrations reduced this competition, indicating effects on the balance of resource allocation between reproduction and vegetative growth in shoots with rising atmospheric CO₂ concentrations.     

Effect of mineral nutrients on cell growth and self-flocculation of Tolypothrix tenuis for the production of a biofertilizer

The influence of mineral nutrients on the growth and self-flocculation of Tolypothrix tenuis was studied. The identification of possible limiting nutrients in the culture medium was performed by the biomass elemental composition approach. A factorial experimental design was used in order to estimate the contribution of macronutrients and micronutrients, as well as their interactions. Iron was identified to be limiting in the culture medium. The micronutrients influenced mainly cellular growth without effects on self-flocculation. Conversely, the self-flocculation capacity of the biomass increased at higher concentrations of macronutrients. The optimization of mineral nutrition of T. tenuis allowed a 73% increase in the final biomass level and 3.5 times higher flocculation rates.     

Impact of nutritional supplements and monosaccharides on growth, oxalate accumulation, and culture pH by Sclerotinia sclerotiorum

Sclerotinia sclerotiorum D-E7 was studied to determine the impact of nutritional supplements and monosaccharides on growth, oxalate accumulation, and culture pH in broth media (initial pH c. 5). Cultures with 0.1% nutritional supplement (tryptone, yeast extract, or soytone) yielded minimal growth, 2-3 mM oxalate, and a final culture pH of 4.2-4.8. In contrast, cultures with 0.1% nutritional supplement and 25 mM glucose yielded significant growth, minimal oxalate (<1 mM), and a final culture pH of 2.8-3.7. Similar trends were observed when glucose in 0.1% soytone cultures was replaced with 25 mM d-mannose, l-arabinose, or d-xylose. With 1% soytone-25 mM glucose cultures, growth and oxalate accumulation ( approximately 21 mM) occurred with little change in initial pH. This was not the case with 1% soytone-250 mM glucose cultures; increased glucose levels resulted in a decrease in oxalate accumulation ( approximately 7 mM) and in final culture pH (3.4). Time-course studies with these cultures revealed that oxalate accumulation was suppressed during growth when the culture pH dropped to <4. Overall, these results indicate that (1) the decrease in external pH (i.e. acidification) was independent of oxalate accumulation and (2) acidification coupled to glucose-dependent growth regulated oxalate accumulation by Sclerotinia sclerotiorum.     

Response of terrestrial microorganisms to a simulated Martian environment

Soil samples from Cape Canaveral were subjected to a simulated Martian environment and assayed periodically over 45 days to determine the effect of various environmental parameters on bacterial populations. The simulated environment was based on the most recent available data, prior to the Viking spacecraft, describing Martian conditions and consisted of a pressure of 7 millibars, an atmosphere of 99.9% CO2 and 0.1% O2, a freeze-thaw cycle of -65 degrees C for 16 h and 24 degrees C for 8 h, and variable moisture and nutrients. Reduced pressure had a significant effect, reducing growth under these conditions. Slight variations in gaseous composition of the simulated atmosphere had negligible effect on growth. The freeze-thaw cycle did not inhibit growth but did result in a slower rate of decline after growth had occurred. Dry samples exhibited no change during the 45-day experiment, indicating that the simulated Martian environment was not toxic to bacterial populations. Psychotrophic organisms responded more favorably to this environment than mesophiles, although both types exhibited increases of approximately 3 logs in 7 to 14 days when moisture and nutrients were available.     

Response of terrestrial microorganisms to a simulated Martian environment.

Soil samples from Cape Canaveral were subjected to a simulated Martian environment and assayed periodically over 45 days to determine the effect of various environmental parameters on bacterial populations. The simulated environment was based on the most recent available data, prior to the Viking spacecraft, describing Martian conditions and consisted of a pressure of 7 millibars, an atmosphere of 99.9% CO2 and 0.1% O2, a freeze-thaw cycle of -65 degrees C for 16 h and 24 degrees C for 8 h, and variable moisture and nutrients. Reduced pressure had a significant effect, reducing growth under these conditions. Slight variations in gaseous composition of the simulated atmosphere had negligible effect on growth. The freeze-thaw cycle did not inhibit growth but did result in a slower rate of decline after growth had occurred. Dry samples exhibited no change during the 45-day experiment, indicating that the simulated Martian environment was not toxic to bacterial populations. Psychotrophic organisms responded more favorably to this environment than mesophiles, although both types exhibited increases of approximately 3 logs in 7 to 14 days when moisture and nutrients were available.     

High cell density and high monoclonal antibody production through medium design and rational control in a bioreactor

A simple feeding strategy was developed and successfully employed for nutritional control in a 2-L fed-batch culture of hybridoma cells. A previously developed stoichiometric model for animal cell growth was used to design a supplemental medium for feeding. Undialyzed fetal bovine serum and trace metals (Fe(2+), SeO(3) (2-), Li(+), Zn(2+), and Cu(2+)) were fed to the cells periodically in addition to the automatic feeding of other nutrients in the supplemental medium. In this study, the maximum viable cell density was increased from 6.3 x 10(6) to 1.7 x 10(7) cells/mL, and the culture span was extended from 340 to 550 hours. The final monoclonal antibody titer achieved was 2400 mg/L. The specific production rates for ammonia and lactate were further reduced from 0.0045 and 0.0048 in our previous fed-batch experiments to 0.0028 and 0.0036 mmol/10(9) cell h, respectively. Only 3.4% of the total glucose consumption was converted into lactate, compared to 67% in a conventional batch culture.     

The Effect of Film Permeability on the Storage Life and Microbiology of Vacuum-packed meat

Joints of beef were stored in packaging films with oxygen permeabilities ranging from 0–920 ml/m²24 h/atm at 25 °C and 100% r.h. The storage life of the 'vacuum-packaged' meat, as assessed by discolouration and the development of putrefactive odours, was inversely related to film permeability; the best results were obtained with meat which received 'zero oxygen' treatment. The growth rates and final counts of Pseudomonas spp. increased with increasing film permeability; the storage life of the meat corresponded with the time taken for the counts of the organism to reach ca. 10⁶/cm² for putrefactive odours to be produced. Although their growth rate was unaffected, the final counts of Brochothrix thermosphacta also increased with increasing film permeability. These results are discussed in terms of the influence of film permeability on the inhibitory effects of Lactobacillus spp., whose numbers were unaffected by the permeability of the film used, and the substrates in the meat available to the bacteria.     

pH-controlled cell release and biomass distribution of alginate-immobilized Lactococcus lactis subsp. lactis

AIMS: To investigate the growth and release of Lactococcus lactis subsp. lactis in gel beads and to affect rates of cell release by changing the growth conditions. METHODS AND RESULTS: The rate of release and the distribution of immobilized L. lactis subsp. lactis in alginate beads were studied in continuous fermentations for 48 h. A change in operating pH from 6.5 to 9.25 initially reduced the ratio of the rates of cell release to lactate production by almost a factor of 105. Compared with fermentations at pH 6.5, growth at pH 9.25 also increased the final internal bead biomass concentration by a factor of 5 and increased the final rate of lactate production by 25%. After 48 h, the ratio of the rates of cell release to lactate production was still 10 times lower than in fermentations at pH 6.5. CONCLUSIONS: A change in the operating pH from 6.5 to 9.25 reduced rates of cell release throughout 48 h of fermentation and increased the final rates of lactate production and internal bead biomass concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: These data illustrate that diffusional limitations and corresponding pH gradients can be exploited in affecting the distribution of immobilized growing cells and their concomitant release.