Cultivation of Sponge Larvae: Settlement,Survival, and Growth of Juveniles

The aim of this study was to culture sponge juveniles from larvae. Starting from larvae we expected to enhance the survival and growth, and to decrease the variation in these parameters during the sponge cultures. First, settlement success, morphological changes during metamorphosis, and survival of Dysidea avara, Ircinia oros, Hippospongia communis, under the same culture conditions, were compared. In a second step, we tested the effects of flow and food on survival and growth of juveniles from Dysidea avara and Crambe crambe. Finally, in a third experiment, we monitored survival and growth of juveniles of D. avara and C. crambe transplanted to the sea to compare laboratory and field results. The results altogether indicated that sponge culture from larvae is a promising method for sponge supply and that laboratory culture under controlled conditions is preferred over sea cultures in order to prevent biomass losses during these early life stages.     

Correlation of rRNA gene amplicon pyrosequencing and bacterial culture for microbial compositional analysis of faecal samples from elderly Irish subjects

Aims: The aim of this investigation was to establish the degree of correlation between measurements from culture‐dependent microbiological techniques and from next generation sequencing technologies. Methods and Results: Data generated by both techniques were collected from faecal samples from 185 elderly Irish people involved in the ongoing ELDERMET study ( http://eldermet.ucc.ie ). The results for three groups of intestinal bacteria were compared. Bifidobacterium sp., Lactobacillus sp. and Enterobacteriaceae were enumerated on selective media through culture‐dependent techniques, whereas proportions of these bacteria were determined through sequencing technology against the background of other bacteria. The Spearman’s rank correlation coefficient determined a good correlation between results from culture‐dependent microbiology and culture‐independent techniques for all three bacterial groups assessed (correlation coefficients for Bifidobacterium sp., Lactobacillus sp. and Enterobacteriaceae were 0·380, 0·366 and 0·437, respectively). Conclusion: Correlation between the two methods implies that a single method is capable of profiling intestinal Bifidobacterium, Lactobacillus and Enterobacteriaceae populations. However, both methods have advantages that justify their use in tandem. Significance and Impact of the Study: This is the first extensive study to compare bacterial counts from culture‐dependent microbiological techniques and from next generation sequencing technologies.     

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.     

Removal of ABS from solutions by a common fungus of sewage

Conclusions Phialophora jeanselmei (Margarinomyces heteromorphum) is very common in sewage treatment systems, and can remove ABS from solution even in the absence of glucose. Because glucose is present in sewage treatment systems either as a pollutant or as a byproduct of the metabolism of other organisms, it appears that, to some extent, ABS is removed by this organism. Additional work is needed to determine whether this organism can be made a nucleus of an artificial population deliberately established to act upon some special types of wastes; whether a mononucleate-monospore culture capable of more rapid and more complete removal of ABS can be developed; and what pathways of metabolism and decomposition of ABS might be involved with the idea of breaking into the chains of reaction with other organisms.In the presence of added glucose,Phialophora jeanselmei was found to be capable of removing ABS from shaken and still culture solutions containing an ABS concentration of 10 ppm. More adsorption on the mycelium took place in still culture than in aerated culture. In shaken culture a greater loss of ABS from solution resulted; this may be due to metabolic processes since the ABS was neither found in solution nor adsorbed on the mycelium. While wild cultures are capable of a demonstrable amount of activity, culture efficiency could be improved if selected monospore-mononucleate cultures were developed.     

应用原位双膜法快速筛选人Flt3配体甲醇酵母高表达转化子

原位双膜法是一种基于免疫原理的快速筛选高表达甲醇酵母转化子的方法,即首先将固体培养基上的菌落转印至醋酸纤维素薄膜上,再利用硝酸纤维素薄膜原位捕获穿过醋酸纤维素薄膜的菌落外泌蛋白,然后用免疫方法检测与硝酸纤维素薄膜结合的蛋白.利用此法筛选到人Flt3配体（hFL）的甲醇酵母高表达转化子,液体诱导表达量约20 mg/L.ELISA结果证明,原位双膜法所得的菌落染色强度与该菌落液体诱导表达水平正相关.蛋白质印迹结果显示,培养上清在25 ku处有明显杂交条带,而对照组杂交呈阴性,且表达量随诱导天数增加.原位双膜法是一种良好的筛选方法,可以快捷、准确地筛选高表达酵母转化子.     

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.     

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.     

Isolation and characterisation of an equol-producing mixed microbial culture from a human faecal sample and its activity under gastrointestinal conditions

Only about one third of humans possess a microbiota capable of transforming the dietary isoflavone daidzein into equol. Little is known about the dietary and physiological factors determining this ecological feature. In this study, the in vitro metabolism of daidzein by faecal samples from four human individuals was investigated. One culture produced the metabolites dihydrodaidzein and O-desmethylangolensin, another produced dihydrodaidzein and equol. From the latter, a stable and transferable mixed culture transforming daidzein into equol was obtained. Molecular fingerprinting analysis (denaturing gradient gel electrophoresis) showed the presence of four bacterial species of which only the first three strains could be brought into pure culture. These strains were identified as Lactobacillus mucosae EPI2, Enterococcus faecium EPI1 and Finegoldia magna EPI3, and did not produce equol in pure culture. The fourth species was tentatively identified as Veillonella sp strain EP. It was found that hydrogen gas in particular, but also butyrate and propionate, which are all colonic fermentation products from poorly digestible carbohydrates, stimulated equol production by the mixed culture. However, when fructo-oligosaccharides were added, equol production was inhibited. Furthermore, the equol-producing capacity of the isolated culture was maintained upon its addition to a faecal culture originating from a non-equol-producing individual.     

The antimicrobial activity of Aspergillus fumigatus is enhanced by a pool of bacteria

In a screening program for new antibiotic producers, a strain of Aspergillus fumigatus was isolated from Brazilian soil samples. A pool of autoclaved bacteria was added to part of the fungus culture on the second day of fermentation to increase antibiotic production. The chloroform extract from the culture broth to which the pool of autoclaved bacteria was added showed an increase of 55%, 63% and more than 100% in activity against Staphylococcus aureus, Candida albicans and Micrococcus luteus, respectively. Also, the HPLC chromatographic profiles of the chloroform extracts from both culture conditions were different. Two active compounds were isolated from the broth of the culture grown in the presence of pooled bacteria and were identified as 3,4-dimethoxyphenol and 1,3,5-trimethoxybenzene.     

In vitro culture of marine trematodes from their snail first intermediate host

The ability to culture parasites outside their host (i.e. in vitro) is essential for several aspects of parasitological research. Here, a culture medium for marine trematode parthenitae was optimized using Philophthalmus sp. rediae from the intermediate snail host, Zeacumantus subcarinatus. The medium was optimized by sequentially testing the suitability of different levels of osmolality, different commercially available media, and different concentrations of supplemented chicken serum, while controlling for genetic variation among cultures. Philophthalmus sp. rediae survived up to 56 days in cultures of the best tested medium, remaining active and continuously shedding cercariae. The broader suitability of the culture medium was tested using five other trematode species from different families (using either the same or other marine snails as first intermediate hosts): Galactosomum sp., Acanthoparyphium sp., Maritrema novaezealandensis, Curtuteria australis, and an undescribed species of the family Opecoelidae. Survivorship of rediae and sporocysts from these species ranged from eight days to 42 days. The culture procedures developed here can therefore be used in the future as a system under which to culture marine trematode parthenitae for experimental studies.     

Growth characteristics of the cyanobacterium Nostoc flagelliforme in photoautotrophic, mixotrophic and heterotrophic cultivation

Nostoc flagelliforme is a terrestrial cyanobacterium with high economic value. Dissociated cells separated from a natural colony of N. flagelliforme were cultivated for 7 days under either phototrophic, mixotrophic or heterotrophic culture conditions. The highest biomass, 1.67 g L⁻¹ cell concentration, was obtained under mixotrophic culture, representing 4.98 and 2.28 times the biomass obtained in phototrophic and heterotrophic cultures, respectively. The biomass in mixotrophic culture was not the sum as that in photoautotrophic and heterotrophic cultures. During the first 4 days of culture, the cell concentration in mixotrophic culture was lower than the sum of those in photoautotrophic and heterotrophic cultures. However, from the 5th day, the cell concentration in mixotrophic culture surpassed the sum of those obtained from the other two trophic modes. Although the inhibitor of photosynthetic electron transport DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] efficiently inhibited autotrophic growth of N. flagelliforme cells, under mixotrophic culture they could grow by using glucose. The addition of glucose changed the response of N.flagelliforme cells to light. The maximal photosynthetic rate, dark respiration rate and light compensation point in mixotrophic culture were higher than those in photoautotrophic cultures. These results suggest that photoautotrophic (photosynthesis) and heterotrophic (oxidative metabolism of glucose) growth interact in mixotrophic growth of N. flagelliforme cells.     

A tissue culture system for different germplasms of indica rice

Agrobacterium-mediated transformation of indica rice has been manipulated in only a limited number of cultivars because the majority of indica varieties are recalcitrant to in vitro response. Establishment of a highly efficient and widely used tissue culture system for indica rice will accelerate the application of transformation technology in breeding programs and the study of the functions of indica-specific genes. By manipulating plant growth regulators, organic components and salts within the culture media, we established two media for callus induction and subculture, respectively, in tissue culture of indica rice. The modified media could guarantee the production and proliferation of a great number of embryogenic calli with high regeneration capacity from mature seeds representing different indica rice germplasms. The calli obtained from this system should be ideal material for Agrobacterium-mediated transformation. The results suggest that this optimized tissue culture system will be widely applicable for the tissue culture of indica varieties. Electronic Supplementary Material Supplementary material is available for this article at The first two authors contributed equally to this work.     

Production of phytoestrogen <Emphasis Type="Italic">S</Emphasis>-equol from daidzein in mixed culture of two anaerobic bacteria

An anaerobic incubation mixture of two bacterial strains Eggerthella sp. Julong 732 and Lactobacillus sp. Niu-O16, which have been known to transform dihydrodaidzein to S-equol and daidzein to dihydrodaidzein respectively, produced S-equol from daidzein through dihydrodaidzein. The biotransformation kinetics of daidzein by the mixed cultures showed that the production of S-equol from daidzein was significantly enhanced, as compared to the production of S-equol from dihydrodaidzein by Eggerthella sp. Julong 732 alone. The substrate daidzein in the mixed culture was almost completely converted to S-equol in 24 h of anaerobic incubation. The increased production of S-equol from daidzein by the mixed culture is likely related to the increased bacterial numbers of Eggerthella sp. Julong 732. In the mixture cultures, the growth of Eggerthella sp. Julong 732 was significantly increased while the growth of Lactobacillus sp. Niu-O16 was suppressed as compared to either the single culture of Eggerthella sp. Julong 732 or Lactobacillus sp. Niu-O16. This is the first report in which two metabolic pathways to produce S-equol from daidzein by a mixed culture of bacteria isolated from human and bovine intestinal environments were successfully linked under anaerobic conditions.     

Proliferation and differentiation of primary bovine myoblasts using Chlorella vulgaris extract for sustainable production of cultured meat

Recently, cultured meat obtained from livestock-derived cells is being considered as a sustainable food source that reduces the use of natural resources. This study aimed to show that nutrients extracted from Chlorella vulgaris were beneficial in the culture of primary bovine myoblasts (PBMs), a major cell source for cultured meat production. Nutrients (glucose, amino acids, and vitamins) present in the animal-cell culture media were effectively recovered from C. vulgaris using acid hydrolysis treatment. On culture in nutrient-free inorganic salt solution, cell death was induced in most PBMs after 6 days of cultivation. However, the addition of C. vulgaris extract (CVE) significantly improved PBM viability, which was comparable to the viability in conventional culture medium (Dulbecco's modified Eagle's medium). Furthermore, by adding horse serum to induce differentiation, the formation of myotubes was confirmed when CVE were used. Together, the results showed that CVE could be used as an alternative to the conventional culture medium for PBMs. These findings will not only lower the environmental risks associated with the establishment of this eco-friendly cell culture system, but also highlight microalgae as a potent nutrient source that can replace conventional grain-dependent nutrient sources.     

Somatic chromosome number doubling of selected potato genotypes using callus culture or the colchicine treatment of shoot nodes in vitro

Experiments were carried out to double the somatic cell chromosome numbers of a monoploid and dihaploid of Solanum tuberosum and a genotype of S. circaeifolium subsp. quimense. Colchicine was used in vitro on shoot nodes from which the axillary meristems had been removed. Plants with doubled chromosome numbers were obtained from shoots grown from the tertiary, sub-axillary meristems of all three genotypes. The callus culture of stem and leaf explants was found to produce more shoots with doubled chromosome numbers than the colchicine treatment in the case of the dihaploid and quimense genotypes but no shoots were obtained from callus culture of the monoploid. Fifty-two % of the shoots from the dihaploid and 63% from the quimense clone were ploidy doubled in the case of the best callus culture system. Using a sub-lethal dose of colchicine, the dihaploid yielded 37% ploidy-doubled shoots whereas all the shoots produced from the monoploid were doubled and the quimense clone produced 27% doubled plants. Callus culture was highly dependent upon the type of growth medium and other, unknown, factors. The colchicine treatment, although yielding fewer products, was more reliable for achieving ploidy doubling in selected clones if the number of plants produced is not important.     

Application of bioreactors for large-scale micropropagation systems of plants

Summary The application of bioreactor culture techniques for plant micropropagation is regarded as one of the ways to reduce production cost by scaling-up and automation. Recent experiments are restricted to a small number of species that, however, demonstrate the feasibility of this technology. Periodic immersion liquid culture using ebb and flood system and column-type bubble bioreactors equipped with a raft support system to maintain plant tissues at the air and liquid interface were found to be suitable for micropropagation of plants via the organogenic pathway. Balloon-type bubble bioreactors proved to be fit for micropropagation via somatic embryogenesis with less shear stress on cultured cells. Several cultivars of Lilium were successfully propagated using a two-stage culture method in one bioreactor. A large number of small-scale segments were cultured for 4 wk with periodic immersion liquid culture to induce multiple bulblets from each segment, then the bulblet induction medium was changed into bulblet growth medium by employing a submerged liquid bioreactor system. This culture method resulted in a nearly 10-fold increase in bulblet growth compared to conventional culture with solid medium. About 20 000 cuttings of virus-free potato could be obtained from 120 singlenode explants in a 20-liter balloon-type bubble bioreactor after 8 wk of culture. The percentage of ex vitro survival and root induction of the cuttings was more than 95%. Other successful results were obtained from the micropropagation and transplant production of chrysanthemum, sweetpotato, Chinese foxglove. Propagation systems via somatic embryogenesis in Acanthopanax koreanum and thornless Aralia elata were established using a liquid suspension of embryogenic determined cells. More than 500 000 somatic embryos in different stages were harvested from a 10-liter balloon-type bubble bioreactor after a 6-wk culture. Further development of these embryos in solid medium and eventually in the field was successful. The bioreactor system could reduce initial and operational cost for micropropagation, but further development of sophisticated technology might be needed to apply this system to plant micropropagation industries.     

Evaluation of a mixed bacterial culture for de-emulsification of water-in-petroleum oil emulsions

A mixed bacterial culture, isolated from a petroleum-contaminated site, was evaluated for its de-emulsification capabilities using a kerosene–water model emulsion system and petroleum oilfield emulsion. The culture exhibited high de-emulsification activity with 96% de-emulsification of a water-in-oil emulsion within 24 h. Nine morphologically distinct pure colonies were isolated from the mixed culture and identified and their de-emulsification capabilities were tested. All three strains of Acinetobacter, i.e. A. calcoaceticus, A. calcoaceticus BV ALC and A. radioresistans were capable of providing > 90% de-emulsification, while Pseudomonas aeruginosa, P. carboxydohydrogena, and Alcaligenes latus showed > 80% de-emulsification. Different de-emulsification patterns were observed between species of Acinetobacter and Pseudomonas. The mixed culture exhibited higher de-emulsifier activity, as compared to the most effective pure culture, Acinetobacter calcoaceticus, when de-emulsification ability was tested on an oilfield water-in-oil emulsion.     

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.     

Acquisition and transmission of potato mop-to furovirus by a culture of Spongospora subterranea f.sp. subterranea derived from a single cystosorus

Potato mop-top virus (PMTV) was detected by ELISA in primary zoospores from four out of six isolates of Spongospora subterranea f.sp. subterranea. One virus-free isolate (N) of S. subterranea was used to acquire PMTV from potato roots and to transmit the virus to healthy plants. A mono-fungal culture of S. subterranea (isolate N) was derived by infecting tomato plant roots with a single cystosorus. The culture was used successfully to acquire PMTV from the roots of infected Nicotiana debneyi plants that had been manually inoculated with virus isolates, and subsequently to transmit the virus to healthy bait plants. These experiments confirm that S. subterranea is a vector of PMTV. Two PMTV isolates that had been maintained by manual inoculation for 19 and 21 passages were also acquired and transmitted by the fungus culture.