3. MATERIALS AND METHODS 72
3.6. Evaluation of a Non-Conventional TPPB System in Pyrene
3.5.3.2. Mixed substrate condition
Pyrene biodegradation efficiency by the Mycobacterium in concomitant presence of other PAHs, naphthalene and anthracene, were also evaluated employing the developed TPPB system. The fermenter operating conditions were identical with those in the single substrate condition, as described earlier. However, organic phase was prepared by dissolving anthracene and pyrene each at previously found optimum concentration of 400 mg l-1 in silicone oil by the use of ultrasonic water bath for 1 h. Both aqueous and organic phases were loaded in the fermenter and autoclaved. Requisite amount of naphthalene was then added after dissolving in a small volume of pre-sterilized silicone oil. Fifty ml of overnight grown M. frederiksbergense culture was aseptically added into the bioreactor as inoculum. This mixed substrate experiment was run for 7 d, and during which duplicate samples of 1 ml each were withdrawn from the fermenter everyday for analyzing the residual PAHs concentrations in the organic phase.
3.6. Evaluation of a Non-Conventional TPPB System in Pyrene
Pyrene was delivered either in silicone oil or water miscible solvent or solubilised in surfactant solution. The encapsulating material used was either alginate or alginate-PVA mixture.
Table 3.6 shows the variations involved while preparing the five different pyrene encapsulated beads. The table shows that the variations were with respect to the solvent used to solubilise pyrene, encapsulating material, presence or absence of additives and composition of the gelling medium. Pyrene at 1 g l-1 was dissolved either in silicone oil (Type I, II, III and V) or DMSO (Type IV) as the organic solvent. The encapsulating material consisted of either alginate (2% w/v) (Type I and IV) or alginate-PVA mixture (2% w/v and 5% w/v respectively) (Type II, III and V), which was further mixed with pyrene containing organic solvent either by adding Triton X-100 for bead types I, II, III and IV or without adding any surfactant in case of the bead type V.
Table 3.6: Variations in the preparation of different pyrene encapsulated bead types in the non-conventional TPPB system.
Bead Types
I II III IV V
Delivery phase Silicone oil Silicone oil Silicone oil DMSO Silicone oil
Alginate (2% w/v) + + + + +
PVA (5% w/v) - + + - +
Surfactant + + + + -
CaCl2 (20% w/v) + + + + +
Boric acid
(Saturated) - - + - +
The mixtures were then emulsified using a high-speed homogenizer (T25, IKA®, Germany) at 7,000 rpm for 10 min in an ice bath, and the resulting o/w emulsion was placed on an orbital shaker set at 150 rpm for 6 h to deaerate. Encapsulated beads were subsequently prepared by extruding the o/w emulsion in a drop wise manner into gently agitated 200 ml of chilled 20% w/v calcium chloride solution containing 0.2% Triton X- 100 using a fine 21-gauge stainless steel needle from a distance of about 6 cm above the surface of the gelling solution. The gelling solution for bead types III and V contained saturated boric acid in addition to calcium chloride and Triton X-100. The prepared beads were allowed to harden for 12 h, washed with distilled water and air dried for 12 h at room temperature.
To estimate the pyrene encapsulation efficiency of the beads, the following relationship was used:
( )
Encapsulation effeciency (%) = M - M⎡⎢⎣ i d Mi⎤×100
⎥⎦ (3.4) where Mi is the initial mass of pyrene present in the hydrogel/mixture solution prior to the drop wise extrusion into gelling medium and Md is the residual mass of pyrene measured in the gelling medium immediately after preparation of the pyrene-loaded beads.
Further, to select the best pyrene encapsulated bead among the five different bead types, pyrene release from the beads was carried out in 250 ml Erlenmeyer flasks each containing 100 ml of 10 CMC Triton X-100 solution. Accurately weighed dried beads known to contain 500 µg pyrene (based on mass balance) were added to the flasks, incubated at 28°C and shaken at 180 rpm in an orbital incubator shaker. Samples (1 ml) were taken from the flasks at regular intervals for 72 h; and for the every withdrawal of
maintain a constant volume of the release medium in the flasks. Concentrations of pyrene in the samples were analyzed and the bead type yielding highest and sustained pyrene release was, therefore, chosen for further characterization study.
3.6.2. Characterization of the bead type V 3.6.2.1. Swelling behavior
Initially prepared beads of this type (V) were weighed (Wi) and placed in a beaker with a dissolution medium containing 10 CMC Triton X-100 solution and was agitated at 180 rpm on a shaker–incubator at 28°C. After every 10 min, beads were removed from the solution, blotted with Whatman® filter paper to remove excess water and re-weighed (Wf). The swollen beads were handled carefully in order to avoid breakage or erosion of the beads. Swelling - the percentage increase in weight of the beads due to the absorbed water was estimated using the following equation:
(
f i)
iSwelling (%) = W⎡⎣ −W W⎤⎦×100 (3.5)
3.6.2.2. Optical and scanning electron microscopy
To clearly distinguish silicone oil droplets in the encapsulated beads, cross- sections of the air dried beads was observed under stereo zoom microscope (Nikon, USA). Prior to this, the cross section of the beads was flooded with coomassie blue stain for 10 min, washed with distilled water to remove excess stain and air dried for 1 h. The morphology of the beads were also observed under a scanning electron microscope (Leo 1430 VP, UK), for which the beads were lyophilized for 12 h, dried under vacuum and
their cross-sections were mounted onto stubs using double-sided adhesive tape for vacuum coating with gold film using sputter coater (Edward, UK).
3.6.2.3. Pyrene release kinetics
To investigate the effect of surfactant concentrations in the release medium on pyrene release, calculated amount of the encapsulated beads containing 500 μg pyrene were taken in 250 ml Erlenmeyer flasks with different concentrations of Triton X-100 (2- 10 CMC) in 100 ml release medium. Experiments were carried out in the same manner, as before for investigating the pyrene release profile from the beads. However, in this particular characterisation study, 0.5 ml silicone oil containing an equivalent amount of pyrene in 100 ml 10 CMC Triton X-100 solution taken in a 250 ml flask served as the control. Samples (1 ml) were collected from the sink medium for 24 h, and an equivalent volume of fresh medium was replaced for every sample withdrawal from the flask.
3.6.2.4. Reusability test of the bead type V
To check the feasibility of reusing the beads for reloading pyrene following its initial exhaustion, virgin silicone oil containing beads (without pyrene) and blank control beads (excluding both silicone oil and pyrene) were prepared as before and accurately weighed to 500 mg. The beads were then soaked in 100 ml unsaturated pyrene solution (50 mg l-1 in 10 CMC Triton X-100 solution) for 48 h in shaking incubator set at 28°C and 180 rpm. At the end of the incubation period, beads were washed with distilled water, vacuum dried and subsequently subjected to reveal its pyrene release behavior.
3.6.3. Pyrene biodegradation experiments using the bead type V
To demonstrate the utility of the developed non-conventional TPPB method for pyrene biodegradation applications, experiments were carried out using M.
frederiksbergense with the pyrene encapsulated bead type V. Calculated amount of beads containing 100 μg pyrene were taken in 250 ml Erlenmeyer flasks with 100 ml BH media inoculated with 5% v/v overnight grown culture of M. frederiksbergense. An abiotic control flask containing an equivalent amount of beads with BH media was also included in the experiments. All the flasks were incubated in a shaker incubator set at 28°C and 180 rpm, and samples were collected at a regular interval for 5 d from the aqueous phase for measuring pyrene concentrations.