microbial production of value-added products from wood hemicellulose prehydrolysate
abstract
hemicellulose, the second most abundant polymer in nature, has the great potential to be used for
the production of biochemicals under the concept of biorefining. as hemicellulose is watersoluble, it can also be easily obtained by the pre-hydrolysis of wood prior to the pulping processes
such as kraft delignification or during the production of dissolving pulp. utilization of these
byproduct streams can play an important role in the development of a circular bioeconomy as it
helps in maintaining the materials and resources for a long period instead of disposing it as waste.
in the biochemical conversion platform for biorefineries, it is crucially important to use all
fermentable sugars of lignocellulosic biomass including both hexose (c-6) and pentose (c-5)
sugars.
the overall objective of this study was to produce bioplastic building blocks from hemicellulose
streams. the specific objective of this work was to investigate the possibility of using wood-based
hemicellulose for microbial production of value-added biochemicals including microbial oil,
biopolyol and poly-γ-glutamic acid. the hemicellulose pre-hydrolysate used in this study was
produced by a proprietary pretreatment process. composition analysis of the hemicellulose prehydrolysate indicated that it had 143.89 ± 1.28 g/l of xylose along with smaller quantities of sugars
like glucose and arabinose.
bioconversion of hemicellulose sugar to microbial oil is one possible way to valorize this industrial
side stream. an oleaginous yeast cryptococcus curvatus (atcc 20509) was selected for this
bioconversion as it is known to accumulate high content of lipids and is able to grow on complex
lignocellulosic hydrolysates/pre-hydrolysates even in the present of impurities. initially, the effect
of xylose concentration and carbon to nitrogen ratio were investigated in order to maximize the
lipid accumulation. the robust yeast strain used was able to produce 13.78 g/l of cell biomass and
5.13 g/l of lipid after 164 hr of fermentation using poplar wood pre-hydrolysate without
detoxification. the obtained microbial oil was characterized to identify its fatty acid profile. oleic
acid (45.86 ± 0.69 wt%) was found to be the main fatty acid present. this fermentation was scaled
up in a batch bioreactor with 1 l capacity. 16.54 ± 0.65 g/l of cell biomass and 6.97 ± 0.58 g/l of
lipid were obtained in the reactor which had better control of environmental conditions.
the microbial oil produced was then used as feedstock for production of bio-based polyol which
has many applications in polymer industry and importantly serves as a precursor for polyurethane
production. the bio-polyol was produced using a two-step approach: epoxidation followed by
ii
ring-opening reaction. lipase enzyme produced by candida antarctica (novozyme 435) and
immobilized on acrylic resin was used as an unconventional catalyst for in situ epoxidation of
microbial oil. 84.55 ± 1.80% conversion was achieved after 12 hours. novozyme 435 was found
to be very stable and can be reused up to 3 cycles efficiently. in the second step, isopropanolamine
was used to open the epoxy ring with the addition of hydroxyl group. hydroxyl value and acid
number of the microbial-based polyol were found to be 299.53 ± 1.24 mg koh/g and 4.93 ± 1.07,
respectively. fourier transform infrared spectroscopy (ftir) and nuclear magnetic resonance
(nmr) were used for structural confirmation of produced bio-polyol. production of biobased
monomers from renewable materials using enzyme catalysts can be considered clean and leads to
energy saving processes. therefore, synthesis of renewable polymers via enzymatic
polymerizations of biobased monomers provides an opportunity for achieving green polymers and
a future sustainable polymer industry.
the production of another useful chemical, poly-γ-glutamic acid (pga) was also studied. pga is
known to have a number of useful applications. in order to make this fermentation efficient, the
detoxification of the pre-hydrolysate was carried out using a method previously developed in our
lab. this detoxification method includes the combination of vacuum evaporation and solvent
extraction procedures as a result of which acetic acid and hydroxy methyl furfural (hmf) were
removed effectively with minimal loss of xylose. bacillus subtilis (atcc 23857), which is a
glutamic acid-dependent strain, was used for this bioconversion. initially, the concentration of pure
xylose and l-glutamic acid were optimized by synthetic medium using response surface
methodology (rsm). 65.40 g/l of xylose and 44.98 g/l of l-glutamic acid were found to be the
optimal concentrations for maximum production of pga. this strain was able to produce 12.93 ±
0.9 g/l of pga after 96 hr of fermentation in pre-treated hemicellulose. such studies on the
production of pga from renewable sources will contribute to further development of biorefineries
and lead to commercial scale production of such products.
overall, the findings of this dissertation will contribute to the utilization of available hemicellulose
streams through fully biobased processes that can lead to the development of a successful
economically feasible circular bioeconomy.