The authors have declared that no competing interests exist.
Plastic materials have been used as packaging materials and also have other numerous applications because of their durability and stability. Plastic products are non degradable and they continue to exist in the environment thereby causing a serious threat to the ecosystem. Bioplastics which are biodegradable plastics are emerging out as a boon to overcome the problem of plastic accumulation. Polyhydroxybutyrate or PHB is a bioplastic that serves as an alternative to synthetic plastics. PHB is a lipid reserve material that gets accumulated within the cell wall of micro organisms under stress conditions. Halophilic microorganisms can be of much use in the production of PHB as it is cost – effective and recovery of PHB is much easier in halotolerant organisms. Hence this study focuses on the isolation of PHB producing halotolerant bacterial organisms from marine sources. Screening of PHB positive isolates was done by Sudan Black B and Nile blue A staining. Large scale production of PHB by the efficient bacterial strain was done by using wastewater as the substrate.
Modernization and progress has its share of disadvantages and one of the main aspects of concern is the pollution it is causing to earth
Soil samples were collected from the proximities of VGP Golden Beach and Neelankarai Beach, Chennai. The samples were collected in sterile zip lock covers and were immediately transferred to the laboratory and stored at 4°C.
1g of soil sample collected was inoculated in Nitrogen-deficient medium without agar
Bacterial smears were made on sterile glass slides, heat fixed and stained with an ethanolic solution of Sudan Black B to visualize the PHB granules. The prepared smears of the cultures were stained for 10 minutes with Sudan Black solution. The slides were then washed with running tap water and air dried. It was then counter stained with 0.5% Safranin for 5 minutes and washed with running water
The bacterial samples were smeared on glass slides, air dried and heat fixed. The smear was flooded with 1% Nile blue A and heated in a water bath at 55°C for 10 minutes. The stain was washed off with water and 8% aqueous solution of acetic acid for 1 minute. The slide was air dried and observed under a fluorescence microscope at an excitation wavelength of 460 nm
After 72 hours of incubation at 37°C in Nitrogen Deficient Medium, the culture broth was centrifuged at 8000 rpm for 15 minutes. The pellet along with 10 ml Sodium hypochlorite solution was incubated at 50°C for 1 hour for lyses of cells. The cell extract obtained was centrifuged at 12000 rpm for 30 minutes and then washed sequentially with distilled water, acetone and absolute ethanol. After washing, the pellet was dissolved in 10 ml chloroform and incubated at 50°C overnight and evaporated at room temperature. After evaporation, 10 ml of sulphuric acid was added to it and placed in water bath for 10 minutes at 100°C. This converts the PHB into crotonic acid, which gives maximum absorption at 235 nm using sulphuric acid as blank
The extracted PHB was quantified by Crotonic acid assay
FT – IR analysis was carried out in an IR Affinity – 1, Shimadzu in Ethiraj College for Women, Chennai. The spectrophotometer was operated in the range of 4000 – 500 cm-1.
The isolated bacterial strains were grown individually on MSM for 24 hours. The bacterial strains in the Mineral Salts Medium were centrifuged at 8000 rpm for 10 minutes and the pellets were immediately re – suspended in 2% Glutaraldehyde with 0.05 M phosphate buffer and 4% sucrose (pH – 7.3). Fixation was obtained overnight at 4°C. After 24 hours the pellets were centrifuged at 8000 rpm for 10 minutes, washed 4 times with distilled water and placed on aluminium foil. The samples were then dehydrated with series of gradient ethanol (10%, 20%, 30% till 90%) air dried and finally the dried flakes were analyzed under the Scanning Electron Microscope (FEI Quanta 200 F). The analysis was performed in IIT Madras, Guindy.
The bacterial strains were morphologically identified by Gram’s Staining and further characterized by various biochemical tests such as Catalase test, Oxidase test, IMViC tests, Triple Sugar Iron Agar test, Starch Hydrolysis Test and Carbohydrate Fermentation test.
The isolated bacterial strains were morphologically characterized and identified by DNA isolation, 16s rRNA sequencing and construction of phylogenetic tree.
To study the production of PHB by fed batch process in wastewater using molasses as a carbon source. The composition of wastewater is given as follows: (mg/l) Glucose – 500, Sodium hydrogen carbonate – 300, Ammonium hydrogen carbonate – 50, Potassium dihydrogen phosphate – 22.5, Magnesium sulphate – 50, Manganese sulphate – 0.03, Zinc sulphate – 0.04, Calcium chloride – 1, Ferric chloride – 0.32 and Yeast extract – 50. Blackstrap molasses was obtained in 500 ml volume from Dhanyam, Chennai.
In a 250 ml Erlenmeyer flask, 100 ml of sterilized wastewater with 4% molasses
For the large scale production of PHB, a bench scale bioreactor of 3L capacity was used for the laboratory study on PHB production under saline conditions. The experimental setup was as follows:
Wastewater – 2.5 litres
Molasses – 100 ml (4% concentration)
Broth culture of bacterial strain – 100 ml (4% concentration)
pH – 7.5 – 8.0
Temperature - 37°C
The bench scale reactor was continuously aerated with constant agitation using the agitator provided in the fermeter at 500 rpm. The wastewaster sample from the reactor was analysed for the physico – chemical parameters such as pH, salinity, TDS using an external pH probe, total protein (Bradford’s procedure), chloride, hardness, Dissolved oxygen
Enrichment of the bacterial cultures was performed in Nitrogen Deficient Medium under halotolerant conditions with 3% NaCl. A total of 16 isolates were obtained, out of which 10 isolates were obtained from VGP soil samples and 6 were obtained from Neelankarai Beach samples. Out of the 16 isolates, 2 bacterial isolates (NFKVG 8 and NFKVG 10) screened positive for PHB production by Sudan Black B staining.
Primary screening was done in order to determine the PHB production of the bacterial strains. These strains were further used for the analysis of PHB polymer. Both the bacterial strains NFKVG 8 and NFKVG 10 accumulated PHB in the form of intracellular granules and these granules were able to absorb the Sudan Black stain and the cell wall of the strains was stained pink as a result of the counter stain used. Under magnification of 100 x it was seen that the granules were stained black against a pink background taken up by the Safranin stain (
The individual bacterial strains were grown in Minimal Salt Medium (MSM) to study the growth pattern and also to determine the PHB yield. It was shown that the individual bacterial strains showed maximum growth for the bacterial strain NFKVG 8 – 0.668 and NFKVG 10 – 0.621 respectively (
|
|
|
NFKVG 8 | 0.997 | 50 |
NFKVG 10 | 0.986 | 37 |
The Sudan black B stained positive bacterial strains were further screened by Nile Blue A, a more specific stain for PHB production which is a more rapid and sensitive method. After staining with Nile Blue A, the PHB accumulating granules showed bright orange fluorescence on irradiation with UV light. Hence the fluorescence of PHB granules was checked from Day 0 to Day 3 to determine the increase in the PHB production. The fluorescence intensity increased with the increase in PHB content of the bacterial strains. The fluorescence was observed under 100 x magnification (
FT – IR spectra of the PHB production predicts the presence of functional groups of PHB
|
|
|
---|---|---|
3411.26 | O – H stretch | Variable, broad |
2923.25 | C – H stretch | Strong |
2860.56 | C – H stretch aldehyde | Variable |
1455.35 | C – H alkane | Variable |
1030.03 | C – O stretch | Strong |
To quantify the production of PHB, Crotonic acid was used as a standard at concentrations ranging from 5 – 50 µg/ml (
|
|
|
|
|
1 | - | 3 | - | 0 |
2 | 50 | 2.950 | 5 | 0.532 |
3 | 100 | 2.900 | 10 | 0.687 |
4 | 150 | 2.850 | 15 | 0.790 |
5 | 200 | 2.800 | 20 | 0.878 |
6 | 250 | 2.750 | 25 | 0.891 |
7 | 300 | 2.700 | 30 | 0.934 |
8 | 400 | 2.600 | 40 | 0.959 |
9 | 500 | 2.500 | 50 | 0.971 |
SEM analysis was carried out to determine the morphological structures of the bacterial strains. The ultra structure of the bacterial strains NFKVG 8 and NFKVG 10 was observed under 11000 x magnification and it was seen that the strains showed rod shaped morphology (
According to the results of the various biochemical tests that were performed, the individual bacterial isolates were classified upto the genus level and they were characterized to belong in the
|
|
|
---|---|---|
|
||
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Alkaline slant and butt | Alkaline slant and butt |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Bioreactor studies for the large scale production of PHB were carried out first in 250 ml and 500 ml Erlenmeyer flasks with the most efficient bacterial strain – NFKVG 8. Water analysis was done for the wastewater which was used for the scale up in the production of PHB (
pH | 7.5 |
Temperature | 29.7°C |
Conductivity | 1413 mScm-1 |
Total Dissolved Solids | 250 ppm |
Salinity | 171 ppm |
Dissolved Oxygen | 1.8 ppm |
Hardness | 16.4 ppm |
Chloride | 61 ppm |
PHB is the most popular and the best characterized polymer belonging to the class of polyhydroxyalkanoates. It could be used for applications similar to those of common plastics and could fit well into new waste-manageable strategies as well. The recovery of PHB from the cell wall of extreme halophiles is much easier when compared with eubacteria. Hence in this study, halotolerant bacterial strains were isolated from marine soil and the PHB production was compared in both the bacterial strains. The efficient bacterial strain was used in the large scale production of PHB using wastewater. The future perspectives of this study would be to employ novel methods of PHB production which are both cost – effective and environmentally friendly. This could be done by commercializing the production of PHB by using wastewater as the substrate for the production of PHB.