Induction and development of potato tubers in a jar fermentor

Potato (Solanum tuberosum L.) tubers were mass propagated in a jar fermentor using a two-step method consisting of a shoot multiplication step (phase 1) followed by a tuber induction and development step (phase 2). Tuberization was observed within 2 weeks of phase 2 and the number of tubers did not increase after this culture period. In contrast, total tuber weight continuously increased for at least 10 weeks. Although the number of tubers and the total tuber weight clearly decreased under the lower temperature (17°C), this weight decrease was partially prevented by changing the temperature from 17°C to 25°C after 2 weeks of phase 2. This result indicates that tuber development can be controlled independently of induction. Lower temperatures influenced the localization and size distribution of tubers in the jar fermentor.     

Large-scale production of anthocyanin by Aralia cordata cell suspension cultures

The suspension culture of high anthocyanin-producing Aralia cordata cell lines, which grow and produce anthocyanin without light irradation, was scaled up from flasks to a 10-1 glass jar fermentor, a 95-1 stainless steel jar fermentor, and finally a 500-l pilot-scale jar fermentor. By the administration of CO₂, cell damage was completely prevented and the anthocyanin content was kept as high as 7.0–17.2% (w/w) of the dried cells. In one of the operations of the 500-l jar fermentor, cells were cultivated for 16 days. During this operation, cell mass was increased by more than 26 times (cell yield: 69.2 kg fresh wt.) and the amount of anthocyanin increased by more than 55 times (anthocyanin yield: 545 g, anthocyanin content: 17.2% of the dried cells). Correspondence to: Y. Kobayashi     

Rhamnolipid production in batch and fed-batch fermentation using<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> BYK-2 KCTC 18012P

The optimization of culture conditions for the bacteriumPseudomonas aeruginosa BYK-2 KCTC 18012P, was performed to increase its rhamnolipid production. The optimum level for carbon, nitrogen sources, temperature and pH, for rhamnolipid production in a flask, were identified as 25 g/L fish oil, 0.01% (w/v) urea, 25 and pH 7.0, respectively. Optimum conditions for batch culture, using a 7-L jar fermentor, were 200 rpm of agitation speed and a 2.0 L/min aeration rate. Under the optimum conditions, on fish oil for 216 h, the final cell and rhamnolipid concentrations were 5.3 g/L and 17.0 g/L respectively. Fed-batch fermentation, with different feeding conditions, was carried out in order to increase, cell growth and rhamnolipid production by thePseudomonas aeruginosa, BYK-2 KCTC 18012P. When 2.5 g of fish oil and 100 mL basal salts medium, containing 0.01% (w/v) urea, were fed intermittently during the fermentation, the final cell and rhamnolipid concentrations at 264 h, were 6.1 and 22.7 g/L respectively. The fed-batch culture resulted in a 1.2-fold increase in the dry cell mass and a 1.3-fold increase in rhamnolipid production, compared to the production of the batch culture. The rhamnolipid production-substrate conversion factor (0.75 g/g) was higher than that of the batch culture (0.68 g/g).     

Studies on the ɛ-Lysine Acylase

Since Achromobacter pestifer EA isolated from soils shows markedly high ?-Iysine acylase activity compared with those of the other microorganisms ever tested, cultural conditions for the production of this enzyme were investigated.

As a result, it was confirmed that simple medium containing 1% peptone, 5% glucose and some inorganic salts is most suitable for the enzyme production and that much more ?-Iysine acylase is produced by shaken culture or submerged culture in jar fermentor than by stationary culture. α-Amino acylase activity in this organism was also studied.     

Fermentative Production of Chloramphenicol-analogs (Corynecins) from Sucrose

Corynecins (N-acyl derivatives of d-(?)-threo-p-nitrophenyl serinol), which were firstly discovered in the culture broth of Corynebacterium hydrocarboclastus KY 4339 grown on n-alkane were also produced when n-alkane was replaced by sucrose. The corynecins production in the sucrose-medium was significantly stimulated by the supplement of molasses. On the basis of the composition of ingredients in molasses, the preferable culture medium was designed for the production of corynecins from sucrose. This semi-synthetic medium is consisted of low concentration of phosphate, high concentration of potassium chloride, inositol, fructose and yeast extract in addition to ordinary mineral salts. By controlling the pH of the medium at the neutral range and keeping the aeration at a relatively high level, approximately 4 g of corynecins (as l-base) per liter of the medium were produced using a 5-liter jar fermentor.     

Acetic acid production by Acetobacter aceti in a silicone tube bioreactor

Summary Continuous acetic acid fermentation was carried out using a column reactor, in which 20 to 200 thin silicone tubes (0.33 mm in outer diameter) were packed to supply oxygen by permeation. The highest value of volumetric oxygen transfer coefficient determined by the sulfite oxidation method was 2,860 h^–1, which was comparable to that of a well agitated and aerated fermentor. The maximum production rate of acetic acid by the bacterial films of Acetobacter aceti M7 grown on the shell-side surface of the tubes was 38.0 g/lh at an acetic acid concentration of 44.5 g/l. This was 29 times that of a continuous culture using a jar fermentor.     

Use of the growth retardant tetcyclacis for potato tuber formation in vitro

The effects of the plant growth retardant tetcyclacis on in vitro tuber formation in potatoes was studied, using two different approaches: 1. tuber formation in various lines that did not or hardly form tubers under control conditions, and 2. tuber formation by the variety Bintje, which readily forms tubers. The ABA-deficient (droopy) lines of S. phureja hardly formed tubers without the addition of tetcyclacis. In the presence of this growth retardant tuberization was nearly 100%, within three weeks of in vitro culture, even in the absence of cytokinin. A series of somatic hybrids between S. tuberosum and S. brevidens, that did not form tubers in field and pot experiments, were tested. They all formed tubers in vitro in the presence of tetcyclacis. Stoloniferous shoots formed on single-node cuttings from in vitro grown Solanum tuberosum var Bintje plantlets were transferred to media containing a high level of sucrose. In the presence of tetcyclacis, tuber formation started after 4 days, reaching a maximum level of 80% at day 7. Tubers formed in the presence of tetcyclacis, accumulated starch and expressed several tuber-specific genes. These effects were fully antagonized by gibberellic acid. It is concluded that the growth retardant tetcyclacis is a potent tool in the study of tuber formation in potatoes.Abbreviations ABA abscisic acid - BAP benzylaminopurine - GA₃ gibberellic acid - STS silver thiosulphate - TET tetcyclacis     

Production of human monoclonal antibodies by heterohybridomas

Summary Mouse human-human heterohybridomas secreting human monoclonal antibodies (MoAb) against tetanus toxoid and hepatitis B virus surface antigen were effectively cultivated in a medium containing a serum substitute called GFS, a 55% to 70% ammonium sulphate fraction of serum from adult cattle. A perfusion culture system using a jar fermentor equipped with a cell sedimentation column with a double jacket was developed and applied to produce human MoAb. In this fermentor, maximum cell density of a heterohybridoma reached 1.2×10⁷ cells/ml and MoAb was continuously accumulated at a constant rate for at least 40 days; this led to the production of more than one gram of human MoAb using a culture vessel with a 1-1 working volume.     

Germination and Growth of Potamogeton pectinatus (L.) at Different Water Depths in Lake Nainital,Uttar Pradesh,India

Data on germination of tubers and subsequent growth of young plants of Potamogeton pectinatus were recorded at different depths of the water column in Lake Nainital. The depth of euphotic zone is about 5 m. Tubers were placed at 1, 2.5, 5.0, 7.5 and 10 m water depth in brass net bags. A highly significant negative correlation was found between depth on one hand and germination percentage of tubers, length of shoots and number of leaves on the other hand. Germination of tubers was positively related to pH, dissolved oxygen and temperature of water.     

Development of food grade media for the preparation of Lactobacillus plantarum starter culture

Based on MRS medium, two types of food grade (FG) culture media (FG medium I and FG medium II) for the preparation of a concentrated starter culture of Lactobacillus plantarum NRIC 0380 to manufacture a new type of instant Chinese noodle, the fermented instant Chinese noodle, were developed using FG materials. FG medium I, which is for normal static culture, contains table sugar (sucrose), Yeast peptone standard type F, Sunsoft Q-17S (emulsifier), sodium acetate, trisodium citrate and MnSO(4).4-5H(2)O. FG medium II was designed to be used for the pH-controlled jar fermentor culture conditions. Therefore, sodium acetate and trisodium citrate as a buffer to prevent acidification of medium were omitted from FG medium I. When L. plantarum NRIC 0380 was cultured under the pH-controlled jar fermentor culture conditions, the kinetics of growth, sugar consumption and lactic acid production in FG medium II were quite similar to those observed in the Difco Lactobacilli MRS Broth. Furthermore, growths of many lactobacilli strains isolated from various fermented foods in FG medium I were also quite similar to those observed in MRS medium. Therefore, simple and practical FG media for the culture of lactobacilli were successfully established.     

Screening of Bacterial Cellulose-producing Acetobacter Strains Suitable for Agitated Culture

Extensive screening for cellulose-producing Acetobacter strains suitable for agitated culture was done by developing the screening conditions. A total of 2096 strains were isolated; isolation from fruits was particularly efficient. The cellulose productivities of 412 isolates were estimated by culturing in two different media under both shaken and static conditions. No correlation between the amounts of cellulose accumulated in shaken and static cultures was observed. Higher cellulose accumulation was obtained in the shaken cultures using a corn steep liquor/fructose-based medium than a conventional yeast extract/peptone/glucose-based one. Many isolates showed higher cellulose accumulation than well-known cellulose-producing strains. The producer that yielded the highest cellulose accumulation in shaken culture was selected and named Acetobacter sp. BPR 2001. Using this strain, cellulose was produced in a jar fermentor.     

Effects of reducing sugar concentration on in vitro tuber formation and sprouting in yam (<Emphasis Type="Italic">Dioscorea cayenensis–D. rotundata</Emphasis> complex)

The effects of reducing sucrose level on tuber formation (% of cultures with microtubers), development (length and fresh weight of microtubers) and sprouting in yam Dioscorea cayenensis–D. rotundata complex in vitro were investigated. Only 29% of the explants showed tuber formation after 3 weeks in the presence of 1% sucrose in contrast to 100% with 3%. After 120 days of culture, the length and the weight of the tubers obtained in the presence of 1% sucrose were less than with 3% sucrose. Addition of sorbitol to keep osmolarity at the same level did not restore normal rate of tuber formation. Similar results were obtained with the use of reduced fructose or glucose level. Microtuber sprouting was also affected by sucrose level incorporated into the tuberisation medium. Tubers obtained on reduced sucrose level sprouted later and the increase of osmolarity with sorbitol did not restore normal sprouting. The bigger tubers obtained on high sucrose media could contain more carbohydrate reserves that could partially explain a higher sprouting rate. These results can be used for optimising in vitro conditions for mass production of microtubers in yam and especially in Dioscorea cayenensis–D. rotundata complex, a very important species in West Africa. They specially showed the importance of tuberisation conditions on precocity of tuberisation, on tuber length and weight and on their further sprouting.     

Direct plantlet regeneration from the tuber of Stachys sieboldii

Development of an efficient in vitro plantlet regeneration system from tubers of Stachys sieboldii (Miq.), a traditional Chinese medicinal plant and vegetable, is described. Adventitious shoots with an average of 9.1 (after 4 weeks of incubation) shoots per explant were obtained from 60.4% of tuber explants cultured on Murashige and Skoog (MS) medium containing 1.0 mg l^–1 thidiazuron (TDZ), and after another 4 weeks of incubation on growth regulator free MS medium, 33.5 shoots per explant were achieved. Subcultures showed results that were similar to those of the initial culture. Excised shoots were rooted both on MS and on half strength MS medium with a frequency of about 95%. Excised shoots were also rooted ex vitro by directly planting them into cups containing sand. Regenerated plants with well developed shoots and roots were successfully transplanted to soil, after which they developed free of abnormalities.     

Tuber surface microorganisms influence the susceptibility of potato tubers to late blight

Potato tubers (cvs Cara and Bintje) were grown in compost in a glasshouse and immature tubers harvested 57, 68 and 78 days after planting. Two moisture levels were imposed after the first harvest by disconnecting the water supply to one of the treatments and allowing the soil in that treatment to dry naturally. Tubers from wetter compost (59.4% moisture holding capacity) were more resistant to Phytophthora infestans than those from drier compost (14.7% moisture holding capacity) 78 days after planting. The potential causes of this difference were investigated. Aqueous extracts of wet compost did not inhibit the growth of P. infestans. The susceptibility of the internal tuber tissue, from which the periderm had been removed, was different to whole tuber susceptibility. The internal tissue of tubers from wet compost was more susceptible (cv. Cara), or as susceptible (cv. Bintje) as that of tubers from dry compost 78 days after planting. Fungi were isolated from the surface of whole tubers and there were no differences between the populations of potentially antagonistic fungal genera on tubers from wet and dry compost. As the experiment progressed, the number of bacteria per gram fresh weight on tubers grown in wet compost increased, whereas that on tubers from drier compost decreased (cv. Bintje) or remained similar (cv. Cara). There were significantly (P= 0.008) more bacteria on the surface of tubers from wet compost 78 days after planting. When P. infestans was co-cultured in Petri dishes with randomly selected tuber surface bacteria, some isolates (≤ 16.7%) inhibited the growth of the fungus. The percentage of the total bacterial population that was antagonistic to P. infestans was not significantly affected by soil moisture level (P= 0.368). The greater numbers of bacteria, of which a proportion are antagonistic to P. infestans, on the surface of tubers grown in wet compost may account for the greater resistance to tuber blight in that instance.     

Features of bacterial cellulose synthesis in a mutant generated by disruption of the diguanylate cyclase 1 gene of <Emphasis Type="Italic">Acetobacter xylinum</Emphasis> BPR 2001

The diguanylate cyclase 1 (DGC1) (dgc1) gene in Acetobacter xylinum BPR 2001—a bacterial cellulose (BC) producer—was cloned and sequenced, and a DGC1 gene-disrupted mutant, strain DD, was constructed. The production and structural characteristics of the BC formed by DD were compared with those of the parental strain BPR 2001. BC production by DD was almost the same as that by BPR 2001 in static cultivation and in shake flask cultivation. However, in a jar fermentor DD produced about 36% more BC than the parental strain. DD produced suspended particle materials that cannot aggregate owing to their random structural characteristics in static cultivation; more uniformly dispersed BC pellicles and smaller BC pellets are produced on average in a jar fermentor, as reflected by the higher BC production by DD than by the parental strain in a jar fermentor. Micrographs of BC produced by DD revealed that the width of cellulose ribbons assemblies decreased as a result of differences in the ultrastructure and mechanism of formation of BC between the two strains. These results reveal that disruption of the dgc1 gene, which catalyzes synthesis of c-di-GMP (an effector of BC synthase), is not fatal for BC synthesis, although it affects BC structure.     

A dense cell retention culture system using stirred ceramic membrane reactor

A novel reactor design incorporating porous ceramic tubes into a stirred jar fermentor was developed. The stirred ceramic membrane reactor has two ceramic tubular membrane units inside the vessel and maintains high filtration flux by alternating use for filtering and recovering from clogging. Each filter unit was linked for both extraction of culture broth and gas sparging. High permeability was maintained for long periods by applying the periodical control between filtering and air sparging during the stirred retention culture of Saccharomyces cerevisiae. The ceramic filter aeration system increased the k(L)a to about five times that of ordinary gas sparing. Using the automatic feeding and filtering system, cell mass concentration reached 207 g/L in a short time, while it was 64 g/L in a fed-batch culture. More than 99% of the growing cells were retained in the fermentor by the filtering culture. Both yield and productivity of cells were also increased by controlling the feeding of fresh medium and filtering the supernatant of the dense cells culture. (c) 1994 John Wiley & Sons, Inc.     

The Role of Polyphenol Oxidase and Peroxidase in Potato Tuber Resistance to Soft Rot Caused by Erwinia carotovora

Tubers from somatic hybrids produced by protoplast fusion between Solanum brevidens, a diploid, non-tuber-bearing wild species, and a tetraploid S. tuberosum showed resistance to decay caused by soft rot Erwinia. Tubers of the S. tuberosum fusion parent and potato cultivar Russet Burbank are susceptible to bacterial soft rot. Tubers of somatic hybrids indicated higher levels of activities of polyphenol oxidase and peroxidase than tubers of the parental line of S. tuberosum and cultivar Russet Burbank. This is true for intact tubers and also for injured or inoculated tubers. Polish commercial potato cultivars indicated a higher susceptibility to soft rot than somatic hybrids. However, there were some differences in susceptibility to soft rot between Polish commercial potato cultivars, only slight differences were observed in the activities of the polyphenol oxidase and peroxidase between Polish cultivars. A relation between soft rot resistance and the activity of each enzyme was not found for intact, injured or inoculated tissue of commercial cultivars. On the contrary, the activities of both enzymes were significantly higher in the periderm than in the medullary tissue of somatic hybrids, the parental line and the commercial cultivars.     

Fruit-body production of a luminous mushroom,Mycena chlorophos

Cultural conditions for fruit-body production ofMycena chlorophos were investigated with the aim of using the mushroom for study of bioluminescence, scientific exhibition, and ecological conservation. A small glass jar having a cap with a microfilter was used as a culture vessel. A compost powder mixed with rice bran in proportion of 20% (fw/fw) and adjusted to 70% (w/w) in moisture content was used as production medium. Casing with 2 g/jar of moistened compost powder was necessary for fruit-body formation. Mycelium was grown in a culture chamber at 27°C, relative humidity (RH) of 80% for 4 wk, then transfered to a culture chamberat 21°C, 90% RH and light intensity of 300–800 lx after casing its, and incubated for 3 wk to produce fruit-bodies. The mean yield was 31 fruitbodies, i.e., 150 mg dry weight per jar.     

Growth dynamics of Potamogeton pectinatus L. in Lake Burullus,Egypt: a modelling approach

In this study, we used a macrophyte model to describe the growth production and the interaction between above‐ and below‐ground organs of Potamogeton pectinatus in Lake Burullus, Egypt. Above‐ and below‐ground biomass of P. pectinatus was sampled on a monthly basis from April to December 2011 at three sites of Lake Burullus. Shoots started to grow in April, reached the maximum biomass in September and then rapidly decreased in October when they moved into the senescence stage. Tubers biomass reduced in August due to the upward translocation to shoots, but sharply increased to the maximum in October by downward translocation from shoots and roots. Potamogeton pectinatus allocated approximately 82.3% of its total biomass to shoots, 15.5% to tubers and 2.2% to roots.     

High-level production of recombinant glutamic acid-specific protease from Bacilllus licheniformis in Bacillus subtilis expression system

To obtain large quantities of glutamic acid-specific protease isolated originally from Bacillus licheniformis (BLase), an expression plasmid was constructed by inserting the BLase gene into a plasmid vector (pUB110) for Bacillus subtilis. B. subtilis strain ISW1214 harboring the resultant recombinant plasmid containing the coding and 5′-promoter and 3′-terminator regions of BLase gene secreted approximately 0.25 g/l of BLase in a culture medium contained in a 90-l jar fermentor, corresponding to nearly 10 times the natural production level and resulting in a stable large-scale production. The amount of BLase in the culture medium accounted for roughly 60% of the total extracellular proteins secreted from the recombinant strain, simplifying enzyme purification.