Cell growth and alpha-amylase production characteristics of Bacillus amyloliquefaciens

Growth and alpha-amylase production characteristics of Bacillus amyloliquefaciens strain F (ATCC 23350) in batch cultures are examined using glucose or maltose as the carbon source. While the cell growth is rapid when glucose is used as the carbon source, higher cell mass, higher total and specific enzyme activities, and higher enzyme production rates are obtained when maltose is used as the carbon source. The overall specific enzyme activity decreases with an increase in the initial concentration of carbon source. The oxygen requirement and carbon dioxide generation vary linearly with the maximum amount of cell mass produced. For experiments conducted using glucose as the carbon source, the kinetics of cell growth and glucose consumption are described using a special form of the Vavilin equation. For a given amount of initial carbon source, the enzyme synthesis capability is retained by the microorganism, although at a substantially reduced level, under severe oxygen limitation.     

Expression of beta-lactamase by recombinant Escherichia coli strains containing plasmids of different sizes--effects of pH, phosphate, and dissolved oxygen

The characteristics of growth and synthesis of plasmid-encoded protein were studied for strains of recombinant E. coli JM103 which carried the beta-lactamase gene on plasmids of different sizes. The plasmids used included the vector pUC8 and its recombinant derivatives containing varying-sized inserts of Drosophila DNA (not expressed in E. coli). Luria broth (LB) and a minimal medium (M9) supplemented in some cases with additional inorganic phosphate were used as growth media. There was no evidence of segregational instability in these experiments, where no antibiotic selection pressure was employed. Responses of the recombinant strains to variations in environmental parameters including pH, phosphate concentration in the medium, and aeration rate were examined. While the cell growth rate in LB decreased with pH in the range 7.0-8.0, the bulk beta-lactamase activity was maximized at an intermediate pH. The recombinant cell growth rate decreases with increasing plasmid size in the minimal medium, while such decrease is not significant when a rich medium such as LB is used. There is an intermediate plasmid size in the range studied (2.7-8.7 kb), at which beta-lactamase activity is maximum. While reduction in aeration rate (which determines the dissolved oxygen level) is detrimental for cell growth, it is beneficial for beta-lactamase synthesis. The bulk beta-lactamase activity therefore exhibits a maximum with respect to aeration rate. Cell growth and beta-lactamase production are affected in a similar manner by phosphate concentration in the minimal medium and therefore both are maximized at the same phosphate concentration. This investigation demonstrates clearly how the production of a recombinant plasmid-encoded protein can be maximized by proper manipulation of culture conditions and how it is affected by plasmid size.     

Induction and elimination of oscillations in continuous cultures of Saccharomyces cerevisiae

Continuous cultures of Saccharomyces cerevisiae are known to exhibit oscillatory behavior in the oxidative region. Important findings of a series of experiments conducted to identify the causes for initiation of and the means for elimination of oscillations in these cultures are reported in this paper. These oscillations are seen to be connected to the growth kinetics of the microorganism and are induced at very low glucose concentrations and at dissolved oxygen (DO) levels that are neither high nor low (DO values between 20 and 78% air saturation at a dilution rate of 0.2 h(-1) and pH of 5.5 at 30 degrees C). The oscillatory behavior is encountered over a range of dilution rates (0.09-0.25 h(-1) at 30 degrees C for pH = 5.5 and DO = 50% air saturation). The oscillations can be eliminated by raising the DO level above a critical value or by lowering the DO level below a critical value.     

Effect of domestic sewage on sand beach meiofauna at Goa,India

Meiofauna of a sewage-polluted sandy beach, where sand alone constituted > 90%, was surveyed. Nematodes dominated the fauna numerically at all stations, followed by harpacticoid copepods. Most of the animals were confined to the top 5 cm of the sediment. A seasonal pattern was observed in the distribution of the fauna. There were significant spatial and temporal variations in mean meiofauna density, attributed to organic discharge via sewage and prevailing environmental conditions in the study area.     

Immobilization of Escherichia coli JM103[pUC8] in kappa-carrageenan coupled with recombinant protein release by in situ cell membrane permeabilization.

Immobilization of Escherichia coli JM103[pUC8] was carried out with kappa-carrageenan as the support matrix. Substantial natural excretion of beta-lactamase, attributable to the less intact membrane of plasmid-harboring cells, was observed in immobilized cell cultures. Nevertheless, a significant portion of the beta-lactamase produced was retained in the cells. As compared to suspension cultures, much higher beta-lactamase activities, especially in the extracellular liquid, and much longer retention of plasmid-bearing cells (improved plasmid stability) were observed in immobilized cell cultures. Further enhancement in excretion of the recombinant protein (beta-lactamase) was achieved by permeabilization of cell membrane by periodic exposure of the immobilized cell cultures to ethylenediaminetetraacetic acid (EDTA). While the presence of EDTA led to some suppression of cell growth in suspension cultures, cell growth in gel beads was not affected by EDTA to the same extent, possibly due to lesser exposure of immobilized cells to EDTA. Exposure of immobilized cell cultures to EDTA presumably inhibited plasmid replication and led in turn to diversion of cellular resources for the support of expression of plasmid genes. Indeed, treatment of the immobilized cell cultures with EDTA resulted in increased production of beta-lactamase when compared to the enzyme production in EDTA-free cultures. More frequent addition of EDTA increased the period of retention of plasmid-bearing cells in these cultures but did not have any noticeable adverse effect on synthesis of beta-lactamase. Improvement in plasmid stability in EDTA-treated immobilized cell cultures was ascribed to the reduction in the growth rate differential between plasmid-free and plasmid-bearing cells, since plasmid-free cells were subject to more reduction in specific growth rate than were plasmid-bearing cells.     

Size-fractionated biomass, photosynthesis and dark CO2 fixation in a tropical oceanic environment

This study examines the spatial distribution and size structureof phytoplankton biomass and productivity in relation to thevertical structrure of the Andaman Sea (northeastern IndianOcean). This region was characterized by low concentrationsof nutrients and high levels of insolation. Nitrogen availabilityappeared to control overall productivity with nitrate-based‘new’ production accounting for 8–24% of thetotal primary production. Euphotic column chlorophyll (chl a)averaged 52.5 mg m^–2 of which a major portion was locatedas a subsurface chl a maximum (SCM) at     

Occurrence of Diaphanosoma excisum (Sars) on a sandy beach at Balramgari (Orissa), India

The occurrence of Diaphanosoma excisum (Sars), a freshwater cladoceran, in benthic samples of an intertidal sandy beach is reported. Population density was seasonal. A relatively high density was recorded from June to September (south-west monsoon season) with a maximum (46 ind 10 cm^–2) in September at a depth of 10–15 cm of sediment. A sudden decline occurred during north-east monsoon (October to January), and in the fair season (February to May), the cladocerans disappeared. Mean density varied (P<0.001) with sediment depth and season and showed a contagious dispersion. Abundance was negatively correlated with salinity (r = –0.76) but positively with POC (r =0.79) and mean grain size of the sediment (r = 0.93). The density of D. excisum was highest in fine sand.     

An alternate method for estimation of cell growth kinetics from batch cultures

An alternative to estimation of cell growth kinetics via continuous culture experiments is proposed in this article. The method employed is based on batch culture experiments with very small inocula (initial cell concentrations being typically less than 5000 cells/mL). Such low initial cell concentrations result in extended exponential cell growth phase during which culture conditions remain unchanged, thereby permitting precise estimation of specific cell growth rates from batch experiments especially for fast-growing microorganisms such as Bacillus species. The effectiveness and utility of this approach are demonstrated via several experiments conducted with a wild-type strain (Bacillus subtilis TN106) and a recombinant strain (B. subtilis TN106[pAT5]). True establishment of exponential growth phase requires insignificant variance of most of the culture conditions during the initial growth phase. Satisfaction of this requirement is demonstrated for microbial systems investigated here. This approach is especially well suited for recombinant microorganisms containing segregationally unstable plasmids, since estimation of growth kinetics of these from continuous cultures is very difficult and highly unreliable due to continual reversion of recombinant ceils to plasmid-free host cells unless some selection pressure is applied at levels sufficient to keep the presence of plasmid-free cells minimal.     

An unusual presentation of a malignant jejunal tumor and a different management strategy

BACKGROUND: Malignant small bowel tumors are very rare and leiomyosarcoma accounts for less than 15% of the cases. Management of these tumors is challenging in view of nonspecific symptoms, unusual presentation and high incidence of metastasis. In this case report, an unusual presentation of jejunal sarcoma and management of liver metastasis with radiofrequency ablation (RFA) is discussed. CASE PRESENTATION: A 45-year-old male presented with anemia and features of small bowel obstruction. Operative findings revealed a mass lesion in jejunum with intussusception of proximal loop. Resection of bowel mass was performed. Histopathological findings were suggestive of leiomyosarcoma. After 3-years of follow-up, the patient developed recurrence in infracolic omentum and a liver metastasis. The omental mass was resected and liver lesion was managed with radiofrequency ablation. CONCLUSION: Jejunal leiomyosarcoma is a rare variety of malignant small bowel tumor and a clinical presentation with intussusception is unusual. We suggest that an aggressive management approach using a combination of surgery and a newer technique like RFA can be attempted in patients with limited metastatic spread to liver to prolong the long-term survival in a subset of patients.