Utilization of Low-Cost Fatty Acid Sources by Bacterial Isolate …
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2 Materials and Methods
2.1 Microorganism and Chemicals
Paper mill wastewater was collected from Raniganj area, West Bengal, India,
from where S. marcescens NITDPER1 was isolated and maintained in laboratory
conditions. All the reagents were from Sigma-Aldrich, Merck and Himedia unless
otherwise mentioned and were of analytical grade.
2.2 Batch Studies for Pigment Production
Five different media, namely, nutrient broth, peptone glycerol broth, peanut powder
broth (2%), peanut oil broth (2%) and palm oil broth (2%) were prepared (100 ml)
and autoclaved at 121 °C, 105 kPa for 20 min followed by inoculation with 5%
overnight-grown bacterial culture. The flasks were kept in an incubator at a shaking
speed of 120 rpm and 30 °C temperature for 96 h. At regular interval, 10 ml culture
was taken out from the flask, and pigment was extracted with 2 ml acidified ethanol
following Suryawanshi et al. (2014). Growth was monitored at regular intervals
spectrophotometrically by withdrawing 1 ml culture volume and drying the pellet
biomass.
2.3 Characterization of the Extracted Pigment
The extracted pigment was characterized by UV–vis spectrophotometery (200–
800 nm) and TLC using chloroform:methanol (9:1) as the solvent system (de Araújo
et al. 2010). The HPLC profile was analysed using acetonitrile: HPLC water (60:40)
as solvent system (Faraag et al. 2017). FTIR spectra were recorded between scanning ranges of 400 and 4000 cm
−1 and
1 H NMR (125 MHz) spectroscopy (Bruker
Advance 500 NMR) was done using chloroform-d2 (CDCl 3 ) as the solvent.
2.4 Kinetic Analysis of Growth and Pigment Production
Growth- and production-associated kinetic parameters such as specific growth rate
(μ), generation time (T G ) and growth-associated (α) and non-growth-associated (β)
kinetic constants for product formation were determined. Production kinetics was
studied using Luedeking–Piret model (Luedeking and Piret 1959), which is given as
d p/dt = α dx/dt + βx
(1)
23
2 Materials and Methods
2.1 Microorganism and Chemicals
Paper mill wastewater was collected from Raniganj area, West Bengal, India,
from where S. marcescens NITDPER1 was isolated and maintained in laboratory
conditions. All the reagents were from Sigma-Aldrich, Merck and Himedia unless
otherwise mentioned and were of analytical grade.
2.2 Batch Studies for Pigment Production
Five different media, namely, nutrient broth, peptone glycerol broth, peanut powder
broth (2%), peanut oil broth (2%) and palm oil broth (2%) were prepared (100 ml)
and autoclaved at 121 °C, 105 kPa for 20 min followed by inoculation with 5%
overnight-grown bacterial culture. The flasks were kept in an incubator at a shaking
speed of 120 rpm and 30 °C temperature for 96 h. At regular interval, 10 ml culture
was taken out from the flask, and pigment was extracted with 2 ml acidified ethanol
following Suryawanshi et al. (2014). Growth was monitored at regular intervals
spectrophotometrically by withdrawing 1 ml culture volume and drying the pellet
biomass.
2.3 Characterization of the Extracted Pigment
The extracted pigment was characterized by UV–vis spectrophotometery (200–
800 nm) and TLC using chloroform:methanol (9:1) as the solvent system (de Araújo
et al. 2010). The HPLC profile was analysed using acetonitrile: HPLC water (60:40)
as solvent system (Faraag et al. 2017). FTIR spectra were recorded between scanning ranges of 400 and 4000 cm
−1 and
1 H NMR (125 MHz) spectroscopy (Bruker
Advance 500 NMR) was done using chloroform-d2 (CDCl 3 ) as the solvent.
2.4 Kinetic Analysis of Growth and Pigment Production
Growth- and production-associated kinetic parameters such as specific growth rate
(μ), generation time (T G ) and growth-associated (α) and non-growth-associated (β)
kinetic constants for product formation were determined. Production kinetics was
studied using Luedeking–Piret model (Luedeking and Piret 1959), which is given as
d p/dt = α dx/dt + βx
(1)