Rotary Incubator Research Articles

Antibacterial Activity of lemongrass plant (Cymbopogan citratus) against pathogens

From the traditional health care system, we use plants as a good source of medicine/drugs. They have been an essential part of traditional health care system as cure or food supplements. Lemon grass (Cymbopogon Flexuosus and Cymbopogon Citratus) is a native aromatic tall sedge/grass belonging to family (Poaceae / Gramineae) with diverse medicinal value and grown in many parts of tropical and subtropical south east Asia and Africa. It was grown in India a century back and now commercially cultivated in different parts of India (Jharkhand, Bihar, UP). The oil has been found to possess bactericidal, anti-bacterial and anti-fungal properties comparable to Penicillin in its effectiveness. The purpose of study was to find the antibacterial activities of the lemongrass plant Cymbopogon citratus. The crushed leaves of selected plant were extracted with methanol and solvent extracts were evaporated for dryness with help of rotary Incubator. Remaining dry residue was dissolved in (E- Ethanol, Cl- Chloroform, A- Acetone, C- Control) and the leaf sample in four different solvents was applied for its antibacterial action. The antibacterial screening of the selected extracts was determined by the Kirby-Bauer’s Disc diffusion technique on the following bacteria- Pseudomonas aeruginosa and Bacillus subtilis. The maximum zone of inhibitions was noted with solvent ethanol (11mm) against plate 1(Pseudomonas aeruginosa) and 12mm against plate 2(Bacillus subtilis). The sample dissolved in ethanol showed the maximum zone of inhibition followed by chloroform and least with acetone against both the pathogens.

In Vitro Evaluation of Antimicrobial Efficacy of the Different Concentrations of Chitosan against Staphylococcus epidermidis in Disinfection of the Root Canal System

During endodontic procedures, along with mechanical instrumentation, irrigants are imperative for pathogeneradication. As the strains of pathogens are becoming antibiotic resistant at a constant ascending pace andalso the potentially harmful effect is associated with synthetic drugs, so studies on natural alternatives forirrigation are being done. Chitosan, a natural alternative, has been evaluated as an antimicrobial medicine.Recently, in cases presenting with persistent infections after endodontic treatment, it was noted that of all themicrobial species identified, the prevalence of s.epidermidis was the most. In this current study, an analysisof the antimicrobial effectiveness of chitosan as a potent root canal irrigant, for disinfecting the root canalwas conducted. Methodology: By the serial dilution method, the minimum inhibitory concentration wasdetermined. Dilution of the test solution was done using 10% DMSO solution. After which, incubationin a rotary incubator, at 180 rotations per minute, the temperature of 37?, for 24 hours was done. Forobtaining the zones of inhibition of different concentration (0.2%, 0.5%, 0.75, 1%) of chitosan irrigant, theagar diffusion method was applied and the resultant clear zone was measured with a ruler. Result: Chitosan(liquid) had promising antibacterial activity against Staphylococcus epidermidis, with a 16 mm zone ofinhibition at 100 µg/ml. Conclusion: Within the limitations of the present study, it was observed that, evenat lower concentrations, chitosan (liquid form) effectively hindered the further growth of S.epidermidis.

Optimization of Xylanase Production by Streptomyces costaricanus 45I-3 Using Various Substrates through Submerged Fermentation

Xylanase is an important hydrolytic enzymes with many application in several industries, but to obtain enzyme derived products is not easy. Thus, the optimization of efficient xylanases production is a great interest for biotechnological application. This study aims to determine the type of substrate, medium composition, and optimum conditions of xylanase production by S. costaricanus 45I-3. Determination of substrate type was done by growing the tested bacteria on birchwood xylan, beechwood xylan, oat spelled xylan, corn cobs xylan, and tobacco xylan substrate, meanwhile the determination of medium composition and enzyme production were done by measuring xylanase activity at various substrate concentration and replacing the carbon, nitrogen, phosphate and surfactants source. The results showed that the highest enzymatic index (EI) produced from corn cob xylan substrate at 3.60 meanwhile the second highest was beechwood xylan substrate at 2.87 EI, however this substrate is purer, thus this substrate was selected and used as xylan sources for further optimization measurement. The best xylanase activity (2.29 U/mL) obtained on eighth day after inoculation on rotary incubator at 120 rpm in 28 ºC. Arabinose as the source of carbon generate the highest activity at 3.161 U/mL meanwhile the most preferred source of phosphate is Na2HPO4 (2.37 U/mL). Both source of nitrogen i.e. nitrogen ammonium sulphate (NH4)2SO4 and yeast extract were able to produce xylanase at 2.57 and 2.36 U/mL. The addition of surfactant in production medium showed addition of SDS surfactant (0.146 U/mL) and Tween 80 (0.438 U/mL) showed a negative response by decreasing the activity. The conclusion showed that the xylanase activity was increased after optimization at various C, N, and P sources, and the use of nitrogen source (NH4)2SO4), become a more economical alternative to replacing a nitrogen source yeast extract so it can lower the production costs of xylanase enzyme.

An experimental and computational study of biosurfactant production from soy molasses

Mathematical modeling was performed of rhamnose production in a batch process, with model validation and optimization of a continuous fixed bed process. The batch experiments were performed for 72 h in a rotary incubator at 27 ± 1.0 °C and 120 rpm, with an initial inoculum concentration of 3 g/L in 150 mL of soy molasses at 100 g/L. Parameter determination was performed using multiple response nonlinear regression, with integration of a set of differential equations, employing the Runge–Kutta algorithm and application of the Contois model. The model was fitted to the experimental data and the sum of squared residuals was 47.61. The fitted parameters were validated in an experiment employing a column operated continuously at a flow velocity of 0.288 cm/h (flow rate of 0.02 mL/min), applying the plug flow reactor (PFR) and axial dispersion models. The optimal conditions were determined by the graphical assessment of the response variable behavior. The best validation results were obtained for the axial dispersion model. The optimum velocity was 0.3 cm/h, with less significant improvements in productivity at higher flow rates, for the substrate concentration range studied.

ETHANOL PRODUCTION AND SUGAR CONSUMPTION OF CO-CULTURE Saccharomyces cerevisiae FNCC 3012 WITH Candida tropicalis FNCC 3033 IN MEDIA CONTAINING INHIBITOR FERMENTATION

Inhibitor fermentation is one of the problems that arise in the ethanol production from lignocellulose waste. This work examined the ethanol yield and sugar consumption of mono and co-culture S. cerevisiae with C. tropicalis in media containing inhibitor fermentation. Furfural and phenol were used for inhibitor fermentation in basal medium with concentrations of 2.0 and 5.0 %, respectively. The basal medium contained 20 g.L-1 glucose, 20 g.L-1 D (+) xylose, 20 g.L-1 arabinose, 4 g.L-1 urea, 3 g.L-1 NaNO3, 3 g.L-1 NH4NO3, 1 g.L-1 KH3PO4 and 0.7 g.L-1 MgSO4·7H2O with pH adjusted to 5.5 with 1 mol.L-1 HCl. After furfural or phenol addition separately, and inoculation by mono and co-culture S. cerevisiae FNCC 3012 with C. tropicalis FNCC 3033, all media were incubated at 28–29 °C, 50% r.h. in the dark for 5 days in a rotary incubator at 60 rev/min. We found yeast colony count, sugar consumption, ethanol yields and efficiency of fermentation by co-culture S. cerevisiae with C. tropicalis higher than mono-culture S. cerevisiae or C.tropicalis in the fermentation media with or without inhibitors. This work indicated that co-culture S. cerevisiae with C. tropicalis were more tolerant to furfural and phenol. Ethanol yield 8.52%, 5.37% and 3.83% obtained from basal medium, basal medium plus 2.5 or 5.0 % furfural, respectively and efficiency of fermentation 27.00%, 17.00% and 12.20%. Ethanol yields 8.13%, 5.62% and 3.19% obtained from basal medium, basal medium plus 2.5 or 5.0 % phenol, respectvely and efficiency of fermentation 28.20%, 20.00% and 14.00%. Co-culture S. cerevisiae FNCC 3012 with C. tropicalis 3033 demonstrated potential as a fermentation process for ethanol production from lignocellulosic media content inhibitors. The use of this co-culture effectively utilizes hexose and pentose sugars in the substrate, increasing the yield and efficiency of fermentation for ethanol production.

English

The use of agricultural by-product as feed stock and co-culture fermentation is a good strategy for improving the efficiency of fermentation and ethanol production. Most rice husks have low protein and nitrogen content and need to be supplemented with nitrogen for fermentation process. This research sought to determine the optimal supplementation of rice husk stream–based fermentation medium with nitrogen and molasses sources, initial pH and incubation time for maximizing ethanol production by co-culturing Saccharomyces cerevisiae with Candida tropicalis. Urea, sodium nitrate and ammonium nitrate were used as nitrogen sources and molasses was used as carbon sources. Co-cultures of S. cerevisiae and C. tropicalis can use different nitrogen sources and molasses for growth and ethanol production. Molasses supplemented with rice husk hydrolysate medium, initial pH and incubation period significantly influenced ethanol yield and content of nitrogen and carbon in distillers grains (DDG). Maximum ethanol yield (20.32 ± 0.42%) with nitrogen (4.40 ± 0.11%) and carbon (9.20 ± 1.01%) content of DDG were obtained in the rice husk hydrolysate medium containing 16.0 g/l urea, 12.0 g/l NaNO3, 12.0 g/l NH4NO3, 1.0 g/l KH2PO4, 0.7 g/l MgSO4•7H2O, 20 ml/l molasses, 1.0 g/l KH2PO4 and 0.7 g/l MgSO4•7H2O with initial pH 5.5 and 6 days incubation period at 28 to 29°C, 50% relative humidity in the dark for 5 d in a rotary incubator at 60 rpm. Key words: Rice husk, Saccharomyces cerevisiae, Candida tropicalis, co-culture, ethanol yield, nitrogen and molasses.

Partial Purification and Characterization of Xylanase from Bacillus weihenstephanensis Strain ANR1 using Watermelon Rind

Aim: The aim of this study is to partially purify and characterize xylanase from Bacillus weihenstephanensis strain ANR1 using watermelon rind as a substrate. Materials and Methods: The soil bacterium, B.weihenstephanensis strain ANR1 was grown at 37°C for 72 h on rotary incubator shaker at 150 rpm in a flask containing 250 ml of medium. The xylanase enzyme was partially purified using ammonium sulfate precipitation and dialysis. It was further purified using DEAE-sepharose chromatography. Molecular weight of the xylanase enzyme was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results and Discussion: The xylanase enzyme was partially purified to a fold of 8.15 and a yield of 83.56%. Molecular weight of the protein was found to be 45 kDa. The purified enzyme was found to be optimally active at pH of 7.0 and at a temperature of 37°C. Conclusion: Based on the results obtained, the purified xylanase enzyme from B. weihenstephanensis strain ANR1 using watermelon rind as a substrate can be effectively used in various food industrial applications

Production of microsclerotia by Brazilian strains of Metarhizium spp. using submerged liquid culture fermentation

We investigated the potential production and desiccation tolerance of microsclerotia (MS) by Brazilian strains of Metarhizium anisopliae (Ma), M. acridum (Mc) and M. robertsii (Mr). These fungi were grown in a liquid medium containing 16 g carbon l⁻¹ with a carbon:nitrogen ratio of 50:1. One hundred milliliters cultures were grown in 250 ml Erlenmeyer flasks in a rotary incubator shaker at 28 °C and 200 rpm for 5 days. Five-day-old MS were harvested, mixed with diatomaceous earth (DE) and air-dried for 2 days at 30 °C. The air-dried MS-DE granular preparations were milled by mortar + pestle and stored in centrifuged tubes at either 26 or -20 °C. Desiccation tolerance and conidia production were assessed for dried MS granules by measuring hyphal germination after incubation for 2 days on water agar plates at 26 °C and for conidia production following 7 days incubation. Yields of MS by all strains of Metarhizium were 6.1-7.3 × 10⁶ l⁻¹ after 3 days growth with maximum MS yields (0.7-1.1 × 10⁷ l⁻¹) after 5 days growth. No differences in biomass accumulation were observed after 3 days growth, whereas Ma-CG168 showed the highest biomass accumulation after 5 days growth. Dried MS-DE preparations of all fungal strains were equally tolerant to desiccation (≥93 % germination) and the highest conidia production was obtained by MS granules of Mc-CG423 (4 × 10⁹ conidia g⁻¹). All MS granules showed similar stability after storage at either 26 or -20 °C for 3.5 months.

SELECTION, ISOLATION AND GROWTH KINETIC STUDY OF A BACTERIAL CONSORTIUM OBTAINED FROM THE POTENGI MANGROVE IN THE PRESENCE OF CRUDE OIL

The selection, isolation and kinetic study of a bacterial consortium obtained from a sample of soil from the Potengi mangrove, located in the city of Natal, Rio Grande do Norte, Brazil, has been carried out using the enrichment culture technique to observe aspects such as the evaluation of main growth parameters. The kinetic study used a rotary incubator shaker at 150rpm, under 30°C. The bacterial consortium isolated from the estuary of the Potengi River showed a good acclimation in minimum mineral medium with 1% (v/v) of oil. The cell concentration reached 2.55 g/L at 16h of cultivation and surface tension dropped. The maximum productivity in cells obtained was of 0.3 g/L.h, the specific velocity of growth was of 0.075h -1 , with a generation time (tg) of 9.24h. This study seeks to demonstrate that the consortium can be used as inoculants in biological treatments, capable of reducing the waste’s degradation time.

Discovery and Characterization of a Peptide Motif That Specifically Recognizes a Non-native Conformation of Human IgG Induced by Acidic pH Conditions

In therapeutic antibody preparation, acidic pH conditions are generally used for elution from Protein A affinity column of IgG or for its viral inactivation. Exposing IgG to low pH conditions induces conformational changes, leading to its functional damage or loss, although the mechanisms have not been fully elucidated. In this study using random peptide T7 phage display libraries, we isolated a unique and novel peptide motif that specifically recognized the non-native conformer (acid conformer) of human IgG that was generated by the low pH treatment, but not the native conformer. We examined the generation conditions and biochemical properties of acid conformer using the peptide motif as an affinity ligand. The acid conformer was easily generated at acidic pH (<pH 3.0) and at moderate temperatures (20-40 degrees C). The conformer was present in a monomeric form functionally maintaining antigen or Fc receptor binding, but showed a tendency to aggregate with a long incubation time at neutral pH (>25 degrees C). The peptides isolated here could contribute to the elucidation of the mechanisms of antibody dysfunction or aggregation during acid exposure as well as storage of human IgG.

Pigeon pea waste as a novel, inexpensive, substrate for production of a thermostable alkaline protease from thermoalkalophilic Bacillus sp. JB-99

Thermoalkaliphilic Bacillus sp. JB-99 was grown in a 250 ml Erlenmeyer flask containing 50 ml medium containing (g/l) Pigeon pea waste 10; NaNO 3, 5.0; K 2HPO 4, 5.0; MgSO 4·2H 2O, 0.2 and Na 2CO 3, 10.0. Incubations were carried out at 50 °C on a rotary incubator shaker for 15 h. A high level of extra cellular thermostable protease activity was observed after 24 h incubation. The optimum temperature and pH for activity were 70 °C and 11, respectively, so this enzyme showed stable activity at high temperature and under alkaline conditions.

Effect of Retting Enzymes on the Structure and Composition of Flax Cell Walls

Commercial enzyme mixtures are tested for their possibly selective degradation of flax ( Linum usitatissimum L.) stem components in relation to the retting process in producing linen. Structural and chemical compositional results from treatments are obtained using scanning electron microscopy, histochemistry, gas-liquid chromatography, 13C cp mas nmr spectrometry, and mid-infrared spectroscopy. Flaxzyme and Ultrazym and an enriched pectinase mixture (epm), which was not developed for flax retting but is included for comparison, are tested for their activity toward cell wall components and used in various concentrations for “enzyme-retting” of flax. Ariane flax stem sections are incubated with enzymes in a rotary incubator and the fibers are manually separated from the residual core. All of the commercial enzyme mixtures have cellulase, pectinase, and hemicellulase activities, but individual enzyme activities vary. Activities against the soluble test substrates do not predict the activity against natural fibers. At about equal protein concentrations, Flaxzyme treatment appears to facilitate bast fiber removal better than the other enzymes, with Ultrazym nearly as effective and epm the least effective. The ranking of effectiveness is generally supported by the amounts of uronic acid, arabinose, and xylose removed from the stems analyzed chemically. Increased enzyme levels generally facilitate removal of matrix carbohydrates from the flax. All enzymes separate bast fibers from the lignified core and partially from the cuticle near the cut surface of the stem sections, but the enzymes do not work far from the exposed ends. Retting quality is defined more by the degree of cell wall degradation and fiber separation than by any differences in kinds of cell walls degraded by the various enzymes. The cuticle remains attached to the fiber at times, apparently reducing access of the enzymes to the matrix polysacchrides and suggesting some recalcitrance of epidermal cells (and therefore loss of cuticle) to biodegradation. Lignin remains in the middle lamellae after enzyme retting and would likely prevent separation of the fiber bundles. Some solubilzation of the inner secondary wall of the flax fiber appears to occur with Flaxzyme. The structural and chemical analyses characterize alterations in flax bast after enzyme retting and would be useful in ranking the specificity and effectiveness of cell wall degradation.

The effects of nerve growth factor on the development of septal cholinergic neurons in reaggregate cell cultures

Recent studies suggest that nerve growth factor is present within the central nervous system where it may exert selective trophic effects on cholinergic neurons. We have measured the effects of nerve growth factor on septal cholinergic neurons in three-dimensional reaggregating cell cultures, a system which closely simulates the cellular environment in situ. Septal cells obtained from 15-day-old mouse embryos were dissociated into a single cell suspension and then allowed to reaggregate in culture in a rotary incubator shaker. After 17 days in culture, half of the reaggregates from a flask were sonicated for measurement of choline acetyltransferase activity, and the remaining reaggregates were processed for acetylcholinesterase histochemistry. Addition of nerve growth factor to medium containing septal reaggregates resulted in greater than a three-fold increase in choline acetyltransferase activity and in the number of acetylcholinesterase-positive cells, as well as an enhancement in the staining of acetylcholinesterase-positive fibers. All of these effects of nerve growth factor could be neutralized by antibodies to nerve growth factor. In order to evaluate the possible role of endogenous hippocampal-derived nerve growth factor, antiserum to nerve growth factor was added to the culture media containing septal-hippocampal coaggregates. After 21 days in culture, the presence of nerve growth factor antibodies did not qualitatively affect the pattern or density of cholinergic fibers observed. Synapse formation between cholinergic axons and hippocampal target cells was still in evidence as revealed by electron microscopy. However, there was a modest decrease in choline acetyltransferase activity (20%) and cholinergic cell number (30%) when compared with coaggregates grown in culture medium either without nerve growth factor antiserum or with non-immune serum. The magnitude of these effects was markedly less than the effects observed when exogenous nerve growth factor was added to septal cells grown alone in reaggregate culture. These results suggest that nerve growth factor may play a role during central cholinergic development, but that additional trophic mechanisms are likely to be required.

Neurotrophic effects of hippocampal target cells on developing septal cholinergic neurons in culture

The influence of hippocampal target cells on the development of cholinergic septal neurons was studied in rotation-mediated reaggregating cell cultures. Brain cells from 15-day-old mouse embryos were obtained from: (i) septum, containing cholinergic cells which project to the hippocampus; (ii) hippocampus which contains target cells for the septal cholinergic neurons; and (iii) cerebellum, containing cells which are not targets for the septal cholinergic cells. The cells were then cultured for 3 weeks in a rotary incubator in the following combinations: (a) septal cells alone; (b) hippocampal cells alone; (c) cerebellar cells alone; (d) septal-hippocampal cells together; and (e) septal-cerebellar cells together. After harvesting, fixation, and embedding, 50 μm sections were cut and processed for visualization of acetylcholinesterase activity. Sections from reaggregates containing either hippocampal or cerebellar cells alone contained only a few acetylcholinesterase-positive cells, but no positive fibers. Sections from septal-hippocampal coaggregates revealed a pattern of well-defined, fine-caliber acetylcholinesterase-positive fibers with extensive arborizations and varicosities suggesting axonal proliferation. In septal-cerebellar coaggregates, acetylcholinesterase-positive fibers appeared to be degenerating and distinct areas were observed which were essentially devoid of acetylcholinesterase fibers. In some experiments, either cerebellar or hippocampal cells were labeled with wheatgerm agglutinin-rhodamine prior to culture in order to identify these cells in the resulting reaggregates. Analysis of sections from these studies showed that acetylcholinesterase fibers were excluded from regions of coaggregates containing cerebellar cells, but were present in regions of coaggregates containing hippocampal cells. Finally, cell counts of acetylcholinesterase-positive cells in the various combinations revealed that these putative cholinergic neurons were significantly more numerous in septal-hippocampal coaggregates ( 271 ± 19 per 10 6 septal cells added) than in septal reaggregates ( 38 ± 6 per 10 6 septal cells added) or septal-cerebellar coaggregates ( 85 ± 29 per 10 6 septal cells added). These results, taken together, suggest that hippocampal target cells influence the development and survival of cholinergic neurons.