Polyhydroxyalkanoate from Bacillus sp.: Its production, isolation and characterization

Synthetic plastics are widely used for the manufacture of packaging
materials, various household and industrially important articles. However they are
recalcitrant to microbial degradation and they persist in the soil leading to
environment pollution. To overcome the problem of plastic pollution, attempts are
being made to replace synthetic plastics by various biopolymers and bacterial
polyhydroxyalkanoate (PHA) is one amongst them. PHA is composed of hydroxy
fatty acids and it represents a rather complex class of storage polymers synthesized
by a various bacteria and are deposited as unique water insoluble cytoplasmic nano
sized inclusions. Bacillus spp. represent industrially important group of organisms
that are used for various metabolite production and hence attempt was made to
isolate this bacterium for polymer synthesis. Based on 16SrRNA, isolated Bacillus
sp. was identified as B. flexus and it was found to posses 97% homology with B.
megaterium it shared the same cluster with B. simplex. This study has led to a finding
of a new unexplored B. flexus strain, which has the ability to produce PHA at 50-
60% level in the biomass. B. flexus grew optimally in media containing different
salts of nitrogen, amino acids, carbon sources, plant oils, free fatty acids, economic
media components such as palm oil effluent, molasses, corn starch, whey, rice and
wheat bran extracts as sources of carbon/nitrogen/nutrients. PHA isolated from the
cells was characterized by FTIR/GC/GC-MS/NMR spectroscopy/DSC.
Homopolymer of polyhydroxybutyrate was synthesized in B. flexus cells fed with
only sucrose as main carbon source and PHA copolymer of polyhydroxy(butyrateco-
valerate-co-octanoate) was produced in palm oil effluent containing medium and
polyhydroxy(butyrate-co-octanoate-co-decanoate) with rice bran oil as co carbon
substrate. This study has shown that based on the fatty acid supplemented in the
medium, medium chain length PHA such as octanoate and decanoate can be
synthesized by Bacillus spp also. Response surface methodology was used for
optimization of nutritional parameters for optimum growth and polymer yield.
Maximum production of biomass (7 g/l) and PHA (2.2 g/l) were obtained with 11.6
g/l of KH2PO4, 4.7 g/l of ammonium phosphate and 31 g/l of sucrose. The growth
kinetics of B. flexus under nutrient limitation was studied by using a simple model
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giving mathematical description of kinetics of microbial growth, substrate
consumption and product formation.
PHAs are accumulated intracellularly and hence their extraction from the
biomass is a critical step for economic production. Numerous separation processes
are employed for the recovery of PHA. These involve extraction by organic solvents,
which is hazardous and explosive. In the present study, novel physical, chemical and
biological methods were assessed for isolation of the intracellular PHA. Analysis of
cell wall of B. flexus indicated that the type of nutrients used for cultivation
significantly influences its composition. Cells grown in inorganic medium contained
lower quantities of amino acids and lacked diaminopimilic acid in the cell wall and
cells lysed easily and this can be further exploited for easier recovery of the
intracellular PHA. Amongst different alkalis tested for cell hydrolysis, NH4OH
efficiently digested non-PHA cellular material at pH 11, to give 50% of PHA with
98% purity. The enzyme produced by Microbispora sp. hydrolysed the cells of B.
flexus to release the intracellular PHA. The enzyme was identified as a protease of
40kDa and it was purified to homogeneity. Aqueous two-phase system was
successfully employed as a non-organic solvent method for the isolation of PHA
from other cellular materials. Exposure of cells to gamma irradiation indicated that
the irradiation resulted in cell lysis leading to easy PHA extractability, low degree of
cross-linking, and improvement in molecular weight as well as tensile strength of the
polymer. Lambda lytic gene was integrated into the amylase gene (amyE) locus of
the chromosomal DNA of B. flexus. This caused autolysis of the recombinant cells
when xylose was supplemented into the medium. The study has focused on
economic production of PHA by a newly isolated strain of bacterium, which is so for
not reported as PHA producer, and newer aspects of PHA isolation methods have
been worked out.