Page 79 - Refining Biomass Residues for Sustainable Energy and Bioproducts

54 Refining Biomass Residues for Sustainable Energy and Bioproducts

Its tale dates back to the 1950s when the lipid inclusions soluble in chloroform
were detected for the first time in Azotobacter sp. (Sudesh et al., 2000). In the ear-
lier 1970s, similar inclusions were identified by Lemoigne (1927) as PHB (polyhy-
droxybutyrate) and its first patent was filed by J. N. Baptist. Following this was the
discovery of HA by Rohwedder and Wallen (Wallen and Rohwedder, 1974) which
caught the attention of many researchers due to extensive applications unlike PHB
(Sudesh et al., 2000). Earlier such storage products were thought to be a part of
only Gram-negative bacteria while the flourishing research later spotted it in many
other Gram-positive bacteria, archaebacteria, and many other microbes and today
we have more than 160 types of existing HAs (Steinbu ¨chel, 1991; Rehm and
Steinbu ¨chel, 1999). There are different possible HA units among which valerate
and butyrate were recognized as predominant constituents while hexanoate was
noted down as a minor component (Findlay and White, 1983). Not until the next
decade, the commercial production and marketing for PHA and PHB was first initi-
ated in 1982 (Anderson and Dawes, 1990). Globally, it is expected that the bioplas-
tics industry will reach a market of approximately 6 billion US dollars in 2021
(Kumar et al., 2016a,b).
The next revolution for PHA was brought up in the field of molecular biology
(1970s) where the genes encoding the synthesis process are well characterized.
Toward the end of 1980s, the PHA synthesis genes were already realized in
Ralstonia eutropha and Escherichia coli. Beginning of the 20th century identified
the presence of raw lipid inclusions which were later identified as PHA. Now, the
bigger picture is to engineer the proteins involved in the synthesis in order to artifi-
cially control the process for a better efficiency.

3.2.1.1 Structural characteristics and analytical techniques
of polyhydroxyalkanoates
PHA is a biocompatible, optically active, and a nontoxic thermoplastic. Their pro-
duction is highly promising to nature as they can be easily produced from renew-
able resources. This polymer exhibits a isotactic structure with piezoelectric
properties. The enzyme governing its production is PHA synthase whose stereo-
specificity is responsible for the R configuration of the HA monomeric units mak-
ing the compound isotactic. Only rare cases show the opposite S configuration
(Haywood et al., 1991; Reddy et al., 2003). Monomeric units of PHA are polymer-
ized to form the structure weighing between 2 and 3 lakh Da depending upon the
growth parameters of the microbe and the involved surrounding (Mobley, 1994).
Owing to a high refractive index, PHA inclusions are easily viewed under phase
contrast light microscope. They appear to be 200 500 nm (diameter) in size, float-
ing in the cell cytoplasm (Dawes and Senior, 1973). Transmission electron micros-
copy is also used to morphologically characterize the PHA granules. Dyes like Nile
red and Sudan black B can be used to visualize these granules as staining confirms
their lipid nature (Burdon 1946; Spiekermann et al., 1999). The process of dye
staining can only confirm the physical presence but its chemical characterization
is done with HPLC (high-performance liquid chromatography), GC MS