A Suspension Culture Method for the Rapid Mass Culture of Cistella japonica Mycelium

Different methods were investigated for the rapid mass culture of Cistella japonica by using water extracts of some nutritional sources. In an agar culture test, there was little difference in mycelial growth in water extracts of wheat bran, rice bran and potato. In suspension culture with wheat bran extract, which is easily and cheaply available, the mycelium of C. japonica increased seven times more than that in agar culture after a month's incubation. C. japonica from suspension culture was pathogenic to Chamaecyparis obtusa. These results suggest that suspension culture in water extract of wheat bran can be adopted for the rapid mass culturing of C. japonica for use in inoculation tests.     

Comparison of culture methods on exopolysaccharide production in the submerged culture of Cordyceps militaris and process optimization

Aims: To improve exopolysaccharides (EPS) production of Cordyceps militaris (C. militaris), effects of different culture method on mycelial biomass and EPS production in the submerged culture of C. militaris were investigated. Methods and Results: A new two‐stage fermentation process for EPS production of C. militaris was designed in this work. Central composite design (CCD) was utilized to optimize the two‐stage fermentation process. The results showed that the two‐stage fermentation process for EPS production was superior to other culture method (conventional static culture and shake culture). CCD revealed that the optimum values of the test variables for EPS production were shaken for 140 h followed by 130‐h static culture. The maximum EPS production reached 3·2 g l^?1 under optimized two‐stage culture and was about 2·3‐fold and 1·6‐fold in comparison with those of original static culture and shake culture. Conclusions: It was indicated that a new two‐stage culture method obtained in this work possessed a high potential for the industrial production for EPS of C. militaris. Significance and Impact of the Study: The fundamental information obtained in this work is complementary to those of previous investigations on the submerged culture of C. militaris for the production of bioactive metabolites.     

Experiences with the on-line measurement of culture fluorescence during cultivation of Bacillus subtilis, Escherichia coli and Sporotrichum thermophile

Bacillus subtilis and Escherichia coli K12 (both transformed for human leukocyte interferon production) and Escherichia coli B/r and Sporotrichum thermophile (a deuteromycete) were cultivated in submersed culture and the culture fluorescence recorded on-line using a fluorometer. During the cultivation of B. subtilis the signal from the fluorometer correlated with cell density and interferon production and thus could be used for process control (interferon production). However, the culture fluorescence of the other organisms did not increase (S. thermophile), was too weak to be measured with the fluorometer used (E. coli transformed for interferon production), or the signal from the fluorometer was not an accurate measure of the culture fluorescence because of the accumulation of a fluorophor in the culture medium (E. coli B/r).     

Production of seedlings from ovules excised at the zygote stage in Lilium spp.

The present study was undertaken to establish a culture system for ovules excised at the zygote stage in Lilium spp. Ovules of Lilium × `Connecticut King' and L. × `Enchantment' were excised together with placental tissue 3, 5, and 10 days after pollination (DAP) and cultured on B5 medium and half-strength B5 medium containing sucrose at different concentrations. In vitro embryo development in ovules cultured at 3 DAP was influenced by the basal media and the sucrose concentration. The half-strength B5 medium with 9% sucrose was the best condition, but only a few ovules isolated from placental tissue developed into seedlings. Application of embryo culture, in which embryos were excised from ovules after 14 weeks of ovule-with-plancetal-tissue culture, greatly improved the production of seedlings. The present study indicates that a two-step culture procedure, ovule-with-placental-tissue culture and embryo culture, make it possible to produce seedlings from ovules just after fertilization.     

Synergism of cellulase enzymes in mixed culture solid substrate fermentation

Sugar cane bagasse was subjected to a mixed culture, solid substrate fermentation with Trichoderma reesei QM9414 and Aspergillus terreus SUK-1 to produce cellulase and reducing sugars. The highest cellulase activity and reducing sugar amount were obtained in mixed culture. The percentage of substrate degradation achieved employing mixed culture was 26% compared to 50% using separate cultures of the two molds. This suggests that the synergism of enzymes in mixed culture solid substrate fermentation have lower synergism than in pure culture.     

Improved growth and acclimatization of somatic embryo-derived Oplopanax elatus plantlets by ventilated photoautotrophic culture

To improve large-scale in vitro production of Oplopanax elatus Nakai, we cultured somatic embryo-derived plantlets under a heterotrophic condition (semi-solid culture with sucrose), photoautotrophic condition (semi-solid culture without sucrose), or modified photoautotrophic condition (liquid culture with forced ventilation). The plantlets grown under the modified photoautotrophic condition had more leaves as well as higher chlorophyll content, and higher net photosynthetic rate than those grown under the conventional conditions. Further, the photoautotrophically grown plantlets acclimatized better and sooner upon ex vitro transplantation than did the conventionally cultured plantlets. Consequently, a photoautotrophic culture method with forced ventilation is effective for enhancing the growth and acclimatization of O. elatus.     

The Utility of <Emphasis Type="Italic">Coccidioides</Emphasis> Polymerase Chain Reaction Testing in the Clinical Setting

Coccidioidomycosis, the fungal infection caused by dimorphic Coccidioides sp., is typically diagnosed by histopathologic identification of spherules in affected secretions and tissues or by culture. These tests are reliable but time-intensive, delaying diagnosis and treatment. To evaluate a polymerase chain reaction (PCR) test developed to detect Coccidioides sp. in clinical specimens, we conducted a retrospective chart review of all patients (N = 145) who underwent Coccidioides PCR at our institution between April 27, 2007, and May 6, 2008, abstracting clinical, microbiologic, serologic, radiographic, treatment, and follow-up data. One hundred fifty-eight PCR tests (153 respiratory; 5 cerebrospinal fluid) produced 5 positive and 153 negative findings. Five of nine patients (56%) with confirmed or highly probable pulmonary coccidioidomycosis had a positive PCR on respiratory specimens, and four of nine (44%) had a positive culture. Among two patients with coccidioidal meningitis, none had a positive PCR, whereas Coccidioides sp. in fungal culture grew for one of two. Among six asymptomatic patients with probable coccidioidomycosis, none had a positive culture or PCR. Compared with culture of respiratory specimens, PCR demonstrated a sensitivity, specificity, positive predictive value, and negative predictive value of 75, 99, 60, and 99%, respectively. Coccidioides PCR appears accurate in identifying negative results, and its sensitivity is similar to that of fungal culture.     

High photosynthetic photon flux and high CO2 concentration under increased number of air exchanges promote growth and photosynthesis of four kinds of orchid plantlets in vitro

Summary In vitro plantlets of Phalaenopsis ‘Happy Valentine’, Neofinetia falcate Hu, Cymbidium kanran Makino, and Cymbidium goeringii Reichb. f. were grown under photoautotrophic [high photosynthetic photon flux (PPF), high CO₂ concentration, and increased number of air exchanges] and heterotrophic (low PPF, low CO₂ concentration, no air exchanges) culture conditions. After 40 d of culture, a significant difference in plantlet growth was observed between the two cultures. Total fresh and dry mass were on average 1.5 times greater in photoautotrophic culture than in heterotrophic culture. Higher net photosynthetic rates were also observed for Phalaenopsis in photoautotrophic culture. In photoautotrophic culture, little difference was observed in air temperature between the inside and outside of the culture vessel, whereas in heterotrophic culture, air temperature inside the culture vessel was 1–2°C higher than that outside the culture vessel. Relative humidity inside the culture vessel was remarkably different between the two cultures: 83–85% in photoautotrophic culture and 97–99% in heterotrophic culture. These results indicated that growth and net photosynthetic rate of in vitro orchid plantlets were susceptible to the culture environments such as PPF, CO₂ concentration, relative humidity (RH), and the number of air exchanges, which would allow a more efficient micropropagation system for these orchid plants.     

Growth of Nitrosomonas europaea in batch and in continuous culture

Summary Techniques are described in this paper for growing Nitrosomonas europea in batch and in continuous culture with apparatus constructed from readily available laboratory materials.The methods employed in batch culture have enabled the collection of cells concentrated in small volumes. Nitrosomonas europaea has been grown successfully in continuous culture and yields of 23 g wet weight were obtained, an average of 0.16 g/l with a flow rate of 300 ml/h over twenty days. Results show that high levels of nitrite do not materially affect the growth of the bacterium. The continuous culture equipment has also been used to grow Azotobacter vinelandii and Thiobacillus concretivorus.     

The effect of dynamic light regimes on Chlorella II. Minimum quantum requirement and photosynthesis-irradiance parameters

Comparisons were made of photosynthesis in three light limited cyclostat cultures (LD = 8:16, dilution rate 0.7 d^–1) of Chlorella pyrenoidosa, differing only in the dynamics of irradiance supply: as a constant rate, i.e. a block culture; as a sine function of the light period, i.e. a sinusoidal culture; as an 8 h sine function superimposed by an 1 h sine function, i.e. an oscillating culture. The sinusoidal culture had a constant minimum quantum requirement for oxygen evolution (QR) of 10.8 over the photoperiod. The OR of the oscillating culture increased from 24 to 37 during the photoperiod. From changes in and P _max we suggest that: (1) photosynthetic units (PSU) of the block and sinusoidal sulture increased in number; (2) increasingly fewer chlorophyll molecules participated in oxygenic photosynthesis with a decreasing turnover time of the PSU's during an oscillating photoperiod. Values of I _k decreased slightly in the block culture, increased slightly in the sinusoidal culture and showed a twofold increase in the oscillating culture. From the ratio of in situ oxygen production (qO₂) and P _max we infer a balanced equilibrium between photosystem activity and electron transport capacity for the block and sinusoidal culture. We hypothesize that the qO₂ values of the oscillating culture underestimated true oxygen production rates due to a nonlinear response at peak light intensities. The results show that a dynamical photoperiod provoked significantly different photosynthetic responses, even though the overall growth rate was unaffected.     

Inhibition of phagocytic activity and nodulation in Galleria mellonella by the entomopathogenic fungus Nomuraea rileyi

Changes in phagocytic activity and nodulation in the greater wax moth, Galleria mellonella (L.) (Lepidoptera: Pyralidae), were examined after treatment with the culture fluid of the entomopathogenic fungus Nomuraea rileyi SH1. When isolated hemocytes of G. mellonella were incubated with the conidia of N. rileyi in vitro, the rates of phagocytosis increased at 4 h after incubation but decreased subsequently. On the contrary, the rates of phagocytosis of isolated hemocytes decreased by 80% after 24 h preincubation with the fungal culture fluid (1/200, 1/100 dilutions). Levels of inhibition of phagocytic activity by the culture fluid depended on dilutions used. Galleria mellonella larvae showed a peak of nodulation at 4 h after injection with conidia. The percentage of nodules in hemolymph did not decrease by preinjection with the culture fluid, whereas the percentage of nodule‐containing conidia decreased, depending on the injected fluid. However, phagocytosis and nodulation in G. mellonella did not change after treatment of the culture fluid with proteinase K, indicating that the culture fluid contained proteinaceous immunosuppressive factors. Electrophoretic analysis of the culture fluid and not the fresh medium without culturing the fungus exhibited protein bands. Therefore, N. rileyi possibly secretes toxic proteins that impair cellular immune responses in G. mellonella larvae.     

Biodegradation of polyethylene glycol by symbiotic mixed culture (obligate mutualism)

Neither Flavobacterium sp. nor Pseudomonas sp. grew on a polyethylene glycol (PEG) 6000 medium containing the culture filtrate of their mixed culture on PEG 6000. The two bacteria did not grow with a dialysis culture on a PEG 6000 medium. Flavobacterium sp. grew well on a dialysis culture containing a tetraethylene glycol medium supplemented with a small amount of PEG 6000 as an inducer, while poor growth of Pseudomonas sp. was observed. Three enzymes involved in the metabolism of PEG, PEG dehydrogenase, PEG-aldehyde dehydrogenase and PEG-carboxylate dehydrogenase (ether-cleaving) were present in the cells of Flavobacterium sp. The first two enzymes were not found in the cells of Pseudomonas sp. PEG 6000 was degraded neither by intact cells of Flavobacterium sp. nor by those of Pseudomonas sp., but it was degraded by their mixture. Glyoxylate, a metabolite liberated by the ether-cleaving enzyme, inhibited the growth of the mixed culture. The ether-cleaving enzyme was remarkably inhibited by glyoxylate. Glyoxylate was metabolized faster by Pseudomonas sp. than by Flavobacterium sp., and seemed to be a key material for the symbiosis.     

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in continuous cultures

The oxidation and growth kinetics of ferrous iron with Thiobacillus ferrooxidans in continuous cultures was examined at several total iron concentrations. On-line off-gas analyses of O₂ and CO₂ were used to measure the oxygen and carbon dioxide consumption rates in the culture. Off-line respiration measurements in a biological oxygen monitor (BOM) were used to measure directly the maximum specific oxygen consumption rate, qO₂,max, of cells grown in continuous culture. It was shown that these reproducibly measured values of qO₂,max vary with the dilution rate. The biomass-specific oxygen consumption rate, qO₂, is dependent on the ratio of the ferric and ferrous iron concentrations in the culture. The oxidation kinetics was accurately described with a rate equation for competitive ferric iron inhibition, using the value of qO₂,max measured in the BOM. Accordingly, only the kinetic constant Ks/K _i needed to be fitted from the measurements. A new method was introduced to determine the steady-state kinetics of a cell suspension in a batch culture that only takes a few hours. The batch culture was set up by terminating the feeding of a continuous culture at its steady state. The kinetic constant K _s/K _i determined in this batch culture agreed with the value determined in continuous cultures at various steady states. Received: 8 February 1999 / Accepted: 17 February 1999     

Properties of ras-amplified recombinant BHK-21 cells in protein-free culture

We compared serum and protein-free cultures ofa ras-amplified recombinant BHK-21 cell line(ras-rBHK-IgG), which hyperproduces a lungcancer specific recombinant human monoclonal antibody. Ras-rBHK-IgG cells were shown to grow well, evenin protein-free medium and to be morphologicallysimilar to cells cultured in serum containing medium. However, the growth rate of ras-rBHK-IgG cellswas considerably slower in protein-free medium, whichresults in a longer maintenance period compared with cells cultured in serum containing medium. In addition, it was found that antibody production in protein-free culture had a ten times higher maximum than cells cultured in serum containing medium. On theother hand, in high density culture, using the hollowfiber bioreactor system, ras-rBHK-IgG cellscould be maintained for a month in protein-freeculture in contrast with serum culture, which onlylasted for half a month. However, the markedincrease of antibody production was not observed. A total amount of about 15 mg of the recombinantantibody, obtained in protein-free culture, was abouttwo times of that obtained in serum culture, and wasshown to be reactive to lung cancer cells in tissue. From these properties in protein-free medium, it isconcluded that protein-free culture of ras-rBHK-IgG cells is suitable for middle scaleproduction of recombinant human monoclonal antibody.     

Optimization of conditions for the production of pullulan and high molecular weight pullulan by Aureobasidium pullulans

Aureobasidium pullulans had a maximum yield coefficient of pullulan (Y _p/s=0.24) with an initial pH of the culture broth of 6.5 in a shake-flask culture. In a batch culture, the maximum pullulan yield coefficient of 0.30 was obtained at the aeration rate of 0.5 vvm. A yeast-like form and mycelial form of cells were found at the culture broth with pH controlled at 4.5 with a maximum yield coefficient of pullulan of 0.27. However, a high portion (35%) of high molecular weight pullulan (M _w>2 000 000) was produced at pH 6.5 with a yeast-like morphology of the cells.     

1,3‐propanediol production from glucose by mixed‐culture fermentation of Zygosacharomyces rouxii and Klebsiella pneumonia

1,3‐propanediol is an important chemical widely used in polymer production. In this study, two strains, Zygosacharomyces rouxii JL2011 and Klebsiella pneumoniae S6, were used as a mixed culture for 1,3‐propanediol production directly from glucose. Two important parameters including inoculation time of K. pneumoniae S6 at stage of mixed culture and initial cell ratio of Z. rouxii JL2011 to K. pneumoniae S6 in mixed fermentation were optimized in culture flasks. In those experiments, the best results were obtained with a yield of 6.8 g/L 1,3‐propanediol from glucose when K. pneumoniae S6 was inoculated after 48 h in the culture of Z. rouxii JL2011 by mixed culture of Z. rouxii JL2011 and K. pneumoniae S6 with initial cell ratio of 1:200. In a 7‐L bioreactor, the maximum 1,3‐propanediol production could reach up to 15.2 g/L. Thus, this study presents an effective process for 1,3‐propanediol microbial production from glucose by using mixed culture of Z. rouxii JL2011 and K. pneumoniae S6. This work does not only demonstrate a new way to produce 1,3‐propanediol from a low‐cost feedstock, but may also make a valuable contribution to the development of a cost‐effective fermentation based on renewable resources.     

On-line fluorescence measurements in assessing culture metabolic activities

Summary NADH fluorescence aided by a stoichiometric metabolic pathway model and culture dynamics was used to elucidate the unobservable intracellular physiological state in two metabolically different phases during culture of Clostridium acetobutylicum. The validity of the theoretical model was examined over a range of culture pH regimes and initial sugar concentrations. The H₂/CO₂ gas concentration ratio was found to be an important process parameter. NADH fluorescence detection was compared with simultaneous enzymatic measurements. The specific fluorescence (fluorescence per biomass, F/X) provided a distinction between oxidative and reductive culture metabolism independent of the pH or substrate concentration changes. A good indicator of the type of culture activity proved to be the dF/dt parameter. The net fluorescence measurements correlated with butanol accumulation under all growth conditions suggesting the possible use of the fluorescence probe as a butanol probe in this fermentation.     

Effect of simple and complex carbon sources,low temperature culture and complex carbon feeding policies on poly-3-hydroxybutyric acid (PHB) content and molecular weight (Mw) from Azotobacter chroococcum 6B

The molecular weight (M _w) of poly-3-hydroxybutyrate (PHB), produced by shake-flask culture of Azotobacter chroococcum showed little variation with increasing glucose concentration as carbon source (being in the range of 400–500 kDa), while M _w increased from 300–400 to 640 kDa when grown with increasing concentration of sugar cane molasses. Molecular weight increased nearly 30% from 48 to 72 h culture time when 5% molasses as carbon source was used, while with glucose the highest M _w was reached at 48 h. Under fermentor cultivation A. chroococcum produced PHB with a relatively high M _w of 1590 kDa at 53 h culture time when grown in modified Burk's medium with glucose as carbon source at an initial C/N ratio (molar basis) of 69 under fermentor cultivation. A batch glucose-grown ammonium-limited fermentor culture was repeatedly fed with sugar cane molasses (initial C/N ratio 69) and it was observed that PHB content curve decreased at a slower rate than in the fed-batch culture in which glucose and sucrose were not consumed in the culture medium after the feed.     

An Extracellular Protein of Propionic Acid Bacteria Inhibits Induced Mutations in Salmonella typhimurium Strains

A culture of propionic acid bacteria grown in a glucose-containing minimal medium, as well as the culture liquid and logarithmic-phase cells obtained from this culture, were found to inhibit the base pair substitution mutations induced by 4-nitroquinoline N-oxide, N-methyl-N"-nitro-N-nitrosoguanidine, and sodium azide and the frameshift mutations induced by 9-aminoacridine. The antimutagenic activity of the culture liquid (CL) was presumably due to the presence of an extracellular thermolabile protein with a molecular mass of no more than 12 kDa, as evidenced by the facts that this activity considerably decreased after the treatment of the CL with pronase, its heating at 92°C, and its dialysis in a cellulose sack, which retains substances with molecular masses greater than 12 kDa. The residual antimutagenic activity of the dialyzed culture liquid was probably related to the interaction of the mutagen with thiols, rather than to the presence of organic acids (acetic or propionic). Thiols may also contribute to the antimutagenic activity of the Propionibacterium shermanii cells.     

A Comparison of Pathogen Isolation in Culture and Injection–infiltration Bioassay of Citrus Leaves for Detecting Xanthomonas citri subsp. citri

Citrus canker [caused by Xanthomonas citri subsp. citri (Xcc)] can cause yield loss of susceptible citrus and result in trade restrictions of fresh fruit. For both regulatory purposes and epidemiological studies, accurate detection and quantification of viable inoculum are critical. Two accepted methods used to detect and quantify Xcc are injection–infiltration bioassay and culture, but these two methods have not been directly compared using field‐obtained samples. The two methods were compared using washates of lesions taken from fruit, leaves and shoots in a commercial orchard in Florida in 2009–2010 and 2010–2011, with bioassay being the assumed standard. Despite some misclassifications, true positives (sensitivity) and true negatives (specificity) were the dominant classes using culture. False positives for lesions from shoots ranged from 13.1 to 21.4% in 2009–2010 and 2010–2011, respectively, and false positives for lesions from fruit and leaves ranged from 4.3 to 15.7%, in the two seasons, respectively. The false positive rate for culture compared with injection–infiltration bioassay was highest (0.16–0.55), due to more frequent recovery of Xcc by culture at ≤10³ colony‐forming units (CFU) Xcc per ml. The false negative rate was consistently lower (0.02–0.21), confirming that in only a few cases did culture fail to detect Xcc when it was present. The area under the curve for receiver operator characteristic analysis ranged from 0.80 to 0.97, confirming that culture provided an accurate diagnosis in most cases. There was a higher frequency of lesions from shoots with a CFU ≤10³ Xcc compared with lesions from fruit or leaves, making culture more effective at detecting these. The data demonstrate that culture is a reliable way to detect and quantify Xcc compared with injection–infiltration bioassay, particularly when the CFU is ≤10³ Xcc per ml.